Embed Size (px)
Citation preview
Sodium Leak Current through a Non-selective Cation
Channel Regulates Spontaneous Activity of Pacemaker Cells
by
Tom Ziming Lu
A thesis submitted in conformity with the requirements
for the degree of Doctorate of Philosophy
Department of Physiology
University of Toronto
© Copyright by Tom Ziming Lu (2012)
ii
Sodium Leak Current through a Non-selective Cation Channel Regulates
Spontaneous Activity of Pacemaker Cells.
Tom Ziming Lu
Doctorate of Philosophy
Department of Physiology
University of Toronto
2012
Abstract
Pacemaker cells are involved in regulating numerous essential biological functions. These cells
exhibit spontaneous activities in isolation, due to the various membrane ion channels.
Background conductances (ILeak) are responsible for the depolarized resting membrane potential
of numerous pacemaker cells, largely due to a high sodium component (INa Leak); however, their
molecular identity remains unclear. NALCN is a non-selective cationic channel that was
suggested as a major contributor to the background Na+ conductance in some neurons. This
thesis investigates whether and how NALCN contributes to the INa Leak of two major pacemaker
systems: neurons and heart. Chapter 4 utilizes the benefits of a simplified respiratory pacemaker
neuron of L. stagnalis to determine whether NALCN has a functional role in regulating
pacemaker cell activity and function. Using an acute gene silencing approach, reduction of
endogenous NALCN orthologue expression in isolated respiratory pacemaker neuron
hyperpolarized the resting membrane potential, due largely to the reduction of INa Leak, which
partially affect respiratory behavioural output. Chapter 5 further determined that the NALCN
orthologue of is a major contributing current to the pacemaker neuron subthreshold conductance.
By using a computation model, developed in collaboration, simulation of spontaneous pacemaker
activity was shown to be more sensitive to Na+ leak than K
+ leak conductance. Together, these
iii
studies indicate that NALCN codes for a fundamentally important INa Leak that is capable of
robust regulation of spontaneous activity. Chapter 6 investigated the role of a NALCN-like
conductance in the establish pacemaker model of the murine sinoatrial node cardiomyocyte.
Given the lethality of NALCN knockout, a combined pharmacological and eletrophysiological
analyses were performed. Results suggest a novel INa Leak that is highly sensitive to Gd3+
and
Co2+
, which is similar to biophysical properties of NALCN channels. Furthermore, functional
NALCN-dependent INa Leak requires an auxiliary subunit co-expression. Interestingly, regional
regulation of NALCN-associating subunits correlates strongly with my electrophysiological
observations. These findings indicate an important role for the NALCN channel in regulating the
spontaneous activity of at least two pacemaking systems.
iv
Acknowledgements
“Pain is temporary. It may last a minute, or an hour, or a day, or a year, but eventually it will
subside and something else will take its place. If I quit, however, it lasts forever.”
-Lance Armstrong
It almost felt like yesterday when I began graduate school here. The quick four years of intense
study opened up potentials, opportunities and challenges that I could not have imagined.
Although sitting at this moment is about the highest point of my exhilaration, I could not help
reflecting back on the countless nights and weekends that I struggled to get to this step. To say it
was my own pure effort would be a selfish one. My drive, optimism, confidence and stamina are
a product of many supportive groups of people. What a daunting task of summarizing those who
contributed to my accomplishments, but I will make mention some of those who have made the
most sacrifices and influences on my own personal growth through my graduate life.
It seems almost cliché to start with my parents, but unconditional care from my parents is,
perhaps, the strongest support that helped me to this point. I will be the first to admit that it isn’t
easy raising an only child; let alone a tenacious man such as me. I feel forever in debt to the
amount of personal and financial sacrifices my parents made. My parents taught me early on that
life isn’t easy. Look back at what they took to get to where they are is something that I will never
be able to fully appreciate. My mom’s personal courage for going through school and getting a
new degree is something that I can only appreciate as I grow older. My dad, being the family
glue that holds everyone together, is someone whom I can learn a great deal from time and time
again. The path they took is a challenging one, and they carved out many opportunities that I
would have never been able to experience. My parents are one of my primary educators, and they
will continue to be.
Next, I feel blessed to have been supervised by Dr. Zhong-Ping Feng. I can’t underscore how
much personal and intellectual influences she has instilled in me throughout my undergraduate
and graduate studies. I appreciate the academic and personal standards she sets for her students
and herself. One of the strongest qualities about her that motivates me in my academic life is her
ability to multitask and follow through with her commitments. She encourages students to “do
their best and commit to as many activity as they can, but never over commit”. I can see this in
her academic career where she is heavily involved in with academic and research work, but she
always manages to complete her obligations. Observing her inspired me to emulate her strong
qualities. Yet time again, I always remember to not only do my best, but also be responsible to
each of my commitments. The lessons and skills I took away from her inspiration will be
treasured for life. Through the mentorship of Dr. Zhong-Ping Feng, I have begun to pursuit an
academic career that was beyond what I initially thought I could achieve. Coming in as a
Master’s student, I initially did not have the intention of going for a Ph.D. She has sparked a
drive for me to aim higher in my academic career. I feel fortunate to have Dr. Feng as my
inspiration. We joke about how difficult it is to keep discussions short of just 30 minutes, but it
demonstrates how much I take away from each conversation, many of which I will always
cherish.
Also inspirational are my supervisory committee members, Dr. Beverley A. Orser and Dr.
Evelyn K. Lambe. Both have offered me examples for what scientists ought to achieve. Their
v
contributions, suggestions, criticisms and words of encouragements helped to fuel my scientific
endeavor. It made me question my findings each and every step. Despite how exhaustive I feel I
have thought about my topic, I knew there will always be new possibilities raised during each
committee meeting. Dr. Orser and Dr. Lambe both helped me understand the intricate means of
being a successful scientist. I am fortunate that I have two excellent role models to emulate.
I also owe a great deal of gratitude to my former and current sidekicks. Dr. Nasrin Nejatbakhsh
(N2!), Dr. Kwokyin Hui, Christine Bae (Master?), Wojciech Kostelecki, Marielle Deurloo, Mila
Aleksic, Andrew Barszczyk, Kathy Li (Stephen!), Roddy Zhou (the things that you say), Gukan
Svoid, Yi Quan and Ammar Alibrahim; you guys have all meant a great deal to me for the past
few years. Having a cohesive group of people to work with is not easy, so I really appreciate the
countless hours of joy and excitement offered during often challenging and difficult times
throughout graduate school. The memories of travelling to conferences, the joy of hanging out
during after hours, the delight of sharing my life’s ups and downs for the past four years is
something that I will not forget.
Over the years there are countless students that have come and go from our lab. I had great
privilege to have worked with many talented people and I want to acknowledge their
contributions to my success. Annie Wang, Ramak Khosravi and Nancy Dong; you guys have all
helped me greatly with experiments and data collection. Without your contribution my project
would not be where it is at right now. There are many more unmentioned individuals, whom
have added to the Feng Lab atmosphere. Thank you all.
Outside of the lab, there are many others whom have made graduate studies all that more interest
and exciting. The members of GASP have made social events fun and engaging, and the
neighbor lab dwellers at MSB whom I can always count on to help me out. Although there are
far too many to name I want to at least make mention to a few individuals. Keith Ho, Tiffany Ng,
Robert Chen, Alex Han and Wayne Huang; you guys make conferences feel like a party! Eliane
Proulx, Irene Lecker, and Paul Luu; MSB wouldn’t be the same without you guys. Last but not
least, Amy Jeon; thank you for sparking happiness in my life.
Finally, I want to thank all of my friends and teammates during my years of dragon boat
paddling throughout graduate school. Although their contribution is not academically related, I
feel their personal contribution to my development helped to shape my academic growth. I
learned a great deal about commitment, courage, patience, perseverance, loyalty and trust. I
enjoyed the excitement of going to races and practices with everyone. The joys and sorrows will
be forever engraved in my mind. As a result, personalities I have developed during this process
seeped through to my study, which helped to mold me into the PhD graduate I am today. As I
conclude one chapter and begin another, I want to express my sincere gratitude to all my friends
and family. You guys are awesome!
vi
Table of Contents
Abstract ........................................................................................................................................... ii
Acknowledgements ........................................................................................................................ iv
Table of Contents ........................................................................................................................... vi
List of Tables ............................................................................................................................... xiv
List of Figures ............................................................................................................................... xv
List of Abbreviations ................................................................................................................. xviii
List of Appendices ..................................................................................................................... xxiii
Chapter 1 Introduction and Background ................................................................................... 1
1 Introduction ................................................................................................................................ 2
1.1 Biological rhythms: physiology, and pathobiology ............................................................ 2
1.1.1 Physiology ............................................................................................................... 2
1.1.1.1 Respiratory rhythm ................................................................................... 2
1.1.1.2 Other rhythmic regions ............................................................................. 6
1.1.1.3 Heart beat and the sinoatrial node ............................................................ 6
1.1.2 Pathophysiology and clinical significance .............................................................. 8
1.2 Generation and regulation of rhythmic activity .................................................................. 9
1.2.1 Rhythmic networks ................................................................................................. 9
1.2.1.1 Synaptic transmission and modulation ................................................... 10
1.2.1.2 Intracellular signaling ............................................................................. 12
1.2.2 Pacemaker cells ..................................................................................................... 13
1.2.2.1 Pacemaker neurons ................................................................................. 13
1.2.2.2 Respiratory pacemaker neurons ............................................................. 14
1.2.2.3 Sinoatrial node pacemaker cells ............................................................. 16
vii
1.2.3 Electrical properties of pacemaker cells ............................................................... 17
1.2.3.1 Resting or basal membrane potential...................................................... 17
1.2.3.2 Intrinsic membrane excitability .............................................................. 19
1.2.4 Rhythmic computation models ............................................................................. 19
1.2.4.1 Network simulations ............................................................................... 20
1.2.4.2 Single-cell simulations ........................................................................... 20
1.3 Ion channel dynamics: genesis and perpetuation of pacemaking ..................................... 22
1.3.1 Voltage-dependent ion channels ........................................................................... 22
1.3.1.1 Sodium channels ..................................................................................... 22
1.3.1.2 Calcium channels .................................................................................... 25
1.3.1.3 Calcium-dependent ion channels ............................................................ 27
1.3.1.4 Potassium channels ................................................................................. 29
1.3.1.5 Hyperpolarizing-activated channels ....................................................... 30
1.3.1.6 Chloride channels ................................................................................... 33
1.3.2 Ligand-gated ion channels, TRP channels and ion exchangers ............................ 34
1.3.2.1 Ligand-gated ion channels ...................................................................... 34
1.3.2.2 TRP channels .......................................................................................... 35
1.3.2.3 Ion exchangers ........................................................................................ 36
1.3.3 Voltage-independent leak and background currents ............................................. 36
1.3.3.1 Potassium leak ........................................................................................ 37
1.3.3.2 Chloride leak .......................................................................................... 39
1.3.3.3 Sodium leak ............................................................................................ 40
1.4 NALCN: a new player in leak conductance ..................................................................... 42
1.4.1 Protein structure and homology ............................................................................ 43
1.4.1.1 Gene and protein structures .................................................................... 43
1.4.1.2 Homology ............................................................................................... 45
viii
1.4.2 Biophysicology and pharmacology ....................................................................... 45
1.4.2.1 Biophysical properties ............................................................................ 45
1.4.2.2 Pharmacological properties .................................................................... 47
1.4.3 Channel regulation ................................................................................................ 48
1.4.4 Expression and distribution ................................................................................... 48
1.4.5 Physiological functions ......................................................................................... 49
1.4.5.1 Rhythmic activity ................................................................................... 49
1.4.5.2 Resting membrane potential and cell excitability................................... 50
1.4.5.3 Synaptic regulation ................................................................................. 50
1.4.5.4 Insulin release ......................................................................................... 51
1.4.5.5 Osmoregulation ...................................................................................... 51
Chapter 2 Rationale, Hypothesis and Objectives ..................................................................... 53
2 Rationale, Hypothesis and Objectives...................................................................................... 54
2.1 Rationale ........................................................................................................................... 54
2.2 General Hypothesis ........................................................................................................... 54
2.3 Objectives and approaches ................................................................................................ 55
2.3.1 Objective 1: Determine whether NALCN-dependent Na+ leak contributes to
neuronal pacemaker activity ................................................................................. 55
2.3.1.1 Major question ........................................................................................ 55
2.3.1.2 Experimental approach ........................................................................... 55
2.3.2 Objective 2: Identify the ionic mechanisms of Na+ leak regulation of
spontaneous pacemaker activity ........................................................................... 56
2.3.2.1 Major question ........................................................................................ 56
2.3.2.2 Experimental approach ........................................................................... 56
2.3.3 Objective 3: Determine whether NALCN contributes to Na+ leak regulation of
pacemaker activity in isolated sinoatrial node cardiomyocytes ............................ 57
2.3.3.1 Major question ........................................................................................ 57
ix
2.3.3.2 Experimental approach ........................................................................... 57
Chapter 3 General Methodologies ............................................................................................. 58
3 General Methodologies ............................................................................................................ 59
3.1 Electrophysiology ............................................................................................................. 59
3.1.1 Solutions ............................................................................................................... 59
3.1.1.1 Internal pipette and saline bath solutions ............................................... 59
3.1.1.2 Na+ free bath solutions ........................................................................... 61
3.2 Pharmacology and blockers .............................................................................................. 61
3.2.1 Pharmacological blockers ..................................................................................... 61
3.2.1.1 Tetrodotoxin (TTX) ................................................................................ 61
3.2.1.2 Tetraethylammonium (TEA) .................................................................. 62
3.2.1.3 4-aminopyridine (4-AP) ......................................................................... 62
3.2.1.4 Spermine ................................................................................................. 63
3.2.1.5 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)
pyrimidinium chloride (ZD7288) ........................................................... 63
3.2.2 Multivalent ion blockers ....................................................................................... 64
3.2.2.1 Gadolinium (Gd3+
) .................................................................................. 64
3.2.2.2 Cobalt (Co2+
) .......................................................................................... 64
3.2.3 Whole-cell patch clamp recordings ...................................................................... 65
3.2.4 Current clamp recordings ...................................................................................... 66
3.3 RNAi designs and principles ............................................................................................ 68
3.3.1 RNAi gene silencing and nonspecific effects ....................................................... 68
3.3.1.1 Innate immunity ...................................................................................... 69
3.3.1.2 Off-targetting .......................................................................................... 69
3.3.2 RNAi for Lymnaea stagnalis ................................................................................ 69
3.3.3 RNAi synthesis ..................................................................................................... 70
x
3.4 Data analysis and statistics ................................................................................................ 70
Chapter 4 A Sodium Leak Current Regulates Pacemaker Activity of Adult Central
Pattern Generator Neurons in Lymnaea stagnalis. ............................................................. 72
4 A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator
Neurons in Lymnaea stagnalis. ................................................................................................ 73
4.1 Abstract ............................................................................................................................. 73
4.2 Introduction and Rationale ................................................................................................ 73
4.3 Hypothesis ......................................................................................................................... 75
4.4 Specific Aims .................................................................................................................... 75
4.5 Materials and Methods ...................................................................................................... 76
4.5.1 Animals and aerial respiratory behavior observation ........................................... 76
4.5.2 Ganglionic RNA preparation and cDNA analysis ................................................ 76
4.5.3 RNAi synthesis and delivery ................................................................................ 76
4.5.4 Primary cell culture ............................................................................................... 77
4.5.5 Electrophysiology ................................................................................................. 78
4.5.6 Data analysis ......................................................................................................... 79
4.6 Results ............................................................................................................................... 79
4.6.1 U-type channel regulates the resting membrane potential and is a prerequisite
for RPeD1 pacemaker activity. ............................................................................. 79
4.6.2 U-type channel conducts an inward Na+ leak current at hyperpolarizing
voltages. ................................................................................................................ 80
4.6.3 U-type channel conductance is pharmacologically similar to reported NALCN
channel conductance. ............................................................................................ 84
4.6.4 Partial U-type knockdown reduces the aerial respiratory behavior in adult
animal in vivo. ....................................................................................................... 89
4.7 Discussion ......................................................................................................................... 90
4.8 Acknowledgements ........................................................................................................... 93
Chapter 5 Robust Regulation of Spontaneous Activity Depends On A Sodium Leak
Current Involving U-type Channel In A Mollusca Pacemaker Neuron Simulation ........ 95
xi
5 Robust regulation of spontaneous activity depends on a sodium leak current by U-type
channel in a molluscan pacemaker neuron simulation. ............................................................ 96
5.1 Abstract ............................................................................................................................. 96
5.2 Introduction and rationale ................................................................................................. 96
5.3 Hypothesis ......................................................................................................................... 97
5.4 Specific Aims .................................................................................................................... 98
5.5 Materials and Methods ...................................................................................................... 98
5.5.1 Experimental animals ............................................................................................ 98
5.5.2 Primary cell cultures, cell isolation and RNAi gene silencing ............................. 98
5.5.3 Bath solutions and chemicals ................................................................................ 98
5.5.4 Electrophysiology ................................................................................................. 99
5.5.5 Data analysis ......................................................................................................... 99
5.5.6 Computer simulation ........................................................................................... 100
5.5.7 Parameters estimation and tuning ....................................................................... 101
5.5.8 Model evaluation ................................................................................................ 103
5.5.9 Variation of leak sodium/potassium currents ..................................................... 103
5.6 Results ............................................................................................................................. 104
5.6.1 U-type channel knockdown does not significantly alter spike profile and
voltage-gated currents ......................................................................................... 104
5.6.2 RPeD1 expresses major voltage-gated Na+, Ca
2+, K
+, and hyperpolarizing-
activated currents ................................................................................................ 104
5.6.3 Simulated RPeD1 action potential profile correctly represents ones from
recording ............................................................................................................. 110
5.6.4 Spiking activity is more sensitive to Na+ leak current compared to K
+ leak
current. ................................................................................................................ 113
5.7 Discussion ....................................................................................................................... 113
5.8 Acknowledgement .......................................................................................................... 116
Chapter 6 Identification of a Novel Background Sodium Current Contributing to
Pacemaker Generation in Adult Mouse Sinoatrial Node Cardiomyocytes. ................... 117
xii
6 Identification of a Novel Background Sodium Current Contributing to Pacemaker
Generation in Adult Mouse Sinoatrial Node Cardiomyocytes. ............................................. 118
6.1 Abstract ........................................................................................................................... 118
6.2 Introduction and rationale ............................................................................................... 118
6.3 Hypothesis ....................................................................................................................... 119
6.4 Specific Aims .................................................................................................................. 119
6.5 Materials and Methods .................................................................................................... 120
6.5.1 Solutions and Chemicals ..................................................................................... 120
6.5.2 SAN region identification ................................................................................... 121
6.5.3 Animals and cardiomyocytes isolation ............................................................... 122
6.5.4 Plasmid preparation ............................................................................................ 122
6.5.5 Cell culture and plasmid transfection ................................................................. 124
6.5.6 Electrophysiology ............................................................................................... 125
6.5.6.1 Voltage-clamp recordings..................................................................... 125
6.5.6.2 Current-clamp recordings ..................................................................... 125
6.5.7 Real-time quantitative PCR (qPCR) ................................................................... 125
6.5.8 Western blotting .................................................................................................. 126
6.5.9 Data analysis ....................................................................................................... 127
6.6 Results ............................................................................................................................. 127
6.6.1 Background Na+ leak current in isolated SAN cardiomyocytes depolarizes
resting membrane potential and is essential for spontaneous pacemaker firing. 127
6.6.2 Na+ leak current in isolated SAN cardiomyocytes is sensitive to Gd
3+ and Co
2+
block. ................................................................................................................... 129
6.6.3 Gd3+
and Co2+
sensitive Na+ leak current is unique from known background
Na+ currents of SAN cardiomyocytes. ................................................................ 131
6.6.4 NALCN is highly expressed in both SAN and RA, but NALCN subunits
expressions are region specific. .......................................................................... 133
6.6.5 Reconstituting NALCN-dependent Na+ leak requires UNC79 coexpression. .... 135
xiii
6.7 Discussion ....................................................................................................................... 138
6.8 Acknowledgement .......................................................................................................... 141
Chapter 7 Results Summary and General Discussion ........................................................... 143
7 Results Summary and General Discussion ............................................................................ 144
7.1 Summary of results ......................................................................................................... 144
7.2 General discussion .......................................................................................................... 145
7.2.1 NALCN regulation of respiratory rhythm and rCPG neuron activities .............. 145
7.2.2 NALCN and other subthreshold currents ........................................................... 148
7.3 Clinical significances ...................................................................................................... 150
7.3.1 Na+ leak fluctuations ........................................................................................... 150
7.3.2 Ion channel regulation ......................................................................................... 152
7.4 Limitations and future directions .................................................................................... 153
7.4.1 Specificity of genetic knockdown ....................................................................... 153
7.4.2 Pharmacological specificities .............................................................................. 153
7.4.3 Synaptic alterations ............................................................................................. 155
7.4.4 Membrane localization ........................................................................................ 156
7.4.5 NALCN conductance with UNC80 .................................................................... 158
7.4.6 General application of computation model findings ........................................... 158
7.5 Concluding remarks ........................................................................................................ 159
Reference List ............................................................................................................................. 160
Appendices A: Additional recordings. ........................................................................................ 211
Appendices B: Permission to Reproduce Previously Published Works ..................................... 214
xiv
List of Tables
Chapter 1: Introduction
Table 1.1 Homologous NALCN channels between different species. ...............................46
Chapter 3: General Methodologies
Table 3.1 Internal and external solutions used in different systems for electrophysiology
recordings. ............................................................................................................................... 60
Table 4.2 Sequences of siRNAs used in the knockdown study. .......................................... 71
Table 4.3 Primer sequences for U-type channel and β-actin. .............................................. 71
Chapter 4: A Sodium Leak Current Regulates Pacemaker Activity of Adult Central
Pattern Generator Nerurons in Lymnaea stagnalis.
Table 4.1 Protein sequence alignments of the U-type pore and S4 regions with NALCNs. ....
.............................................................................................................. ...................... 74
Chapter 5: Robust Regulation of Spontaneous Activity Depends On A Sodium Leak
Current Involving U-type Channel In A Mollusca Pacemaker Neuron Simulation.
Table 5.1 Parameters used in establishing the simulation of isolated RPeD1. .................. 102
Chapter 6: Identification of a Novel Background Sodium Current Contributing to
Pacemaker Generation in Adult Mouse Sinoatrial Node Cardiomyocytes.
Table 6.1 Transfection treatment conditions for tsA201 overexpression study. ................ 124
Table 6.2 Real-time qPCR primer sequences for NALCN channel, UNC79, UNC80, and
GAPDH. ................................................................................................ ................... 126
Table 6.3 Different splice variant of NALCN, Unc79 and Unc80 subunits in C. elegans, D.
melanogaster, M. musculus, R. norvegicus and H. sapiens. ............................................... 142
xv
List of Figures
Chapter 1: Introduction
Figure 1.1 Schematic diagram of Lymnaea stagnalis rCPG network regulation of respiratory
motor output. ................................................................................................................. 5
Figure 1.2 Current components contributing to spontaneous activity in pacemaker cells of
neurons and heart. ........................................................................... .............................. 23
Figure 1.3 Molecular channels known to contribute to background membrane currents of
excitable cells. ............................................................................................................ ... 37
Figure 1.4 Schematic diagram of functional NALCN complex in neurons and pancreatic beta
cells. ............................................................ ................................................................ 44
Chapter 3: General Methodologies
Figure 3.1 Whole-cell current in individual RPeD1 neurons isolated from naïve control
animals. ....................................................................................................................... 6 7
Chapter 4: A Sodium Leak Current Regulates Pacemaker Activity of Adult Central
Pattern Generator Neurons in Lymnaea stagnalis.
Figure 4.1 Effects of the U-type dsRNA on rhythmic firing and intrinsic membrane
properties in RPeD1 neurons. ......................................................................................... 81
Figure 4.2 U-type RNAi knockdown reduces inward hyperpolarizing leak current in RPeD1
neurons. ........................................................................................................................ 83
Figure 4.3 ILeak conducted by the U-type channel in RPeD1 is carried by Na+. ................... 85
Figure 4.4 Gd3+
partially blocked ILeak via the U-type channels in RPeD1 neurons. ............ 87
Figure 4.5 Low extracellular Ca2+
depolarizes the membrane potential by enhancing U-type
channel activity in RPeD1 neurons. ................................................................................. 88
xvi
Figure 4.6 Acute U-type dsRNA knockdown suppresses aerial respiratory behavior in adult
L. stagnalis in vivo. ....................................................................... ............................... 90
Chapter 5: Robust Regulation of Spontaneous Activity Depends On A Sodium Leak
Current Involving U-type Channel In A Mollusca Pacemaker Neuron Simulation.
Figure 5.1 U-type knockdown does not significantly affect voltage-dependent current or
action potential profile. ......................................................................................................... 105
Figure 5.2 Characterization of Na+ currents in the RPeD1 neuron. .................................... 107
Figure 5.3 Characterization of K+ currents in the RPeD1 neuron. ...................................... 108
Figure 5.4 Characterization of Ca2+
currents in the RPeD1 neuron. ................................... 110
Figure 5.5 Characterization of hyperpolarization-activated current in the RPeD1. ............ 111
Figure 5.6 Simulated action potential fitted to natural variation of the spontaneous action
potential recorded in isolated RPeD1 neuron. ....................................................................... 112
Figure 5.7 Rhythmic spiking during RPeD1 simulation variation of gLNa and gLK. ........... 114
Chapter 6: Identification of a Novel Background Sodium Current Contributing to
Pacemaker Generation in Adult Mouse Sinoatrial Node Cardiomyocytes.
Figure 6.1 Characterization of electrical properties of SAN and right atrial cardiomyocytes.
................................................................................................................................................ 121
Figure 6.2 Vector constructs map for pcDNA3-RnNCA and pcDNA-Unc79-2. ............... 123
Figure 6.3 Morphological and electrophysiological properties of SAN and right atrial
cardiomyocytes in isolation. .................................................................................................. 128
Figure 6.4 Na+ leak current regulates resting membrane potential and pacemaker activity in
SAN cardiomyocytes. ............................................................................................................ 130
Figure 6.5 The membrane potential and leak current of isolated SAN cardiomyocytes are
sensitive to Gd3+
and Co2+
. .................................................................................................... 132
xvii
Figure 6.6 Gd3+
and Co2+
sensitive background current is unique from known candidates of
background current. ............................................................................................................... 134
Figure 6.7 NALCN is highly expressed in both SAN and RA, but NALCN subunits
expression is region-specific. ................................................................................................ 136
Figure 6.8 NALCN-dependent Na+ leak current reconstituted in tsA201 overexpressed with
NALCN and UNC79. ............................................................................................................ 137
Chapter 7: Results Summary and General Discussion.
Figure 7.1 Working model of U-type conductance contributing to rCPG rhythmic output and
respiratory behavior. .............................................................................................................. 147
Figure 7.2 Proposed model of NALCN channel function in regulating pacemaker activity. ....
................................................................................................................................................ 151
Figure 7.3 Proposed working model of NALCN channel in SAN and atrial cardiomyocytes. .
................................................................................................................................................ 157
xviii
List of Abbreviations
5-HT 5-hydroxytryptamine
ADP Adenosine diphosphate
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ATP Adenosine triphosphate
AVN Atrioventricular node
BSA Bovine serum albumin
cAMP Cyclic adenosine monophosphate
CaSR Calcium-sensing receptor
Cav Voltage-gated calcium channel
cDNA Complementary deoxyribonucleic acid
cGMP Cyclic guanosine monophosphate
CGN Cerebellar granule neurons
CM Conditioned-medium
CPG Central pattern generator
CRG Central ring ganglia
CT Crista terminalis
Cx30.2 Connexin30.2
Cx43 Connexin43
Cx45 Connexin45
DEKA Aspartic acid – glutamic acid – lysine – alanine
DHP 1,4-dihydropridine
DM Defined-medium
DMEM Dulbecco’s modified eagle medium
DMSO Dimethyl sulfoxide
Dmα1U Drosophila melanogaster α1 subunit unique
DNA Deoxyribonucleic acid
DRG Dorsal root ganglia
dsRNA Double-stranded ribonucleic acid
ECG Electrocardiogram
ECl Chloride equilibrium potential
ECL Enhanced chemiluminescence
EEEE Glutamic acid – Glutamic acid – Glutamic acid – Glutamic acid
EEG Electroencephalogram
EEKE Glutamic acid – glutamic acid – lysine – glutamic acid
eGFP Enhanced green florescent portein
EGTA Ethylene glycol tetraacetic acid
EK Potassium equilibrium potential
EMG Electromyogram
ENa Sodium equilibrium potential
xix
FBS Fetal bovine serum
GABA gamma-aminobutyric acid
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GDP Guanosine diphosphate
GI Gastrointestinal
GPCR G-protein coupled Receptor
GTP Guanosine triphosphate
GYG Glycine – tyrosine - glycine
HCN Hyperpolarization-activated cyclic nucleotide-gated
HEK-293 Human embryonic kidney-293
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HRP Horseradish peroxidase
ICa L-type L-type voltage-gated Ca2+
current
ICa N-type N-type voltage-gated Ca2+
current
ICa P/Q-type P/Q-type voltage-gated Ca2+
current
ICa R-type R-type voltage-gated Ca2+
current
ICa T-type T-type low voltage-activated Ca2+
current
ICl Leak Cl--dependent leak current
If “Funny” current
Ih Hyperpolarizing-activated current
IK Leak K+-dependent leak current
IK Leak K+-dependent leak current
IKATP ATP-sensitive K+ current.
IK-Ca Ca2+
-dependent K+ current
IKv Voltage-gated K+ current
ILeak Leak current
INa Leak Na+-dependent leak current
INa Na+ current
INa-P Voltage-dependent persistent Na+ current
INav Voltage-gated Na+ current
INCX Na+/Ca
2+ exchanger current
IP3I Input 3 interneuron
Iq “Queer” current
Ist TTX insensitive sustain Na+ current
IVC Inferior vena cava
K2P Two-pore domain potassium channels
KB solution Modified Kraftbrühe solution
KV Voltage-gated potassium channel
LV Left atrium
M3R M3 muscarinic receptor
MAGUK Membrane-associated guanylate kinase
mRNA Messenger ribonucleic acid
xx
NALCN Na+ leak, non-selective
Nav Voltage-gated sodium channel
NCA Putative nematode calcium channel
NCX Na+/Ca
2+ exchanger
NMDA N-methyl-D-aspartic acid
NMDG N-methyl-D-glucamine
PAF Platelet activating factor
PBS Phosphate buffered saline
PEI Polyethylenimine
pFRG Parafacial respiratory group
PKC Protein kinase C
PLC Phospholipase C
P-loop Pore-forming loop
preBotC pre-Botzinger Complex
qPCR Quantitative real-time Polymerase Chain Reaction
RA Right Atrium
rCPG Respiratory Central Pattern Generator
RMP Resting Membrane Potential
RNA Ribose Nucleic Acid
RNAi Ribonucleic Acid Interference
RPeD1 Right Pedal Dorsal 1
RPM Rate per minute
RTN Retrotrapezoid nucleus
SAN Sinoatrial node
SCN Suprachiasmatic nucleus
SFK Src-family kinase
SH3 SRC homology 3
siRNA Small interfering ribonucleic acid
SP Substance P
SR Sarcoplasmic reticulum
SVC Superior vena vava
TACR1 Tachykinin receptor 1
TASK TWIK-related acid-sensitive potassium channel
Tbx T-box transcription factor 18
TEA Tetraehylammonium
Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol
TRP Transient receptor potential
TRPA TRP ankyrin
TRPC TRP canonical
TRPM TRP melastatin
TRPML TRP mucolipin
TRPN TRP no mechanoreceptor potential C
xxi
TRPP TRP polycystin
TRPV TRP vanilloid
tsA201 Human embryonic kidney, SV40 transformed
TTX Tetrodotoxin
TVGXG Threonine – valine – glycine – X – glycine (where X could be either tyrosine (Y)
or phenylalanine (F))
TWIK Two pore domain weakly inward rectifying potassium channel
UNC Uncoordinated family member
U-type Unidentified-type channel
VD4 Visceral dorsal 4
VGCNL1 Voltage-gated channel like protein 1
VRG Ventral respiratory group
VTA Ventral tegumental area
ZD7288 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride
ω-AGA ω-agatoxin
ω-CTX ω-conotoxin
Units Abbreviations
% percent
°C degrees Celsius
A amp(s)
Å angstrom(s)
Da dalton
dB decibel(s)
F farad(s)
g gram(s)
hr hour(s)
Hz hertz
L litre(s)
M molar (mol/L)
min minute(s)
mol mole(s)
s second(s)
S siemen(s)
V volt(s)
Ω ohm(s)
Prefixes
M mega- (106)
xxii
k kilo- (103)
c centi- (10-2
)
m mili- (10-3
)
µ micro- (10-6
)
n nano- (10-9
)
p pico- (10-12
)
xxiii
List of Appendices
APPENDIX A: Additional recordings. ............................................................................ 211
Appendix A1 TTX regulates voltage-gated Na+ channel of L. stagnalis RPeD1 neuron in
a dose-dependent manner. ...................................................................... 211
Appendix A2 Na+ free subsitution reveals a Na
+-independent transient inward current.
................................................................................................................. 212
Appendix A3 Partial knockdown of U-type channel reduces inward hyperpolarizing Na+
current in RPeD1 neurons. ..................................................................... 213
APPENDIX B: Permission to reproduce previously published material. .................... 214
1
Chapter 1
Introduction and Background
Part of the text presented in this chapter is reproduced with permission (see appendix)
from the following publication:
Lu TZ, Feng Z-P (2012) Molecular Neurobiology. 45(3): 415-423.
2
1 Introduction
1.1 Biological rhythms: physiology, and pathobiology
Biological rhythm is the periodic oscillation of physiological responses. The generation and
regulation of the rhythmic output is dynamic, capable of intrinsic coordination or extrinsic
modifications from environmental cues. The importance of rhythm is characterized by the many
fundamental behaviors that it regulates, including heartbeat (Mangoni and Nargeot, 2008),
respiration (Feldman and Del Negro, 2006;Garcia, III et al., 2011), locomotion (Harris-Warrick,
2010;Kiehn et al., 2010), gastrointestinal motility (Sarna, 2008), hormonal release (Comunanza
et al., 2010) and circadian rhythms (Bell-Pedersen et al., 2005). Despite the importance of
understanding the regulatory mechanisms behind biological rhythms, there are still questions
remain unaddressed about the intrinsic mechanisms that drive these biologically essential
functions. Chiefly, the complexity, ambiguity, and heterogeneous nature of many rhythmic
centers limit the specificity of scientific inquiries. Therefore, many studies turned to invertebrate
systems to address many fundamental principles given the many levels of conserved cellular,
electrical, molecular, and genetic principles found in many invertebrate models (Bell-Pedersen et
al., 2005).
1.1.1 Physiology
1.1.1.1 Respiratory rhythm
Breathing in advanced vertebrates is fundamental to normal physiology. Normal breathing is
rhythmic, characterized by the periodic oscillation between inspiration and expiration. The
central role of breathing is to regulate the reservoir of bodily O2 and CO2, but it also serve as an
important regulator of pH and temperature. This behaviour is also observed in invertebrate aerial
respiration with fundamentally similar principles (JONES, 1961). Although normal breathing
rhythm can be temporarily interrupted by voluntary control, most of the respiratory behavior is
generated and regulated by the involuntary motor neurons.
In mammals, motor output that controls involuntary respiration is located in the brain stem
region of medulla and pons. It involves a complex network of interneurons regulating both
inspiratory and expiratory motor neurons. The collective group of interneurons located within
3
this region are capable of generating rhythm without sensory feedback (Feldman and Del Negro,
2006;Smith et al., 1991), thus they are a conserved example of the central pattern generator
(CPG). The exact mechanism underlying rhythmic pattern generation is not completely
understood, but it involves multiple interacting regions within the brainstem. Rhythmic behavior
is thus largely dependent on the synaptic and intrinsic membrane properties of the respiratory
CPG (rCPG) neurons located within the various regions (Del Negro et al., 2002;Pena et al.,
2004;Pena and Ramirez, 2004;Tryba and Ramirez, 2004). The current mammalian model for
rCPG rhythm generation involves two mutually inhibiting respiratory groups, one in the pre-
Botzinger Complex (preBotC) (Smith et al., 1991), and the other in the retrotrapezoid nucleus
(RTN) (Connelly et al., 1990) and parafacial respiratory group (pFRG) (Onimaru et al.,
1987;Onimaru et al., 1988;Onimaru and Homma, 2003). The preBotC is responsible for
inspiration (Feldman and Del Negro, 2006;Janczewski and Feldman, 2006a;Smith et al., 1991)
and the RTN/pFRG responsible for expiration (Feldman and Del Negro, 2006;Janczewski and
Feldman, 2006b;Janczewski and Feldman, 2006a).
Two concepts currently describe the mechanism for respiratory rhythm generation. The
pacemaker hypothesis posits that a single respiratory pacemaker region is the sole driving
generator of the rhythmic behaviour. The preBotC has been considered as the principle
pacemaker generator, as isolated brainstem and spinal cord of neonatal rat could generate fictive
respiratory rhythm (Smith et al., 1991). In addition, synaptic inhibition in en bloc and in slice
preparations does not block in vitro respiratory rhythm (Brockhaus and Ballanyi, 1998;Gray et
al., 1999b;Onimaru et al., 1990). Evidences also indicate that both preBotC and RTN/pFRG
exhibit pacemaker activities (Del Negro et al., 2002;Pena et al., 2004;Pena and Ramirez,
2004;Tryba and Ramirez, 2004), however preBotC has been suggested to be the dominant
rhythmic generator under resting conditions (Janczewski and Feldman, 2006a). Synaptic
connectivity between bursting rCPG neurons recruits additional burst-generating intrinsic
currents, is the group-pacemaker hypothesis. In the preBotC, modulators regulate network
activity and shape rhythm patterns. Rhythm generation also requires many neurotransmitters
release (Doi and Ramirez, 2008), and inhibition of intrinsic pacemaker activity is insufficient to
abolish respiratory rhythm (see section 1.2.2.2) (Del Negro et al., 2005;Pena et al., 2004). Given
the highly complex nature of the vertebrate rCPG network, many studies have adopted a
4
simplified rCPG network to understand the fundamental mechanism of respiratory
rhythmogenesis.
The great pond snail, Lymnaea stagnalis (L. stagnalis), is an important model in numerous rCPG
studies and contributed greatly to our current understanding of the rCPG network properties
(Spencer et al., 1999;Syed et al., 1990;Taylor and Lukowiak, 2000). The beauty of adopting this
reductionist model is the ability to address many fundamental questions pertaining to rhythm
generation, since the snail rCPG network retains many of the key features found in their
mammalian counter parts. The snail is a bimodal breather (JONES, 1961) and its aerial
respiratory activity can be easily described by examining the frequency and duration of opening
of the respiratory gas-exchange orifice (pneumostome). L. stagnalis aerial respiration is
controlled by a simple well-described rCPG network consisting of 3 large identified neurons
(Syed et al., 1992;Syed and Winlow, 1991;Winlow and Syed, 1992). Two mutually inhibiting
interneurons (input 3 interneuron (IP3) and visceral dorsal 4 (VD4)) regulating antagonistic
motor neuron output (expiration and inspiration, respectively), and a pacemaker neuron (right
pedal dorsal 1 (RPeD1)) that regulates and initiates rCPG rhythmic activity (Figure 1.1). This
network connectivity is capable of generating physiological appropriate fictive rhythm in culture
(Feng et al., 1997;Feng et al., 2002;Syed et al., 1990) and in situ, with (Winlow and Syed, 1992)
or without peripheral input (Syed and Winlow, 1991). Synaptic specificity between the rCPG
neurons in culture allows for direct study of the synaptic interactions within the rCPG network.
In semi-intact preparations where the peripheral connections are retained, the intrinsic rhythmic
generation in RPeD1 is largely suppressed (Inoue et al., 2001;Inoue et al., 1996). However, in
cell culture and isolated ganglia preparations, RPeD1 exhibits rhythmic activity characterized by
intermittent action potential bursts (Syed et al., 1990;Taylor and Lukowiak, 2000). Axonal and
single-cell ablation of respiratory neuron (RPeD1) in whole organism provided direct evidence
of the necessity and sufficiency for the rCPG to generate respiratory activity (Haque et al., 2006).
The power of addressing question at single-cell, network and whole-animal level makes the L.
stagnalis rCPG network a popular model for studying the cellular mechanisms underlying rCPG
rhythmic activities.
5
Figure 1.1. Schematic representation of the Lymnaea stagnalis respiratory central pattern generator (rCPG)
network. RPeD1, IP3 and VD4 forms the three interneuron rhythm generator. VD4 and IP3 forming mutual
inhibitory connections that regulates corresponding motor neuron controlling inspiration and expiration,
respectively. RPeD1 is the pacemaker neuron that initiates network activity and regulates rhythmic outputs. Adopted
from Syed et al., 1992. RPeD1 right pedal dorsal 1, IP3 input 3 interneuron, VD4 visceral dorsal 4, VK visceral K
cell, VI/J visceral I/J cells.
6
1.1.1.2 Other rhythmic regions
Many neurons can also periodically secret hormone that regulates a rhythmic downstream effect
on animal homeostasis. These neuroendocrine loop exhibit dynamic feedback mechanisms that
oscillate based on external sensory input to further regulate internal neuronal and humoral
responses. Circadian rhythm in vertebrates is a prime example of this neuroendocrine loop
(Cassone and Menaker, 1984). Central to the vertebrate circadian clock is the oscillators found in
mammals, light activation of the retina projects to the neurons of the suprachiasmatic nucleus
(SCN) via the retinohypothalamic tract. Inhibitory signals project from the SCN to the pineal
gland which secretes serotonin (5-HT) that regulates melatonin level (a key hormone that
modulates wake/sleep pattern). Increase melatonin level at night inhibits SCN output and affects
various peripheral oscillators that express melatonin receptors (Cassone, 1998). Projections from
SCN regulate many homeostatic mechanisms within the CNS that also determine peripheral
oscillator behavior. This oscillation can be self-sustaining; however it requires photonic input to
prevent temporal-dependent dampening of oscillation amplitude (Bell-Pedersen et al., 2005).
Various electrical and molecular mechanisms regulate this neuroendocrine loop and much of the
similar principle can be found in many photo-sensitive invertebrates.
Beyond the CNS, rhythmic activity can also be observed in gastrointestinal motility. Located
within the muscular network of the gastrointestinal (GI) tract is a type of pacemaker cell called
the interstitial cells of Cajal (see review Sarna, 2008). These cells exhibit spontaneous electrical
activity characterized by slow-wave oscillations of membrane potential, which are responsible
for the rhythmic propagation and contractility of the gastrointestinal smooth muscle cells.
Beyond rhythmic regulation, interstitial cells of Cajal also mediate autonomic nervous system
signals and regulate rhythmic activity through mechanic-stretch.
1.1.1.3 Heart beat and the sinoatrial node
Heart beat is another rhythmic behaviour that is essential to normal animal physiology.
Combined with stroke volume, heart beat determines the overall cardiac output. Rhythmic
cardiac chamber contraction helps to circulate fluid around the body which provides tissue
oxygenation, metabolites removal, nutrient transport, endocrine responses, osmotic regulation,
temperature distribution and immune response. Regulating heartbeat depends on many extrinsic
7
and intrinsic feedback signals. However, contractility is intrinsic to the organ as an isolated heart
can continue to beat for hours when maintained in physiological solution.
In most vertebrate systems, specialized pacemaker cardiomyocytes are the principle generator of
spontaneous cardiac chamber contraction. Fundamentally, three regions located in the heart
forms the pacemakers that are capable of generating and perpetuating heartbeats: sinoatrial node
(SAN) located at the junction between the right atrium and the superior vena cava (SVC),
atrioventricular node (AVN) located between the atria and the ventricles, and the Purkinje fibre
network located on the endocardial surfaces of the septum and free wall (Boyett et al.,
2000;Boyett et al., 2003;Morgado-Valle and Feldman, 2004;Silverman et al., 2006). The
electrical signal, originating in the pacemaker cells of the SAN propagates through the right
atrium and through Bachmann’s bundle to the left atrium, stimulating the myocardium of the
atria to contract. The impulse is then delayed at the AVN, allowing for ventricular filling, after
which it travels through the His-bundle and rapidly transmitted to each of the left and right
bundle branches and Purkinje fibre network (Silverman et al., 2006). As the original source of
the pacemaker rhythm, the intrinsic contractility rate of the SAN is much higher than the
conduction network, which functions to suppress the pacemaking rhythms of the AVN and
Purkinje network. By this nature, the SAN is called the primary pacemaker region of the heart,
whereas AVN and the Purkinje fibre network are called the secondary pacemakers. AVN can
take over pacemaker function during SAN failure or blockade of SAN to AVN conduction
(James, 2003;James and Nadeau, 1963;Urthaler et al., 1974). Likewise, the Purkinje network can
also function as a pacemaker region during AVN and SAN failure.
Human SAN were anatomically described over 100 years ago by (Keith and Flack, 1907). Its
apparent role as a pacemaker was not known until few years later (Lewis, 1910). The crescent-
shaped SAN tissue is located on the intercaval region extending toward the endocardial side of
the cristae terminalis (Boyett et al., 2000;Boyett et al., 2003;Dobrzynski et al., 2005a). Its region
is defined by the cristae terminalis, left and right sinoatrial ring bundles, the superior vena cava
and the interatrial septum. A heterogeneous mixture of cells is found within this region, which
includes the pacemaker cells, atrial cells, fibroblasts and adipocytes (Boyett et al.,
2000;Dobrzynski et al., 2005c). This region contains higher amount of collagen and have
relatively slow conduction velocity in comparison to other non-pacemaker regions of the heart
8
(Opthof et al., 1987). Recently, a “paranodal region” has been identified in human to be located
within the cristae terminalis (CT), but not anatomically continuous with the SAN region
(Chandler et al., 2009). The cells are diffused and exhibit intermediate electrical and molecular
properties to SAN and atrial myocytes (Chandler et al., 2011;Chandler et al., 2009). Functionally
paranodal region is currently unknown, but it has been hypothesized to facilitate action potential
exiting from the SAN due to its intermediate electrical characteristics (Chandler et al., 2011).
1.1.2 Pathophysiology and clinical significance
Rhythmic activity of many neuronal networks underlies numerous fundamental biological
behaviours, thus disease can arise from network irregularities. These include the inability of the
CNS to integrate and synchronize rhythmic input from multiple regions. The cause for these
network dysfunction ranges from the molecular to network level. The progression and
development of disease due to rhythmic dysfunction of a neural network is highly complex;
nonetheless, evidences show that regulation of network through therapeutic intervention could
limit disease progression. For example, rhythmic firing and susceptibility of voltage-gated Ca2+
channels in basal ganglia neurons has been link to the development of Parkinson’s disease
(reviewed by Hurley and Dexter, 2012), and therapeutic intervention with limiting rhythmic
firing by blocking channel activation has been shown to slow Parkinson’s disease progression
(Becker et al., 2008;Chan et al., 2007). Therefore, understanding the fundamental physiology of
rhythm generation is essential for the potential development of novel therapeutic approaches.
In GI tract, loss of functional interstitial cells of Cajal has been linked to numerous
gastrointestinal diseases. Since interstitial cells of Cajal also mediate inputs from enteric motor
neurons, animals with either lost or abnormal interstitial cells of Cajal result in dysfunctional
motor neuron control of the GI tract, which is a major contributor to irritable bowel syndrome
(Eshraghian and Eshraghian, 2011;Sarna, 2008). Furthermore aberrant network of interstitial
cells of Cajal is thought to be a critical cause of chronic intestinal pseudo-obstruction (De et al.,
2004).
Similarly in heart, rhythmic dysfunction can develop with often life-threatening consequences.
Impulse is generated at SAN and the pacemaker activity, also called automaticity, which is a
prerequisite for propagation of the impulse throughout the heart. Genetic alteration of ion
9
channels can result in SAN dysfunction, and patients with these genetic channel mutations, often
require implantation of electronic pacemaker devices to compensate (Lamas et al., 2000).
Development of SAN dysfunction has been linked to conditions such as heart failure and cardiac
ischemia (DiFrancesco and Camm, 2004;Gaul et al., 1996;Kannel et al., 1994;Ornato and
Peberdy, 1996). These conditions often lead to arrhythmias and sudden death (Kannel et al.,
1994). A slow SAN rate has been shown in heart failure patients (Janse, 2004) and in rabbit heart
failure model (Opthof et al., 2000). In cardiac ischemia, bradycardia (slowing of heart rate) often
develops and it is associated with resuscitation after cardiac arrest or ventricular fibrillation
(Ornato and Peberdy, 1996), SAN artery stenosis or occlusion (Alboni et al., 1991;Chiba et al.,
1976), or ischemia-reperfusion (Moffat, 1987). Much of the disease development involves
modification of the ionic conductances, which alters the excitability and rhythmic generating
properties of the tissue. However, the ionic mechanism that underlies rhythm generation is
complex and currently not completely understood.
1.2 Generation and regulation of rhythmic activity
Emergence of rhythmic output depends on a multitude of properties exhibited by the cells and
their respective network connectivity. These properties include the cell-to-cell connections and
the intrinsic properties of individual cells. Often, these properties are co-dependent on each
other, thus blurring the distinctions into a complex circuitry that proves difficult to understand.
Therefore, numerous studies have used reductionist approach through simplified animal models
to understand the fundamental principles of rhythm generation and apply the principles to
complex organisms.
1.2.1 Rhythmic networks
In understanding how a rhythm is generated it is necessary to first consider how cell-to-cell
connectivity influences the activity of one cell to the next. Most rhythmic output focuses on
network-based generators established on half-center oscillator principle, where rhythmogenic
ability is established following synaptic coupling of non-rhythmogenic cells (Brown,
1922;Harris-Warrick, 2010). In the nervous system half-center oscillator regulates the rhythmic
generation of many CPG networks and these principles are observed in both vertebrate and
invertebrate systems (Dickinson, 2006;Selverston, 2010). Classical reductionist CPG networks
10
include the leech heartbeat (Kristan, Jr. et al., 2005;Roffman et al., 2012) and crustacean pyloric
network (Harris-Warrick, 2010;Russell, 1979) in invertebrate, lamprey locomotion in vertebrate
(Cohen and Wallen, 1980). In mammalian networks, CPGs that regulates locomotion (Kiehn et
al., 2010), chewing (Lund and Kolta, 2006), and respiration (Garcia, III et al., 2011) are currently
amongst the most well studied rhythmic networks.
1.2.1.1 Synaptic transmission and modulation
The importance of synaptic connectivity was recognized from neurophysiology experiments
conducted over 100 years ago (Brown, 1911;SHERRINGTON, 1910). Observations correlating
the spinal cord activity with the stepping movement of felines and canines helped to derived the
principle of intrinsic neuronal connectivity of two mutually antagonistic control centers, the
flexors and extensors (Brown, 1911;SHERRINGTON, 1910). These observations helped to
establish the principle of the half-center oscillator, which was one of the earliest models for
central pattern generation. This concept eluded to the importance of synaptic connectivity;
namely, that rhythmic properties could be established from arrhythmic neurons (Brown, 1922).
Respiratory motor output is a well-defined example of synaptic and neuromodulatory regulation
of a rhythmic network. Mammalian respiratory network are modulated by numerous regions of
the brain stem (Doi and Ramirez, 2008;Feldman and Del Negro, 2006;Garcia, III et al., 2011).
rCPG generation areas receive multiple inputs from local regions within the brain stem as well as
distant projections of the cerebrum. In addition, the rCPG generation regions such as the preBotC
also extend projections to many brain regions with respiratory related functions. A multitude of
neuromodulators are found in all levels of integration from local to brain-wide networks,
indicating the complex nature of network integration and modulation in the mammalian system
(reviewed by Doi and Ramirez, 2008). At the heart of rhythmic generation in the preBotC,
synaptic transmission is necessary in respiratory motor rhythm initiation. For example, depletion
of excitatory neurotransmitters, Substance P and glutamate, within the preBotC impeded
respiratory rhythm in neonatal rat brain stem slices (Morgado-Valle and Feldman, 2004).
Inhibitory postsynaptic currents from GABA and glycine have also been shown to influence
respiratory rhythm by shaping the respiratory pattern (Brockhaus and Ballanyi, 1998;Gray et al.,
1999c;Shao and Feldman, 1997). Neurokinin-1 receptor neurons (Gray et al., 2001a) and/or
somatostatin-expressing neurons (Tan et al., 2008) within the preBotC region were also found to
11
be necessary for generation of breathing in young adult rat. The various neuromodulators
involved in rhythm generation and modulation suggest that the mammalian respiratory network
is highly robust and dynamic.
Alteration of pattern generation also depends on synaptic modulators inducing or suppressing
neuronal types. Cellular activity depends on the activation and inactivation of ion channels,
which can be influenced by their local extracellular milieu. Neuromodulators regulate bursting
properties of many CPG neurons, which correspond to changes in respective rhythmic output.
These includes: serotonin (Pena and Ramirez, 2002), norepinephrine (Viemari and Ramirez,
2006), substance P (Del Negro et al., 2005;Morgado-Valle and Feldman, 2004;Pena and
Ramirez, 2004), Orexin A (Ayali and Harris-Warrick, 1999) and Ach through muscarinic
acetylcholine receptors (Shao and Feldman, 2005;Shao and Feldman, 2009b). In the respiratory
network, endogenous released serotonin (5-HT) is required for generation of bursting in a group
of respiratory pacemaker neurons known for their cadmium insensitivity (Pena and Ramirez,
2002). Under hypoxia-induced fictive gasping activity, 5-HT is also required in modulating
respiratory rhythm within the ventral respiratory group (Tryba et al., 2006).
Neurotransmitters also regulate cardiac rhythm through activation of the vagal parasympathetic
pathway. Vagal transmission decreases heart rate and contractility; to date, four of the five
muscarinic receptor subtypes, namely M1 to M4, have been found in heart in various species (rat
(Krejci and Tucek, 2002), mouse (Dhein et al., 2001), canine (Shi et al., 1999a), and human
(Brodde and Michel, 1999;Peralta et al., 1987)). Gi/o-coupled M2 receptor (M2R) is the main
subtype in SAN pacemaker cells and negatively regulates heart automaticity (Brodde and
Michel, 1999;Dhein et al., 2001), by interacting with various ion channels, such as potassium
acetylcholine-activated current (IKACh), hyperpolarizing-activated or funny current (If) and L-type
voltage-gated calcium channel (ICaL) (Caulfield, 1993;Felder, 1995). M3 receptor gene is highly
expressed in cardiac myocytes (human atria and ventricles (Brodde and Michel, 1999), canine
atria (Shi et al., 1999a)) and intrinsic cardiac ganglia (rat (Dhein et al., 2001;Krejci and Tucek,
2002)). M3 receptors in atrial myocytes from canine and guinea pig are coupled to K+ channels
(Wang et al., 1999). Neuropeptide, substance P, may regulate heart rate through interaction with
autonomic nerve system at SAN (Dzurik et al., 2007;Smith et al., 1992) and has been implicated
in myocardial hypertrophy and heart failure (D'Souza et al., 2007).
12
1.2.1.2 Intracellular signaling
Modulators are capable of activating various intracellular signaling cascades that modulate
membrane ion channel properties to regulate cellular physiology. These pathways represent the
flexibility of the networks to generate, strengthen, and modify rhythmic patterns. Short-term
signaling effects include ion channel modification, membrane localizing and sequestering of
proteins, post-translational modifications and vesicle release. Long-term effects involve gene
expression changing various membranous and non-membranous proteins. Modulation of network
activity by G-protein coupled receptors (GPCRs) has been implicated in numerous rhythmic
networks both in vertebrate and invertebrate systems (Dahdal et al., 2010). Typically, a ligand
binds to a GPCR and activates associated G-proteins by exchange of bound GDP with GTP
resulting in a dissociation of G-protein into β, γ and α subunits. The activated G-protein subunits
will exert their intracellular functions by activating either cAMP or phosphatidylinositol
signaling pathway. In invertebrates, activation of GPCR was implicated in the generation of
rhythmic output in molluscan (Angers et al., 1998;Gerhardt et al., 1996), nematode (Xie et al.,
2005) and arthropoda (Tanoue et al., 2008). Likewise in the vertebrate system, activation of
GPCRs is involved generation and regulation of numerous rhythmic networks (Civelli et al.,
2006;Doi and Ramirez, 2008), including the respiratory network. Typically, respiration under
normal physiological condition involves activation of GPCR coupled with to Gαq/11 proteins,
which are mainly lead to facilitation in respiratory motor output. Activation of Gαi/o proteins are
typically involved in inhibition of respiratory activity (summarized by (Doi and Ramirez, 2008)).
For example, 5-HT2A receptors were identified to be critical for respiratory rhythm by activation
of PKC pathway (Pena and Ramirez, 2002). 5-HT2 are known for their facilitating role as they
are coupled to Gαq/11 and linked to PLC (Fink and Gothert, 2007). In addition, activation of
Gαq/11 is also known to activate mitogen-activate proteins, phosphatidylinositol 3-kinase,
extracellular-regulated kinase and protein kinase B(Cowen, 2007), suggesting possible long-term
modifications to rhythmic activity.
Calcium is also an important signaling molecule in cell physiology. Its charged properties and
regulated low intracellular concentration make Ca2+
entry a potent electrical stimulus. Beyond
acting as a stimulus, Ca2+
also serve an important role as a signaling molecule. In neurons and
secretory cells, intracellular Ca2+
activates mechanisms for vesicle fusion and release of content
13
into the extracellular space. In myocytes, Ca2+
is an essential signal to induce muscle contraction.
In addition, calcium also acts as an activating ligand for many other ion channel and transporters
(see section 1.3.1.3). The multifaceted roles of Ca2+
make it an important regulator of rhythmic
activity, illustrated by its extensive involvement in multiple rhythmic tissues in various
organisms (see reviews by Mangoni et al., 2006a, Nakayama et al., 2007, and Whalley, 2011).
1.2.2 Pacemaker cells
Pacemaker cells are identified by their intrinsic ability to generate spontaneous activity. Under
experimental conditions, true pacemaker cells are capable of electrical activity following
physical or pharmacological isolation. The specific types of pacemaker activity vary from tissue
to tissue, but include tonic or burst firing in neurons of the CNS (Feldman and Del Negro,
2006;Pena et al., 2004;Puopolo et al., 2007;Steriade and Timofeev, 2003), spontaneous
contractions of pacemaker cells in heart (Mangoni and Nargeot, 2008) and periodic slow wave
oscillations in the pacemaker cells of the gut (Thomsen et al., 1998). Although cells of different
pacemaker regions adopt various methods of generating and regulating pacemaker activities,
many fundamental mechanisms are conserved between tissues.
1.2.2.1 Pacemaker neurons
Pacemaker neurons have been identified in numerous neuronal regions that regulate rhythmic
activities. These includes the neocortex (Le Bon-Jego and Yuste, 2007), ventral tegumental area
(Liu et al., 2002), raphe nucleus (Farkas et al., 1996), basal ganglia (Amini et al., 1999;Harris et
al., 1994;Harris and Constanti, 1995;Lee and Tepper, 2007;Mrejeru et al., 2011;Puopolo et al.,
2007), thalamus (Jahnsen and Llinas, 1984;McCormick and Bal, 1997;Steriade and Timofeev,
2003), locus coeruleus (Shen and North, 1992b;Shen and North, 1992a), hypothalamus
(Ghamari-Langroudi and Bourque, 2002) and hippocampus (Maccaferri and McBain, 1996). The
physiological importance of pacemaker neurons is an area of continuous research. It is believed
their central role is to regulate rhythmic properties and modulate network activities within the
CNS. Their functional roles are to modulate oscillation behavior of the respective neural network
through their ability to generate various rhythms. Indeed many pacemaker neurons are highly
plastic, capable of alternating between different patterns. For example, the alternation between
tonic and bursting properties in thalamic pacemaker neuron is an essential determinant of the
14
sleep-wake cycle (Jahnsen and Llinas, 1984;McCormick and Bal, 1997;Steriade and Timofeev,
2003). This is also observed in various other brain regions (Rubin et al., 2009;Wolfart and
Roeper, 2002).
Beyond the regulation of network activity, pacemaker neurons also serve as the major integrator
of external and internal cues that drive and modify network rhythm. Pacemaker neurons allow
the circadian rhythm of D. melanogaster to integrate various internal molecular or cellular
signals, along with environmental cues, to determine the phase and timing of diurnal clock
(Grima et al., 2004;Im et al., 2011;Peng et al., 2003;Stoleru et al., 2005). This type of integration
is also observed in the mammalian system, such as the integration of appetite signals in the
neuropeptide Y and agouti-related protein pacemaker neurons (van den et al., 2004). In other
neuronal regions, integration of pacemaker activity is both physiologically and pathologically
important. As demonstrated by the pacemaker neurons of the substantia nigra, where L-type
calcium channel activation leads to susceptibility of potential development in Parkinson’s
disease(Becker et al., 2008;Chan et al., 2007). It is clear from many lines of evidence that
pacemaker neurons are involved in different aspect of modulating neuronal output.
1.2.2.2 Respiratory pacemaker neurons
As evident from studies in respiratory neurophysiology, contribution of pacemaker neurons to
respiratory rhythm generation is unclear. Respiratory pacemaker neurons were identified in the
heterogeneous structure of preBotC through pharmacological isolation. They were characterized
by their intrinsic bursting abilities (Thoby-Brisson and Ramirez, 2000). Within this group of
pacemaker neurons, various pharmacological sensitivities and electrical properties further
distinguishes between the different cell types. Cells there classified as type I or type II based on
different bursting duration and sensitivity to TTX and Cd2+
(Thoby-Brisson and Ramirez, 2000).
Further pharmacological distinctions were established based on the expression of persistent
sodium current (INaP) and calcium-sensitive non-selective cationic current (ICAN). Type I
pacemakers generate their activity through INaP, whereas type II rely largely on ICAN as the source
of pacemaker generation (Del Negro et al., 2005;Pena et al., 2004;Tryba et al., 2006).
Developmentally, type I pacemakers are largely present throughout postnatal respiratory network
development, whereas type II pacemakers emerge during the developing process (Del Negro et
al., 2005;Pena et al., 2004).
15
Respiratory pacemaker neurons are also sensitive to various neuromodulators. Functional type I
pacemaker activity depends on the expression of 5-HT2A receptors (Pena and Ramirez,
2002;Tryba et al., 2006) and its activation has been linked to regulation of INaP. This 5-HT
dependent activation on pacemaker generation is not observed in type II neurons. Substance P is
also well known to enhance preBotC activity through activation of a TTX-insensitive inward
current (Pena and Ramirez, 2004), identified as ICAN (Ben-Mabrouk and Tryba, 2010).
Regulation of type I and type II activity by substance P is to facilitate the bursting activity (Pena
and Ramirez, 2004). The molecular constituent was determined to be partially contributed by
conductrance from the transient receptor potential (TRP) channel family (Ben-Mabrouk and
Tryba, 2010) (see section 1.3.2.2). Noradrenaline also modulates pacemaker activity similar to
substance P, which depolarizes neurons and increases bursting frequency (Viemari and Ramirez,
2006). Both substance P (Ben-Mabrouk and Tryba, 2010;Pena and Ramirez, 2004) and
noradrenaline (Viemari and Ramirez, 2006) increase type I pacemaker bursting frequency, and
effectively facilitate bursting activity of type II pacemakers. The dependence of modulators to
affect pacemaking generation brought forward the fundamental question of whether pacemaker
neurons are the generator or regulator of respiratory rhythm.
Much remains unclear of whether pacemaker neurons in the preBotC are the source of rhythm
generation. Evidence suggests differential roles of pacemaker neurons in regulating and
modifying their bursting proportions in generating different fictive respiratory motor outputs
(Pena et al., 2004;Pena and Aguileta, 2007;Tryba et al., 2006). Under eupnea condition,
inhibition of either INaP or ICAN does not abolish respiratory rhythm (Del Negro et al., 2002;Del
Negro et al., 2005;Paton et al., 2006;Pena et al., 2004), but combined application eliminates
spontaneous respiratory activity (Del Negro et al., 2005;Pena et al., 2004). This finding is
confirmed in both in vitro (Pena et al., 2004) as well as in vivo observations (Pena and Aguileta,
2007). Pharmacological evidences also indicate that rhythm can be restored and maintained
provided continuous application of substance P during INaP and ICAN block (Del Negro et al.,
2005), though the finding is contested (Ben-Mabrouk and Tryba, 2010;Tryba et al., 2006).
Despite evidences suggesting the essential role of other neuromodulators in generation of rhythm
of adult mammalian respiratory rhythm, respiratory pacemaker neurons still form an essential
component in generating rhythm during post-natal development.
16
1.2.2.3 Sinoatrial node pacemaker cells
Unlike neurons, cardiac pacemaker cells are well studied for their fundamental roles in
generating cardiac rhythms. The pacemaker myocytes found in the adult SAN region are small,
pale and poor in sacromeres and myofilaments (Mangoni and Nargeot, 2008). Discoveries of
developmental markers such as T-box transcription factor 3 (Tbx3) (Mommersteeg et al.,
2007;Wiese et al., 2009) and 18 (Tbx18) (Wiese et al., 2009) helped to delineate the SAN region
during development. In addition, developed SAN region shows relatively higher expression of
low-conductance connexin45 (Cx45) and connexin30.2 (Cx30.2) rather than high-conductance
connexin43 (Cx43) (Davis et al., 1995;Kreuzberg et al., 2005). In addition, expression of gap
junction proteins appear to be lower in abundance in SAN than atrial tissues, which along with
slow-conductance channel expression, contributes to its slow conduction velocity and poor cell-
to-cell electrical coupling (Davis et al., 1995;Kreuzberg et al., 2006b;Kreuzberg et al., 2006a).
This has been thought as a necessary membrane resistive barrier to protect the SAN from the
large electrical oscillations of much more hyperpolarized adjacent atrial tissues (Joyner and van
Capelle, 1986;Watanabe et al., 1995).
Despite the heterogeneious nature of the SAN region, attempts have been made to describe the
organization of the SAN largely relying on the electrical properties of the pacemaker cells.
Currently, two models exist. The first outlined by Boyett et al. (2000) called for a “gradient
model” where there is a gradual transition between periphery SAN and central SAN in terms of
cell sizes, upstroke velocity, and pacemaking rate. Consistent with this hypothesis are the
observations of electrophysiological differences between the pacemaker cells isolated from the
center versus the periphery of the SAN region (Boyett et al., 2000;Boyett et al., 1998;Kodama et
al., 1999;Kodama and Boyett, 1985;Nikmaram et al., 1997). A mosaic model had also been
proposed by Verheijck et al. (1998), which describes non-preferential distribution of pacemaker
cells of different electrophysiological profiles within the SAN region. Evidence largely supports
gradient organization of the SAN as cells of the periphery is found to contain higher expression
of Cx43 and is found to be more electrically coupled to atrial cells than central SAN (Boyett et
al., 2006). This sets the periphery SAN as a possible the exit point for the pacemaker signal
originating from the central SAN (Boyett et al., 2006;Dobrzynski et al., 2005b). Although
pacemaker rate of the periphery is higher than the central SAN, the central SAN dominates over
17
the periphery, acting as the determinant of the pacemaker rhythm. This is largely attributed to the
inhibition of periphery SAN from the large hyperpolarizing activities of the atrial myocytes
(Bleeker et al., 1980;Kodama and Boyett, 1985). Larger pacemaker cells of the periphery SAN
expresses higher Ih and INa, which seems to account for the higher upstroke velocity (Kodama et
al., 1997;Mangoni and Nargeot, 2008;Nikmaram et al., 1997).
1.2.3 Electrical properties of pacemaker cells
The spontaneous activity of pacemaker cells is determined by the electrical properties arising
from the ion channels. Although not all pacemaker cells share the exact molecular determinant of
these properties, many fundamental principles of regulating electrical properties of pacemaker
cells are highly conserved.
1.2.3.1 Resting or basal membrane potential
The fundamental importance of the resting membrane potential (RMP) has been described over
60 years ago during the initial characterization of the action potential in giant squid axon
(Goldman, 1943;Hodgkin and Huxley, 1947;Hodgkin and KATZ, 1949). The RMP is defined as
the membrane potential of a quiescent cell or “resting” cell. Although pacemaker cells are not at
rest, the resting or basal membrane potential of spontaneously active cell describes the average
basal membrane potential (no external stimulation) without contribution of action potential. The
ionic determinant of the resting membrane potential involves many different ionic components.
As determined by Goldman-Hodgkin-Katz equation (or commonly known as the Goldman
equation) (equation 1.1), the resting membrane potential
(
∑
∑
∑
∑
) (equation 1.1)
can be mathematically calculated as the summation of ionic gradient and permeability of all ionic
species across the cell membrane. In most cells, Na+, K
+, and Cl
- are the major ionic components
that determine the resting membrane potential (equation 1.2). The permeability to individual
(
-
-
-
-
) (equation 1.2)
ionic species is largely determined by the functional ionic channels that form conducting
18
membrane pores (see section 1.3). RMPs are largely determined by the background currents of
the cell (see section 1.3.3).
The resting membrane potential is one of the essential factors that control spontaneous activity. It
regulates cell excitability through two mechanisms: 1) A depolarized RMP reduces the
depolarizing voltage necessary to bring cell membrane potential to the threshold of action
potential firing, and 2) RMP regulates voltage-dependent ion channel activation and thus helps
shape rhythmic patterns and profiles. For example, bursting in the respiratory CPG pacemaking
neurons requires activation of various subthreshold channels, which depends on the RMP
(Feldman and Del Negro, 2006;Koizumi and Smith, 2008). Tonic firing or burst firing patterns
of respiratory pacemaker neurons depends on the basal membrane potential (Feldman and Del
Negro, 2006). In most cells that do not exhibit spontaneous activity, a negative RMP helps to
maintain a quiescent state. This is typically observed in both neurons and myocytes where most
RMP of non-pacemaker cells is approximately between -90 mV to -70 mV. Pacemaker cells on
the other hand are generally much more depolarized with resting or basal membrane potential
between the ranges of -60 mV to -35 mV. In neurons, highly depolarized basal membrane
potential maintains tonic or burst firing in many pacemakers (Amini et al., 1999;Farkas et al.,
1996;Ghamari-Langroudi and Bourque, 2002;Harris et al., 1994;Harris and Constanti,
1995;Jahnsen and Llinas, 1984;Le Bon-Jego and Yuste, 2007;Lee and Tepper, 2007;Liu et al.,
2002;Maccaferri and McBain, 1996;McCormick and Bal, 1997;Mrejeru et al., 2011;Puopolo et
al., 2007;Shen and North, 1992a;Shen and North, 1992b;Steriade and Timofeev, 2003). In SAN
pacemaker cardiomyocytes, inhibiting rhythmic firing by interfering with intracellular [Ca2+
]
reveals a resting membrane potential of approximately -40 mV (Ju and Allen, 1998). In the gut
pacemaker cells, maximal hyperpolarization of spontaneous slow wave oscillation recorded from
interstitial cells of Cajal is approximately at -60 mV with average resting membrane potential at
approximately -55 mV (Connor et al., 1974;Dahdal et al., 2010;El-Sharkaway and Daniel,
1975;Kim et al., 2012;Sanders et al., 2006a). Likewise in the chromaffin cells of the adrenal
gland, average resting membrane potential was established to be at approximately -45 mV at
basal condition (Marcantoni et al., 2010) and during inhibition of spontaneous activity by
voltage-gated Ca2+
and Na+ channel blockers (Marcantoni et al., 2010). These principles of basal
membrane potential between pacemaker and non-pacemaker cells are also found in invertebrate
19
systems (Moccia et al., 2009;Staras et al., 2002), indicating that regulation of resting membrane
potential is a fundamental determinant of pacemaker activity in almost all pacemaker cell types.
1.2.3.2 Intrinsic membrane excitability
Beyond resting membrane potential, intrinsic membrane excitability also regulates electrical
activity. There are a number of membrane parameters that determine membrane excitability;
some of the most common are the input resistance, temporal summation and length constant. A
fundamental principle of regulating membrane excitability is the input resistance, a D/C resistive
property of the membrane to current flow. Based on principles of Ohm’s law, stimulating current
will cause a greater depolarization on cell with high membrane resistance than one with low
membrane resistance. Temporal integration refers to the integrative ability of multiple synaptic
inputs to significantly alter action potential activity. These integrative properties of cells
modulate their firing pattern, firing ability, and sensitivity to extrinsic inputs. Length constant
can also affect intrinsic excitability, by modulating the passive conductance of electrical signal
through spatial summation. This depends on the resistance of the neuron membrane and the
intrinsic resistivity of the cyotosolic compoartment. In axons, resistivity is inversely proportional
to the diameter of the axon.
Another mean of regulating membrane excitability is to regulate the intrinsic properties of the
ion channels. Channel properties could be modified through post-translation modifications,
subunits association, or alternative splice variations. Altering these properties has been shown to
affect the conductance, gating, activation and inactivation kinetics, and membrane protein-
coupling (see section 1.3). These modifications can effectively alter the functional contribution
of the ion channel and limit their contribution to membrane excitability.
1.2.4 Rhythmic computation models
Understanding the mechanism of many levels of cellular, sub-cellular and network interaction
often is beyond the means of practical experimentations; hence many investigations adopt a
mathematical approach toward understanding the principle of rhythm generation and
perpetuation.
20
1.2.4.1 Network simulations
Network simulations are aimed at approximating the oscillating behavior observed from
experimental recordings. The underlying objectives are to understand the regional connectivity
that can generate rhythm. In neurons, network simulations have grown in complexity to
mathematically explain how rhythm can be generated from combinations of rhythmic and non-
rhythmic neuronal populations. Various models of half-oscillation centers and CPG networks
were established to explain network output from intrinsic and synaptic variations (Hill et al.,
2001;Perkel and Mulloney, 1974;Prinz et al., 2004;Skinner et al., 1994). Numerous models were
also established to replicate CPG output based on known network connectivity, including the
rCPG network (Del Negro et al., 2010;Rybak et al., 2007;Smith et al., 2007). The spatially
organized rCPG network model proposed by(Rubin et al., 2009) consists of 5 neuronal
populations described by activity-based parameters. This simplified model of the rCPG network
aims to investigate the synaptic coupling mechanisms between the various rCPG regions, namely
the Botzinger Complex and preBotzinger Complex, and retrotrapezoid nucleus and parafacial
respiratory group as reconstructing the oscillatory patterns. In heart, spatial organization has also
been simulated for the SAN (Dobrzynski et al., 2005b) as well as large scale model of the
cardiac tissue (Henriquez, 1993;Potse et al., 2006) to determine how pacemaker signal is
conducted throughout cardiac tissue as well as rhythmic regulation under pathological
conditions. Models such as Rubin et al (2009) rely on a combination of single-cell simulations
linked with mathematical parameters from experimental observations to approximate network
behaviour.
1.2.4.2 Single-cell simulations
At the single-cell level, pacemaker generation varies from cell to cell. Although many
fundamental principles persist between different pacemaker cell types, huge variations in
rhythmic pattern and firing activity are observed in recordings. In order to study and understand
the current dynamics of rhythmic firing, mathematical models are employed to simulate and
describe how ionic conductance influence pacemaker activity. Beyond integration of ionic
conductance, many simulations further integrate different mechanisms such as regional ionic and
signaling dynamics with compartment models (Kurata et al., 2002;Toporikova and Butera,
2011;Wilson and Callaway, 2000). Cellular regions are segregated into compartment with its
21
own distinct oscillating characteristics, which are linked through mathematic parameters and
equations. Features such as this are important in explaining anomalies in experimental
recordings.
The typical description of neuronal activity is a conductance-based model, such as the classic
Hodgkin-Huxley model from the characterization of the squid giant axon action potential
(Hodgkin and Huxley, 1952). Hodgkin and Huxley identified 3 types of currents carried by
voltage-dependent Na+ current, voltage-dependent K
+ current and a leak current carried by
largely K+ and Cl
-. The model separates different conductance and used mathematical equation
to describe current-voltage relations. The final output voltage is a combination of all 3 types of
currents. Based on this model, numerous variants followed suit with changes and addition of
different currents to describe the respective cell conductance (Connor and Stevens, 1971;Morris
and Lecar, 1981). For pacemaker cells, integration of various identified currents that contributes
to pacemaker activities (section 1.3) has been highly successful in describing the generation of
pacemaker rhythm (Amini et al., 1999;Amini et al., 2005;Butera, Jr. et al., 1999;Rose and
Hindmarsh, 1985). In thalamic neurons, mathematical models were a useful to describe the
contribution of calcium current in regulating intrinsic oscillation between tonic and bursting
activities (McCormick and Huguenard, 1992). A similar application has been used to investigate
the mechanisms of ionic currents and their distributions that contribute to various rhythmic
patterns of respiratory neurons (Dunmyre et al., 2011;Toporikova and Butera, 2011). However,
due to the heterogeneity of network composition, most pacemaker models describe pacemaker
mechanism of a cell population, rather than an identifiable single-cell.
Current cardiac pacemaker models are inherently more complex due largely to the extensive
characterization of various electrical, molecular and physical properties. Cardiac pacemaker
requires multiple currents regulating voltage-dependent and calcium-dependent activation for
pacemaker activity (reviewed by Mangoni and Nargeot, 2008 and Wilders, 2007). Initial
characterization of cardiac rhythm was also based on Hodgkin-Huxley principle (Noble,
1960;Noble, 1962). Since then, the model was continuously revised and modified with the
inclusion of newly identified currents (Wilders, 2007). Development of conductance-based
single-cell models began in the 1980s (Bristow and Clark, 1982;Yanagihara et al., 1980) and
continues to this day. Recently, models for SAN pacemakers have even been incorporating
22
information of membrane capacitance variation of SAN pacemaker cells (Zhang et al., 2000a),
Ca2+
dynamics and buffering (Kharche et al., 2011;Kurata et al., 2002;Maltsev et al., 2004) and
cell shape and volume dynamics (Matsuoka et al., 2003;Sarai et al., 2003;Takeuchi et al., 2007).
In addition, new and old currents are still continuously being described and refined (Alig et al.,
2009;Demion et al., 2007;Huang et al., 2009), ensuring that continuous revision of SAN
pacemaker models will provide a more accurate quantitative description of the pacemaking
mechanism.
1.3 Ion channel dynamics: genesis and perpetuation of pacemaking
In pacemaker cells, different ion channel dynamics represent a major component contributing to
pacemaking (Figure 1.2). These currents regulate and shape action potential properties, as well
as determine the oscillation and bi-stability conditions (Bean, 2007). In this respect, many similar
currents have been identified in pacemaker cells in different tissues. These similarities are further
conserved between many invertebrate and vertebrate system. For this reason, identification and
interpretation using many invertebrate models has been useful in understanding the fundamental
ionic mechanisms of pacemaker activity.
1.3.1 Voltage-dependent ion channels
1.3.1.1 Sodium channels
Voltage-dependent sodium channel family is ubiquitously known for their role in shaping and
propagating action potentials of neurons and myocytes (Catterall, 2000;Ruan et al., 2009;Yu and
Catterall, 2003). The family of α pore-forming subunits consists of 10 known members with 4
linked repeats of 6 transmembrane domains (Catterall, 2000;Catterall et al., 2005;Yu and
Catterall, 2003). The voltage-dependent properties are largely derived from the voltage sensor
located on the 4th segment (S4) of each transmembrane domain, which contains positive-charged
residues at every third position (Catterall et al., 2005;Stuhmer et al., 1989;Yu and Catterall,
2003). During activation, depolarization causes opening of the channel pore through the S4
sensor and the channel quickly inactivates (within milliseconds) through a hinge-lid located on
the intracellular loop between the III and IV transmembrane domains (Rohl et al., 1999;Vassilev
et al., 1989;Yu and Catterall, 2003). The physiological gating properties of voltage-dependent
23
Figure 1.2. Current components contributing to spontaneous activity in pacemaker cells of neurons and heart. (A) In
neurons, action potential firing depends on voltage-dependent persistent Na+ current (INa-P) depolarizes membrane
potential to threshold activating voltage-gated Na+ current (INav). Rapid depolarization of membrane potential
activates various voltage-gated Ca2+
channels (ICaN,P/Q,R,L) and voltage-gated K+ current (IKv). After hyperpolarization
is mediated by the Ca2+
dependent K+ current (IBK,SK), followed by hyperpolarizing-activated current (Ih) and T-type
low voltage activated Ca2+
current (ICaT). K+ leak current (IK Leak) determines resting membrane potential along with
Na+ leak current (INa Leak) and Cl- leak current (ICl Leak). Maintinance of bursting various between different neurons
but it involves various activations of different ligand-gated currents (IGABA, Igly, Iglu, IAch, I5-HT) and Ca2+
dynamic
through voltage-gated calcium channel activating various Ca2+
dependent ion channels such as IBK and ISK and
calcium-dependent non-selective cationic current (ICAN). (B) In heart, pacemaker potential of SAN cardiomyocyte
dependent on resurgent depolarizing inward currents generated from funny current (If) sustained sodium current (Ist),
Na+/Ca
2+ exchanger (INCX) and chloride current (ICl). Action potential profile is largely dictated by INav and ICaL
for depolarization and IKv for hyperpolarization. Similarily, IK Leak, INa Leak and ICl Leak contributes to the background
conductance that determines the resting membrane potential.
24
sodium channels are also highly dependent on their auxiliary subunits (Isom et al., 1992;Isom et
al., 1994). Co-expression of β subunits is required to reestablish the native sodium current in
vitro (Isom et al., 1994). This is further highlighted by mutation of the β1 subunit can result in
epilepsy, due to modified gating mechanics (Wallace et al., 1998).
The sodium channels are also highly selective for sodium ions, which is largely attributed to the
pore selectivity motif located within the inner ring. The pore forming loop (P-loop) sequence,
DEKA (in single-amino acide codes), are highly conserved between sodium channels, hence the
derived name “selectivity filter” (Heinemann et al., 1992). Voltage-dependent sodium channels
can be typically blocked by extracellular application of an alkaloid-based toxin such as
tetrodotoxin (TTX) (Narahashi et al., 1964). Classical identification of different voltage-
dependent sodium channels can be made through their pharmacological sensitivity to TTX, with
TTX-sensitive channels being completely blocked within the nanomolar range, and TTX-
resistant channels blocked in the micromolar range (Elliott and Elliott, 1993;Ogata and
Tatebayashi, 1993). Despite their role in action potential initiation, various isoforms of voltage-
dependent sodium channels has been identified to be crucial for pacemaker generation.
Through a combination of pharmacology and genetics, numerous studies have shown voltage-
gated sodium channels to be an important element in pacemaker activity. In the cardiac system,
early neonatal SAN pacemaker has shown high expression of Nav 1.1 isoform (Baruscotti et al.,
1997). However, pacemaker function in adult SAN is largely regulated by both TTX-sensitive
Nav 1.1, Nav 1.6 and TTX-resistant Nav 1.5 (Du et al., 2007;Lei et al., 2004). In neuron, Nav 1.6
has been found to be important in fast spiking activity in Purkinjie cells (Khaliq et al.,
2003;Raman et al., 1997) and pacemaker neurons of globus pallidus (Mercer et al., 2007). Nav
1.6 contributes to resurgent depolarizing current that maintains cells at fast spiking frequency
(Khaliq et al., 2003;Raman et al., 1997). However, this functional role is not consistently
observed in all pacemaker neurons (Do and Bean, 2004). Many studies have investigated the
functional physiological role of Nav 1.6 isoform in neurons, largely due to evidence that it
contributes to voltage-gated persistent sodium current found in many pacemaker neurons and
rhythmic networks.
25
Persistent voltage-dependent sodium current has been characterized early on as subthreshold
non-inactivating sodium current (Colmers et al., 1982;Crill, 1996;French et al., 1990;Hotson et
al., 1979). This type of current has been described initially by Hudgkin and Huxley (Hodgkin
and Huxley, 1952), which later studies have also found this current in numerous neurons of
vertebrate (Cummins et al., 1994;French et al., 1990;Stafstrom et al., 1982) and invertebrates
(Colmers et al., 1982;Davis and Stuart, 1988;Nikitin et al., 2006) organisms. Subsequent
pharmacological classification of the persistent voltage-dependent sodium current indicate high
sensitivity to TTX (Colmers et al., 1982;Crill, 1996) and riluzole (Song et al., 1997;Urbani and
Belluzzi, 2000). Unlike most voltage-dependent sodium currents, persistent sodium current
activate at “subthreshold” voltage (approximately 10 mV lower) and exhibit slow inactivation
kinetics (Crill, 1996). This sustained subthreshold activation state allows it to be a major
contributor of a resurgent depolarization current found in many pacemaker neurons (Koizumi
and Smith, 2008;Le Bon-Jego and Yuste, 2007;Tazerart et al., 2008). Currently, the exact
isoform of voltage-dependent sodium channel contribute to the persistent sodium current is
unclear. However, evidence suggests that Nav 1.6 is highly linked to the persistent sodium
current, given that mull mutation of the Nav 1.6 isoform reduced persistent sodium current
amplitude in Purkinje and prefrontal cortex pyramidal neurons (Maurice et al., 2001;Raman et
al., 1997;Vega-Saenz de Miera EC et al., 1997). Nevertheless, residual persistent sodium current
is found in Nav 1.6 mull mice, suggesting possible contribution from other channels such as Nav
1.1 and Nav 1.2 (Maurice et al., 2001;Raman et al., 1997;Van and Matthews, 2006). In
respiration, involvement of persistent sodium current to rhythm generation is complex and not
completely elucidated with evidence both supporting (Koizumi and Smith, 2008;Rybak et al.,
2004) and dismissing (Del Negro et al., 2005;Pace et al., 2007) its involvement in rhythm
generation. Complex role of persistent sodium current suggest that it may function in a dynamic
role to regulate both intrinsic and network contributions.
1.3.1.2 Calcium channels
Similar to voltage-dependent sodium channels, calcium channel activation regulates cell
excitability and action potential profile. In addition, calcium channels also regulate cell
contractility, gene expression, intracellular signaling and vesicle release. Many similarities are
shared between voltage-dependent sodium and calcium channels. For one, α1 pore-forming
26
subunit of voltage-dependent calcium channels is also a single protein with 4 homologous
domain repeats (I-IV) of 6 transmembrane α-helices (Catterall, 2011). Voltage-dependent
activation relies on the positive residues on the S4 voltage-senor of domains I-IV (Adams et al.,
1990;Catterall, 2011). Pore-forming and ion selectivity motif is also determined by 4 residues
located on the P-loop, with EEEE for high Ca2+
selectivity (Ellinor et al., 1995;Yang et al.,
1993). Calcium channels also form multimeric complex with membrane expressed auxiliary
subunits including α2δ, β1-4 and γ (Arikkath and Campbell, 2003;Catterall, 2011). The α2δ
subunit class is formed from two separated subunits produced from 1 gene linked in disulfide
bonds (De Jongh et al., 1990;Jay et al., 1991), and they function to regulate α1 channel gating,
kinetics, conductance and blocker binding sites (Bangalore et al., 1996;Felix et al., 1997;Itagaki
et al., 1992;Klugbauer et al., 1999;Shirokov et al., 1998;Shistik et al., 1995;Singer et al.,
1991;Williams et al., 1992). The β subunits cytosolic membrane-associated guanylate kinase
(MAGUK) -like proteins with a src homology 3 (SH3) domain. Their functional role largely
involves plasma membrane trafficking of the α1 (Bichet et al., 2000;Chien et al., 1995;Pragnell
et al., 1994), regulation of conductance and activation/inactivation kinetics (Feng et al.,
2001;Stea et al., 1993). The γ subunit is the smallest of the subunit within the multimeric
complex and it in channel physiology remains a topic of continuous investigation. Currently,
evidences suggest γ subunit is not directly involved large regulation of channel conductance or
trafficking (Black, III, 2003;Wei et al., 1991).
At least 10 isoforms of the calcium channels has been identified, with five different types
distinguished by their voltage-activation, tissue expression and sensitivity to blockers. Among
the high and medium voltage-activated calcium channels, classification and physiological
dissection are made through testing sensitivity to 1,4-dihydropridine (DHP), ω-conotoxin (ω-
CTX), and ω-agatoxin (ω-AGA) (Catterall, 2011;Tsien et al., 1988). Four different types have
been identified from these toxin combinations, with L-type (Cav 1.1-1.4) being sensitive to DHP
block (Tsien et al., 1988), P/Q-type (Cav 2.1) sensitive to ω-AGA (Llinas and Yarom,
1981;Llinas et al., 1989;Randall and Tsien, 1995), N-type (Cav 2.2) sensitive to ω-CTX
(Nowycky et al., 1985), and R-type (Cav 2.3) resistant to all three blockers (Randall and Tsien,
1995). The last type of calcium channel is the low voltage-activated T-type channel (Cav 3.1-
3.3) characterized by its low threshold of activation and rapid inactivation (Perez-Reyes, 2003).
The overall biophysical function of the calcium channel complex is to regulate Ca2+
27
conductance, and since Ca2+
is both a signaling molecule and an excitatory ion. Many voltage-
dependent Ca2+
channels are found to be involved in regulating pacemaker rhythm in neurons,
myocytes, and neuroendocrine cells.
Currently a large body of evidence indicates direct involvement of L-type calcium channel in
pacemaker generation of many excitable tissues. Specifically, Cav 1.3 and Cav 1.2 contribute to
basal calcium oscillation, which Cav 1.3 is also the major L-type dependent calcium current
(Mahapatra et al., 2011). This has been attributed to the biophysical properties of Cav 1.3, which
has faster activation at lower voltages and slower voltage-dependent inactivation compared to
other L-type calcium channels (Helton et al., 2005;Koschak et al., 2001;Platzer et al., 2000;Xu
and Lipscombe, 2001). Cav 1.3 contributes to pacemaker generation in the substantia nigra
(Amini et al., 1999;Puopolo et al., 2007), the SAN (Mangoni et al., 2003;Zhang et al., 2002),
AVN (Marger et al., 2011) of cardiac pacemakers, and the basal oscillations of chromaffin cells
(Marcantoni et al., 2010;Vandael et al., 2010). Genetic loss of functional Cav 1.3 has been linked
to deafness, SAN dysfunction and bradycardia in both mouse (Mangoni et al., 2003;Platzer et al.,
2000) and human (Baig et al., 2011). Susceptibility of Cav 1.3 conductance has also been linked
to possible development of Parkinson’s disease (Chan et al., 2007) and chronic stress disorder
(Vandael et al., 2010).
Low voltage-activated, T-type calcium channels are also important in the generation of
pacemaker rhythm (Perez-Reyes, 2003). In heart, functional expression of Cav 3.1 contributes
largely to pacemaker rhythm regulation (Mangoni and Nargeot, 2008). Cav 3.1 null mice show
reduced heart rate and prolonged PR interval on ECG telemetry (Mangoni et al., 2006b). In the
CNS, Cav 3.1 is necessary to generate burst firing in thalamic neurons (Kim et al., 2001). Outside
of the cardiac tissues, T-type current has been observed and suggested for possible contributions
to pacemaker rhythms in portal veins (Loirand et al., 1989;Perez-Reyes, 2003), anterior pituitary
gland (Cota, 1986;Perez-Reyes, 2003) and pancreas (Barnett et al., 1995;Perez-Reyes,
2003;Satin et al., 1994).
1.3.1.3 Calcium-dependent ion channels
The dual nature of calcium as both an excitatory ion and a signaling molecule is demonstrated in
many pacemaker cells with calcium coupled SK- or BK-induced bursting properties. Calcium-
28
dependent potassium channels are highly sensitive to intracellular [Ca2+
] (Salkoff et al., 2006).
They share many structural similarities to voltage-dependent potassium channels, hence their
conductance is voltage-dependent (Vergara et al., 1998). Identification of these channel types are
based on their conductance, with BK for big conductance and SK for small conductance (Salkoff
et al., 2006;Vergara et al., 1998). In CNS, coupling of an excitatory depolarizing voltage-
dependent Ca2+
channel with fast repolarizing BK or SK conductance is found to contribute to
bursting rhythm in many different neurons (Bowden et al., 2001;Marrion and Tavalin, 1998;Shah
and Haylett, 2000;Wolfart et al., 2001). The flexible selective coupling allows some pacemaker
neurons to switch between pacemaker and bursting activity (Wolfart and Roeper, 2002). Similar
coupling has been found in chromaffin cells between Cav 1.3 and BK channel (Marcantoni et al.,
2010;Vandael et al., 2010). These properties make voltage-dependent calcium channel a key
target of investigation as a pacemaker channel.
In addition, many rhythmic firing neurons within the CNS also express a calcium-dependent
non-selective cationic current (ICAN). This current was first identified in the pacemaker neurons
of the mollusk, Helix pomatia (Swandulla and Lux, 1985). ICAN is characterized by an inward
current that is activated by increase in intracellular [Ca2+
] (Del Negro et al., 2005;Lee and
Tepper, 2007;Partridge and Valenzuela, 2000). This current has been identified in hippocampal
pyramidal (Caeser et al., 1993;Crepel et al., 1994;Partridge and Valenzuela, 2000) and hilar
mossy cells (Hofmann and Frazier, 2010), substantia nigra GABAergic (Lee and Tepper, 2007)
and dopaminergic neurons (Mrejeru et al., 2011), respiratory pacemaker neurons of preBotC
(Del Negro et al., 2005) and manocellular neuroendcrine cells of the hypothalamus (Ghamari-
Langroudi and Bourque, 2002). ICAN has been suggested to be an important component to
regulate spontaneous activity and bursting properties. Evidences suggest transient receptor
potential (TRP) channels (see 1.3.2.2) as the molecular identity of ICAN, with some groups
suggesting TRPC channels in some region of the brain (Ben-Mabrouk and Tryba, 2010), while
others suggest TRPM channels (Lee and Tepper, 2007).
Intracellular calcium also activates a family of chloride permeable ion channels, the CLCA
channels (Eggermont, 2004;Jentsch et al., 2002). The calcium-dependent chloride current has
been identified in neurons (Andre et al., 2003;Currie et al., 1995;Frings et al., 2000;Scott et al.,
1995), heart (Papp et al., 1995;Sipido et al., 1993;Zygmunt and Gibbons, 1991;Zygmunt and
29
Gibbons, 1992) and smooth muscle cells (Large and Wang, 1996). This chloride current can
regulate cell excitability, particularly in neurons where calcium-dependent chloride current can
generate after-depolarization (Mayer, 1985). In cardiac cells, calcium-dependent chloride current
can also regulate cell excitability and membrane repolarization (Hume et al., 2000). CLCA
channels are also regulated by various calcium signaling molecules, pH, G-proteins, and other
chloride channels (reviewed by Hartzell et al., 2005).
1.3.1.4 Potassium channels
The most recognized physiological role of voltage-dependent potassium channels is to repolarize
membrane potential following depolarization during action potential spiking. Their fundamental
importance is demonstrated by the large family of diverse functional membrane proteins. In
human, over 40 known voltage-dependent potassium channels have been identified. They are
grouped into 12 families (Kv 1-12) based on similar functions (Gutman et al., 2005). The basic
channel structure is in many ways similar to voltage-dependent Na+ and Ca
2+ channels.
Functional pore forming channel consist of 4 individual α subunits, each containing 6
transmembrane segments (Gutman et al., 2005). Voltage-sensor is found on the 4th segment of
the transmembrane helices and P-loop, making up the selectivity filter. However, unlike voltage-
dependent Na+ and Ca
2+ channels, voltage-dependent potassium channels are formed from 4
separate α subunits of either homomultimers or heteromultimers (Gutman et al., 2005). Certain
families of α subunits (Kv 5, 6, 8, and 9) do not form functional homotetramer channels but
rather work as modifiers of other families (Kv 2) (Gutman et al., 2005). In addition, selectivity
motif of potassium channels is TVGXG where X could be either tyrosine (Y) or phenylalanine
(F) (Gutman et al., 2005;Heginbotham et al., 1992;Heginbotham et al., 1994). Gross
classification of potassium channel types can be made based on pharmacological and biological
properties, there are 5 different voltage-dependent potassium currents based on rectification
and/or activation/ inactivation kinetics. 1) Delayed rectifiers are slow inactivating/non-
inactivating, 2) A-type is fast activating and inactivating, 3) Outward-rectifiers conduct larger
current in outward direction and is non-inactivating, 4) inward rectifiers conduct larger current in
the inward direction, and 5) slowly activating group of potassium channels (Gutman et al., 2005).
The molecular determinants of potassium channel subunits that determine these currents are
highly complex given the formation heterotetrameters, modifier proteins, accessory subunits, and
30
splice variants (Nitabach et al., 2002). However, most potassium currents are the primary
reporlarizing conductance during excitation, thus voltage-dependent potassium channels are
expressed in a wide array of cells and are found in almost every excitable tissue (Gutman et al.,
2005).
In many pacemaking systems, voltage-dependent potassium channels have been identified to be
important in not just action potential repolarization, but also establishing and perpetuating
pacemaker activity. The primary role of voltage-dependent potassium channel is to repolarize
membrane potential follow action potential depolarization. In addition, they also function to
regulate cell excitability through secondary recruitment of other voltage-dependent current. In
the cardiac system, partial blocking of the delayed rectifier current slows pacemaker rhythm, due
largely to reduce activation of depolarizing currents such as Ih, ICa T-ype and ICa L-type (Clark et al.,
2004). In contrast, complete block of delayed rectifier current abolished pacemaker activity
(Matsuura et al., 2002;Ono and Ito, 1995). In the brain, A-type current has been identified to be
important for regulation of pacemaker activity in substantia nigra (Liss et al., 2001) and
tuberomammillary nucleus (Jackson and Bean, 2007) neurons. As demonstrated in numerous
computation models (Zhang et al., 2007), incorporation of multiple voltage-dependent potassium
channels in necessary in establishing a functional simulation of the physiological pacemaker.
1.3.1.5 Hyperpolarizing-activated channels
Hyperpolarizing-activated cyclic nucleotide-gated (HCN) channels are often referred to as
pacemaking channels since their initial discovery as the major conductance responsible for
perpetuating pacemaker activity. The current has been identified to be important for pacemaker
rhythm in the heart (Barbuti et al., 2007;Mangoni et al., 2003;Yanagihara and Irisawa, 1980) and
brain (Maccaferri et al., 1993;Maccaferri and McBain, 1996;McCormick and Pape, 1990;Thoby-
Brisson et al., 2000). In mammals, 4 gene isoforms encode HCN channels (HCN1-4). Like many
of the voltage-gated pore-loop channels, HCN channels are formed from a tetrameric
conductance pore consisting of subunits with 6 transmembrane repeats (Biel et al., 2009;Wahl-
Schott and Biel, 2009). In animals, homotetramers (Ishii et al., 1999;Ludwig et al., 1998;Ludwig
et al., 1999b;Santoro et al., 1998) or heterotetramers (Altomare et al., 2003;Ulens and Tytgat,
2001;Whitaker et al., 2007) can form, giving rise to channels of different biophysical properties.
Fundamentally to HCN channels, is voltage-activation at hyperpolarizing membrane potentials
31
and it is not inactivated by voltage-dependent manner (Macri et al., 2002;Macri and Accili,
2004;Proenza et al., 2002). The S4 voltage-sensor of the HCN channel is similar to other
voltage-gated channels, with positive charged amino acids distributed evenly at every third
position (Chen et al., 2000;Vaca et al., 2000). However, inward motion during hyperpolarization
induces channel opening, whereas it would lead to closure in voltage-gated potassium channels
(Mannikko et al., 2002). Also similar to the voltage-gated potassium channel is the HCN
selectivity filter with GYG as the residue motif (Yu and Catterall, 2004), which partially explain
its higher conductance to K+ (Biel et al., 2009). However, HCN reversal potential is at -40 to -25
mV, and substitution studies indicate it is also permeable to Na+ in endogenously expressed
channel (Dekin, 1993;Ho et al., 1994;Kiehn and Harris-Warrick, 1992).
In addition to voltage-dependent activation, an increase in concentration of cAMP and cGMP
can increase HCN current amplitude and facilitate channel activation (Biel et al.,
2009;Yanagihara and Irisawa, 1980). This is achieved by a cyclic-nucleotide binding domain on
the c-terminus of the channel protein (Zagotta et al., 2003), which upon binding of cyclic
nucleotide, can shift activation curve to more depolarized level and increase activation kinetic
(Banks et al., 1993;Brown et al., 1979;DiFrancesco and Tortora, 1991;Larkman et al.,
1995;Tokimasa and Akasu, 1990). This represents significant flexibility in cAMP-dependent
regulation (Brown et al., 1979;DiFrancesco et al., 1989). Pharmacologically, HCN channels are
highly sensitive to extracellular Cs+ block and many bradycardic agents such as alindine,
ZD7288, zatebradine, and more recently with ivabradine (Biel et al., 2009). These highly
conserved properties of HCN current has been identified in numerous vertebrate (Ishii et al.,
1999;Ludwig et al., 1998;Seifert et al., 1999) and invertebrate (Gauss et al., 1998;Krieger et al.,
1999;Marx et al., 1999) organisms, suggesting the important functional role of this channel.
Initial classification of the HCN current in heart identified it as the “funny current” with the
label, If (Brown et al., 1979) or Iq (Halliwell and Adams, 1982). Expression profiles indicate all
four HCN isoforms are widely expressed in the heart of rabbit (Ho et al., 1994;Ishii et al., 1999),
mouse (Ludwig et al., 1999a;Mangoni and Nargeot, 2001), and human (Ludwig et al.,
1999b;Seifert et al., 1999). However, the exact molecular tetrameric channels responsible for the
If current has not been identified. Currently, molecular determinant of the cardiac pacemaker
region is the prevalent expression of HCN4 in cardiac SAN and AVN node (Marionneau et al.,
32
2005;Shi et al., 1999b). Functional determinant of HCN conductance is conferred through tests
with pharmacological blockers (reviewed by DiFrancesco, 2005 and DiFrancesco, 2006). These
investigations determined that HCN is the only voltage-dependent current activated at
hyperpolarizing voltages, and it functions to regulate diastolic depolarization rate (Mangoni and
Nargeot, 2008). Beyond pharmacology, genetics have also demonstrated importance of HCN
toward pacemaking in heart. HCN2 knockout mouse displayed SAN arrhythmia with a 30%
reduction in If (Ludwig et al., 2003). HCN4 knockout is embryonic lethal (Stieber et al., 2003),
but HCN4 inducible knockout demonstrated 75% reduction in If characterized by frequent SAN
pauses under ECG measurements (Herrmann et al., 2007). These studies help to shed light on the
inherited HCN channelopathies in humans, where currently 4 mutations are known (Milanesi et
al., 2006;Nof et al., 2007;Schulze-Bahr et al., 2003;Ueda et al., 2004). All 4 mutations are
characterized by sinus bradycardia similar to results seen from murine genetic studies. These
studies demonstrate the importance of If and HCN channels to normal cardiac pacemaking
function.
In the brain, HCN channels have been identified to conduct Ih current that contributes to rhythm
and oscillation of many neural circuitries. All 4 HCN isoforms are expressed in the brain with
different regional specificity (Biel et al., 2009). In knockout studies, deletion of HCN1 gene in
forebrain resulted in increase in the theta frequency oscillation due to partial regulatory role in
resisting contributing waveforms at below theta frequencies (Nolan et al., 2004). In addition,
oscillating role of Ih has also been identified in hippocampus (Fisahn et al., 2002), inferior olive
(Bal and McCormick, 1997) and entorhinal cortex (Dickson et al., 2000;Haas et al., 2007). At the
single-cell level, Ih has been identified in the thalamus to be essential in generating the bursting
behaviour observed during non-REM sleep (Jahnsen and Llinas, 1984;Steriade and Timofeev,
2003). In this model, Ih provides the depolarizing drive that activates T-type calcium channel,
which generates a series of slow spikes (McCormick and Bal, 1997). This Ih component has been
found to be largely dependent on the expression of HCN2 (Ludwig et al., 2003). In addition, Ih
has also been found to be important in generation of pacemaker rhythm within the hippocampus
(Maccaferri and McBain, 1996) and substantia nigra (Harris et al., 1994;Harris and Constanti,
1995). At the network level, Ih has been identified to be important in regulating respiratory
frequencies (Thoby-Brisson et al., 2000). These findings suggest that Ih and HCN channels also
exert their important role in both neurons and heart.
33
1.3.1.6 Chloride channels
Chloride channels are the major membrane channel that conducts anions. Of the 9 different CLC
chloride channel genes, 4 are largely membrane expressed channels (Jentsch et al., 2002).
Functional CLC chloride membrane channel consist of 2 subunits of homo or hetero dimmers.
Evidence suggests a “double barrel” model where two pores are formed by the functional
channel by each individual subunit (Dutzler et al., 2002;Fahlke et al., 1997;Miller, 1982;Miller
and White, 1984). Although most chloride channels are sensitive to voltage activation, no
conspicuous voltage sensor has been identified (Jentsch et al., 2002). Activation of the chloride
channel appears rapid, with τ in 10 ms range; inactivation is orders of magnitude slower at 10s to
100s (Jentsch et al., 2002). Gating of the channel depends highly on intracellular [Cl-], pH,
temperature and other anions (Jentsch et al., 2002). In addition, the channel can also be activated
or regulated by cell swelling or membrane stretch (Duan et al., 1997b;Duan, 2009;Duan et al.,
2000;Jentsch et al., 2002). Although pacemaker roles of CLC channels in neurons are unclear,
their roles in pacemaker activities of cardiovascular and smooth muscles systems are well
known.
In myocytes, CLC channels contribute to a large part of the anionic conductance that regulates
pacemaker activity. Early studies of the heart found that replacing Cl- with impermeable anions
resulted in a transient increase in heart beat followed by a drastic long-term slowing of
pacemaker activity (HUTTER and Noble, 1961). In SAN cells, a hyperpolarizing-activated
inward rectifying chloride current has been identified to contribute to pacemaker activity both in
vivo (Seyama, 1977) and in vitro (Duan et al., 2000). This was hypothesized to contribute to the
inward current responsible for the diastolic depolarization when membrane potential is below
that of the reversal potential for chloride (HUTTER and Noble, 1961;Noma and Irisawa, 1976).
Genetic cloning identified CLC-2 as the candidate channel contributing to the hyperpolarizing-
activated inward rectifying chloride current (Duan et al., 2000). It is activated by
hyperpolarization, hypotonicity and acidosis (Duan et al., 2000;Huang et al., 2009). Although
CLC-2 conductance under isotonic condition is small, the channel can increase its conductance
under hypotonic or acidic conditions (Duan et al., 2000;Isom et al., 1992). These include
decreasing action potential duration, amplitude and increase diastolic depolarization rate (Duan,
2009). In addition, an outward-rectifying volume sensitive chloride current has also been shown
34
to regulate cardiac rhythm (Duan et al., 1997a). However, the exact identity of the CLC channel
has not been confirmed, although CLC-3 channel has been proposed to be the likely candidate
(Duan et al., 1997a;Duan et al., 1997b;Duan, 2009;Hume et al., 2000). Beyond the cardiac
system, inward rectifying chloride current has also been identified in the intestinal pacemaker,
interstitial cell of Cajal (Huizinga et al., 2002;Zhu et al., 2005), CLC-2 was hypothesized as the
contributing channel.
1.3.2 Ligand-gated ion channels, TRP channels and ion exchangers
1.3.2.1 Ligand-gated ion channels
Ligand-gated ion channels form an essential component within the neurons to regulate and
modulate rhythmic firing and alternating between different bursting behaviours. Some of the
classic ligand-gated receptors that regulate pacemaker neuron activity includes the anionic
channels of GABAA and glycine receptors (Hayar et al., 1996;Morgado-Valle and Feldman,
2004;Tepper et al., 1995;Wu et al., 2008), cationic subtypes of nicotinic acetylcholine (nAChR)
(Quik and Wonnacott, 2011;Shao and Feldman, 2009a), 5-HT3 (Legendre et al., 1989), Kainate
(Artinian et al., 2011;Mereu et al., 1997), AMPA and NMDA receptors (Johnson, 2007;Mereu et
al., 1997;Paarmann et al., 2000;Paarmann et al., 2005). Many catonic channels are non-selective
and often conduct Na+, K
+, and Ca
2+ during activation. These results in not only immediate
depolarization of membrane potential, but may also cause secondary activation of calcium-
dependent ion channels and other calcium-dependent signaling pathways. In respiratory
pacemaker neurons, GABAAR (Shao and Feldman, 1997), glycine (Morgado-Valle and Feldman,
2004;Shao and Feldman, 1997), nAchR (Shao et al., 2008;Shao and Feldman, 2009a), NMDAR
(Paarmann et al., 2005) and AMPAR (Paarmann et al., 2000) have all been identified to be
important in regulating membrane excitability. Similar mechanisms have also been identified in
the invertebrate respiratory network of L. stagnalis. GABA inhibit spontaneous pacemaker
activity of the respiratory pacemaker neuron, RPeD1, via activation of GABAA receptors
(Moccia et al., 2009); whereas glutamate and acetylcholine depolarize and modulate bursting
through activation of AMPA-like receptors (Moccia et al., 2009) and nAChR (Bell et al.,
2007;Woodin et al., 2002), respectively. Ligand-gated ionotropic receptors generally do not play
a significant role in modulating cardiac pacemaker activity.
35
1.3.2.2 TRP channels
Transient receptor potential (TRP) channels constitute a large family of non-selective cationic
membrane channels with diverse functional roles in regulating cellular physiology, including
pacemaker generation and regulation. TRP channels were initially cloned from Drosophila
melanogaster recognized by their transient response to light (Cosens and Manning, 1969). Now,
TRP channel constitute a major family of non-selective cationic channels with over 28 channel
subunits divided into two large groups (Clapham et al., 2003). Group 1 includes: TRP canonical
(TRPC), TRP vanilloid (TRPV), TRP melastatin (TRPM), TRP no mechanoreceptor potential C
(TRPN) and TRP ankyrin (TRPA), which all share sequence identity. Group 2 are distantly
related to group 1 and include channels such as TRP polycystin (TRPP) and TRP mucolipin
(TRPML). Each TRP channel type differs in their activation stimuli, voltage-dependency, ion
selectivity and gating mechanisms (Montell, 2005;Nilius et al., 2005b;Ramsey et al., 2006).
Much of the details of these mechanisms have been extensively reviewed (Cosens and Manning,
1969;Miller and White, 1984;Nilius et al., 2005b;Nilius and Owsianik, 2010;Ramsey et al.,
2006).
The functional role of TRP channels in different pacemaker system is still a topic for continuous
research. Much of the evidence suggesting TRP channel involvement in pacemaker generation
has been identified in pacemaker cell of the gut (Holzer, 2011). Initially, TRPC current was
characterized as having pacemaker-like properties (Walker et al., 2002). TRPC-like current has
been implicated to be important for pacemaking in invertebrate neurons (Wicher et al., 2006).
However, subsequent studies have identified TRPM7 has the major candidate responsible for
pacemaker activity within the interstitial cells of Cajal (Kim et al., 2005). In the heart, TRPC
channels have been identified to be involved in cardiac contractility and possible pacemaker
activity (Ju et al., 2007;Sabourin et al., 2011). In addition, TRPM4 channels have also been
identified in the cardiac pacemaker. Although its physiological function remains unclear, many
have suggested that it may potentially mediate pacemaker generation through Ca2+
-dependent
activation (Demion et al., 2007) or background conductance (Demion et al., 2007;Guinamard et
al., 2010;Mangoni and Nargeot, 2008). In neurons, TRP channels were thought to contribute
toward ligand-activated inward currents (Crowder et al., 2007;Mironov, 2008) and calcium-
36
dependent non-selective cationic currents (Ben-Mabrouk and Tryba, 2010). All of which, have
been implicated to be important in generation of pacemaker activity.
1.3.2.3 Ion exchangers
Ion exchangers also represent an important component in regulating pacemaker activity. They
use the concentration gradient of one ionic species to move another ion into or out of the cell.
Much of their functional role is to maintain ionic homeostasis, which can indirectly regulate cell
excitability and contribute to pacemaker regulation. Na+/Ca
2+ exchanger (NCX) is a major
component regulating Ca2+
homeostasis (Annunziato et al., 2004;Bers, 2002b). Increase
intracellular [Ca2+
] activates NCX, which uses the electrochemical gradient of sodium to remove
one Ca2+
for every 3 Na+. Under normal physiological condition, NCX can account for 30% of
total reduction of intracellular [Ca2+
] (Eisner et al., 2000). The net result is an inward current
resulting in depolarization of the membrane potential (Bers, 2002a). In the cardiac pacemaker
cell, NCX current (INCX) induced by sacroplasmic reticulum release of Ca2+
contributes to an
important component to pacemaker cell depolarization (Ju and Allen, 1998). Subsequent
activation of voltage-dependent Ca2+
channels increases intracellular [Ca2+
], thus further
activating INCX to remove intracellular calcium, and contributes to pacemaker activity (Eisner et
al., 2000;Mangoni and Nargeot, 2008). In addition, NCX also regulates intracellular [Ca2+
],
which in absence of INCX can result in a built-up of intracellular [Ca2+
] and eventual abolition of
pacemaker activity (Bogdanov et al., 2001;Ju and Allen, 1998;Sanders et al., 2006b). Similarly
in neurons, NCX mediate ionic homeostasis and controls cell excitability, firing activity and
transmitter release (reviewed by Annunziato et al., 2004).
1.3.3 Voltage-independent leak and background currents
One type of current that regulates resting or basal membrane potential is the linear leak current.
Background conductance exists in all cells and is a combination of leak channels and sustained
current from voltage-gated and ligand-gated channels. Numerous studies have showed these
background currents to be important in network oscillation (Koizumi and Smith, 2008;Pang et
al., 2009;Zhao et al., 2010), cell excitability, (Brickley et al., 2007b;Rekling et al., 2000) and
pacemaker regulation (Farkas et al., 1996;Hagiwara et al., 1992;Jackson et al., 2004;Liu et al.,
37
2002;McCormick and Huguenard, 1992;Pena and Ramirez, 2004;Ptak et al., 2009;Shen and
North, 1992b;Shen and North, 1992a) (Figure 1.3).
1.3.3.1 Potassium leak
Potassium forms the major background leak conductance in almost all excitable cells. This
concept was established over 60 years ago by the classical work of Goldman, Hodgkin, Huxley,
and Katz (Goldman, 1943;Hodgkin and Huxley, 1947;Hodgkin and KATZ, 1949), based on
initial observations of a high potassium conductance hypothesized to be contributed largely by a
potassium selective channel that lack voltage-dependent activation. The molecular component of
this conductance was not identified until almost 50 years later, when a tandem pore domain
potassium channel (TWIK-1) was cloned that outlined the fundamental structure of an entire
Figure 1.3. Molecular channels known to contribute to background membrane currents of excitable cells. Channels
contributing to Cl- leak current (ICl Leak) includes the Na-K-Cl cotransporters (NKCC transporter), K-Cl cotranspoters
(KCC transpoter) and CLC chloride family channels. Channels contributing to Na+ leak current (INa Leak) includes the
38
sodium leak non-selective (NALCN) channels, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels,
voltage-dependent Na+ (Nav) channels, transient receptor potential (TRP) channels, sustained-inward sodium current
(Ist) channels and Na+/Ca
2+ exchangers. Channels contributing to K
+ leak current (IK Leak) includes the two-pore
domain potassium (K2P) channels, HCN channels, TRP channels and NALCN channels.
class of potassium channel family (Lesage et al., 1996). Now it is understood that TWIK-1 is one
of fifteen different members within a family of regulated potassium selective channel called two-
pore domain potassium channels (K2P). The name is derived from the unique molecular structure
of the channel, which consists of subunits with 4 transmembrane domains each with 2 pore
forming loops (Enyedi and Czirjak, 2010;Goldstein et al., 2001;Lesage et al., 1996). Functional
channels form from dimerization of two separate α subunits (Enyedi and Czirjak, 2010;Lopes et
al., 2001). Similar to the voltage-gated potassium channels, ion selectivity of K2P channels is
determined by the GYG selectivity motif (Enyedi and Czirjak, 2010). Despite being described as
a leak channel, some members of the K2P channels display voltage-dependent activation, such as
the outward-rectifying properties of TREK-1 channels (Fink et al., 1996). This was found to be
regulated by extracellular concentration of Mg2+
and Ca2+
. Many mechanisms exist to regulate
potassium leak conductance. These includes: temperature (Maingret et al., 2000a), membrane
tension (Maingret et al., 1999a;Maingret et al., 1999b), pH (Cohen et al., 2008;Cohen et al.,
2009b;Lopes et al., 2001;Maingret et al., 1999b), phospholipids (Lopes et al., 2005;Maingret et
al., 2000b), cyclic nucleotides (Torres et al., 2003), anesthetics (Patel et al., 1999), protein
kinases and phosphatases (Lopes et al., 2007;Murbartian et al., 2005;Zilberberg et al., 2000),
oxygen concentration (Buckler et al., 2000), G-proteins (Lesage et al., 2000;Mathie, 2007) and
neurotransmitters (Dedman et al., 2009;Honore, 2007). The detailed biophysical,
pharmacological, molecular, and functional mechanisms of different K2P channels have already
been extensively reviewed (Cohen et al., 2009a;Enyedi and Czirjak, 2010;Goldstein et al.,
2001;Lesage, 2003). However it is important to note that through regulation of potassium leak,
different cells have adopted various mechanisms to regulate their resting membrane potential and
excitability.
39
In pacemaker tissues, potassium leak conductance also represents an important regulatory
component to pacemaker rhythm. Regulation of membrane expression or permeability of the
channel can directly affect many pacemaker cell activities. Under physiological condition,
potassium leak has been implicated to directly regulate pacemaker activity of neurons (Koizumi
and Smith, 2008;Nitabach et al., 2002) and heart (Barbuti et al., 2002;Seyama, 1977). In neurons
of D. melanogaster, silencing of pacemaker neuron through targeted overexpression of a
potassium leak channel resulted in deficit of free-running circadian clock (Nitabach et al., 2002).
In the mammalian respiratory network, potassium leak along with persistent sodium current has
been hypothesized to be important in generation of emergent respiratory rhythm (Koizumi and
Smith, 2008). In heart, a TWIK-related acid-sensitive potassium channel (TASK-1) type current
has been found to be inhibited by platelet activating factor (PAF) that results in increase activity
in quiescent cells and cardiac arrhythmia (Barbuti et al., 2002). Since being identified for its
essential role in the first action potential simulation by Hodgkin and Huxley, potassium leak
current has become a fundamental feature of all pacemaker simulations in order to quantitatively
describe pacemaker rhythm and ion channel dynamics.
1.3.3.2 Chloride leak
Another important leak conductance regulating resting membrane potential is the chloride leak
current. Background chloride conductance has been identified in many excitable cells, including
those of in the brain (Rinke et al., 2010;Sacchi et al., 1999) and heart (Duan, 2009;Seyama,
1977). Fundamentally, chloride conductance represents a unique background conductance as it
can both regulate membrane hyperpolarization and depolarization. This is largely due to the
differences in the equilibrium potential of Cl- (ECl) under physiological condition, the which can
often fluctuate in between different tissues, cellular milieus, development stages and pathological
conditions (Hubner et al., 2001;Jin et al., 2005;Rivera et al., 1999). In many developed neurons,
equilibrium potential varies -90 mV to -60 mV (Jin et al., 2005;Pignatelli et al., 2009). In
cardiomyocytes, reversal potential of Cl- is generally more depolarized, ranging from -60 mV to
-35 mV (Baumgarten and Fozzard, 1981;Desilets and Baumgarten, 1986;Seyama, 1979).
Chloride background current can facilitate membrane depolarization or hyperpolarization when
membrane potential is either more negative or positive than the ECl, respectively.
40
Beyond the chloride equilibrium potential, membrane permeability to chloride ion is also an
important regulator of cell excitability. Initially outlined by the Goldman – Hodgkin – Katz
equation, chloride permeability accounted for more than 30% of all background conductance
necessary to establish the resting membrane potential (Goldman, 1943;Hodgkin and KATZ,
1949). However, comparison of mammalian permeability of K+, Na
+ and Cl
-, chloride
contributes to a considerably smaller portion of the background current. For example, in
pacemaker cells of the heart approximately 9% of resting conductance is carried by Cl- (Seyama,
1977). These currents although small, are nonetheless important regulating activation of many
different voltage-gated ion channels.
Most background chloride current dependents on transporters such as the sodium-potassium-
choride cotransporters (NKCC) and the potassium-chloride cotransporters (KCC) (Gillen et al.,
1996;Payne et al., 1995); However, chloride ion channels invariably participate in regulating
background chloride leak. Different tissues exhibit different chloride channel expression,
suggesting there are different fundamental mechanisms involved in regulation of chloride
background currents (Jentsch et al., 2002). CLC channels have been identified as major
candidates for many background chloride ion channel conductances. Many CLC channels exhibit
incomplete inactivation with slow inactivation kinetics (Jentsch et al., 2002). Therefore, under
physiological conditions, many CLC channels function to conduct background chloride current.
CLC-1 has been identified to provide bulk of the resting conductance in muscles (Koch et al.,
1992;Steinmeyer et al., 1991). This was largely identified due to a mutation of CLC-1, which
results in severe myotonia (Koch et al., 1992;Steinmeyer et al., 1991). In addition, CLC-1 was
also hypothesized to carry background chloride current in neurons (Sacchi et al., 1999). CLC-1 is
an outward rectifier, but conducts mostly linear current at hyperpolarizing voltages. CLC-2 on
the other hand is inward rectifier, and it contributes to the background chloride current in certain
neurons (Rinke et al., 2010) and cardiomyocytes (Duan, 2009).
1.3.3.3 Sodium leak
Under physiological condition, Na+ is one of the most important ions regulating depolarization of
the resting membrane potential. As recognized during the initial description of the squid axon ion
dynamics, sodium represented a significant component that drives depolarization of the basal
membrane potential beyond the equilibrium potential of potassium (Hodgkin and KATZ, 1949).
41
Using the Nernst equation, the potassium equilibrium potential (EK) of most mammalian cells is
between -100 mV to -90 mV. Sodium equilibrium potential (ENa) on the other hand is between
+40 mV to +60 mV. As described, the RMP of most pacemaker cells is much more depolarized
than the reversal potential of potassium, suggesting other permeable ions. Background chloride
conductance is not highly expressed in many mammalian excitable cell types. In addition,
intracellular chloride concentration variation can sometimes drive chloride equilibrium potential
to a similar value as potassium. Therefore, sodium permeability is a major driving force that
determines the resting membrane potential.
Regulation of sodium permeability is essential for determining the electrical behavior of the cell.
Quiescent cells generally have lower sodium permeability, with the bulk of the background
conductance driven by potassium. Pacemaker cells are more permeable to sodium ions, resulting
in a RMP that is somewhere between equilibrium potential of potassium and sodium. This is
most evident in the heart, where RMP of non-pacemaker cardiomyocytes are largely determined
by potassium with small sodium component (Kiyosue et al., 1993). Pacemaker cells on the other
hand have depolarized RMP, which is determined by a higher permeability to sodium (Hagiwara
et al., 1992). In fact, sodium contributes to almost 30% of the background permeability in
pacemaker cardiomyocytes of the SAN (Seyama, 1977). Similarly in neurons, background
sodium permeability helps to establish the RMP of pacemaker neurons (Atherton and Bevan,
2005;Jackson et al., 2004).
Many neurons are capable of regulating their RMP through modulation of a Na+ leak current.
Studies of many different excitable cells indicate an ionic component contributing to the
background sodium permeability is contributed by a Na+-dependent leak current (INa Leak) that is
voltage-independent and TTX-insensitive (Atherton and Bevan, 2005;Despa et al.,
2002;Eggermann et al., 2003;Jackson et al., 2004;Jones, 1989;Khaliq and Bean, 2010;Lesauter et
al., 2011;Pena and Ramirez, 2004;Raman et al., 2000;Russo et al., 2007). Excitatory
neurotransmission can increase sodium leak conductance to drive higher neuronal activity. This
mechanism of an apparent INa Leak-dependent excitation has been identified in ventral tegumental
area (VTA) neurons (Liu et al., 2002), dorsal raphe nucleus (Farkas et al., 1996), locus coeruleus
(Shen and North, 1992a;Shen and North, 1992b), and the preBotC (Pena and Ramirez, 2004;Ptak
et al., 2009). Alternatively, suppression of neuronal activity has also been identified to inhibit INa
42
Leak. In the retinorecipient neurons of the suprachiasmatic nucleus, light exposure resulted in a
hyperpolarization of the RMP caused by a suppression of the INa Leak (Lesauter et al., 2011).
Fundamentally, INa Leak is important component in driving the spontaneous or burst activity of
many pacemaker cells.
The identity of a sodium leak channel has been shrouded in mystery up until a few years ago.
Since the description by Hodgkin and Katz (1949), numerous hypotheses were proposed to
explain the channel that could describe the observed sodium leak. These include the ion
exchangers that conduct Na+, the “window current” carried by persistent sodium current (INaP)
channels in neurons or sustained inward current (Ist) channels in heart, HCN channels, and non-
selective cation channels such as the TRP channels. However, HCN, window current of INaP and
Ist, and many TRP channels exhibit strong voltage-dependent activation and/or inactivation. In
addition, HCN is also not expressed in some animals, such as C. elegans (Biel et al.,
2009;Robinson and Siegelbaum, 2003). Finally, much of the major component of the
background sodium current found in many pacemaker neurons (Atherton and Bevan,
2005;Eggermann et al., 2003;Jackson et al., 2004;Jones, 1989;Khaliq and Bean, 2010;Lesauter et
al., 2011;Pena and Ramirez, 2004) and heart (Despa et al., 2002;Hagiwara et al., 1992;Kiyosue
et al., 1993) is voltage-independent and TTX-insensitive INa Leak. Although many of these
channels can contribute toward a background sodium current at rest, the proposed solutions are
insufficient to account for the experimental observations. Recent description of a sodium leak
non-selective (NALCN) channel (Lu et al., 2007) has shed light on the molecular identity of this
INa Leak.
1.4 NALCN: a new player in leak conductance
NALCN is a newly characterized channel that demonstrates many promising biophysical,
pharmacological, and molecular properties toward rhythm generation in excitable tissues (Jospin
et al., 2007;Kim et al., 2012;Lu et al., 2009;Lu et al., 2007;Lu et al., 2010;Swayne et al.,
2009;Yeh et al., 2008). Many studies have found that NALCN, along with UNC79 and UNC80,
forms a protein complex that is involved in regulating intrinsic membrane and synaptic activities
(Jospin et al., 2007;Lu et al., 2009;Lu et al., 2010;Wang and Ren, 2009;Yeh et al., 2008).
Although this field of research is still in early development, evidence from numerous groups
43
currently suggest NALCN channel functions in regulating cell excitability and pacemaker
activity.
1.4.1 Protein structure and homology
1.4.1.1 Gene and protein structures
Human NALCN (formerly VGCNL1) is located on chromosome 13, at location 13q32.3. It is
encoded by 44 exons spanning approximately 6.9 kb of genomic sequence. Processed NALCN
mRNA of 5.2kb encodes a large membrane channel protein of 1738 amino acids. In rodents,
mouse and rat NALCN genes are located on chromosome 14 at 14E5;14 and chromosome 15 at
15q25, respectively. Genomic transcript of mouse and rat NALCN are 7.1kbp and 6.7kbp,
respectively. Processed mouse and rat NALCN mRNAs are 5.2kbp and 4.3kbp, respectively.
NALCN proteins in all three mammalian systems are approximately 1700 residues in length.
The predicated structure of mammalian NALCN is similar to α1 subunits of voltage-gated Na+
and Ca2+
channels (Lee et al., 1999;Lu et al., 2007). It has four homologuous repeats (domains I -
IV) with 6 transmembrane segments (S1-S6) (Lee et al., 1999;Lu et al., 2007) (Figure 1.4A &
1.4B). Four pore forming loops (P-loops) spanning from S5 - S6 make up the ion selectivity
filter. Unlike voltage-gated Na+ and Ca
2+ channels with pore selectivity filters motif of DEKA
and EEEE, respectively, NALCN has a hybrid putative selectivity filter with an EEKE motif (Lu
et al., 2007). In addition, in comparison to the voltage-gated Na+ and Ca
2+ channels, the fourth
transmembrane segment that typically functions as a voltage-sensor (S4), lacks many positively
charged amino acids. Less is known about the structural importance of NALCN cytosolic loops,
N- and C-terminus. Although mutation analysis of the NALCN orthologue, NCA, in C. elegans
identified two gain-of-function alleles located in the S6 segment of domain II and the cytosolic
interconnecting loop of domain I and II (Yeh et al., 2008). NCA has also been shown to
genetically interact with UNC-7, an invertebrate gap junction protein (Bouhours et al., 2011).
Mammalian cytosolic link between loop I and II of NALCN interacts with M3 muscarinic
receptor when coexpressed in human embryonic kidney-293 (HEK293) cells (Swayne et al.,
2009) (Figure 1.4A). Further mutation analysis of the distal C-terminus showed an interaction
domain necessary for modulation of NALCN current by extracellular Ca2+
, possibly through
association with cytosolic subunit, UNC80 (Lu et al., 2010) (Figure 1.4A).
44
Figure 1.4. Schematic diagram of functional NALCN complex in neurons and pancreatic beta-cells. (A) Schematic
diagram of hypothesized NALCN transmembrane structure. It has four homologous repeats (domains I–IV) with six
transmembrane segments (S1–S6). Four pore forming loops (P-loops) spanning from S5–S6 make up the ion
selectivity filter. Mutation analysis identified a putative Unc80 binding domain located in the distal C-termius of
NALCN. M3R interacts with NALCN via the cytosolic loop between domain I and II. (B) NALCN has a putative
selectivity filter with an EEKE motif, which contributes to the non-selective cation channel properties. NALCN
permeability profile is as follows: Na+ ≈ K
+ > Ca
2+. (C) NALCN indirectly interacts with Unc79 via Unc80. Unc80
protein expression requires Unc79. Unc80 function as a scaffolding protein for SFK-coupled signaling from
receptors. In neurons, one of the identified receptor in this pathway is activation of TACR1 by SP. Both Unc80 and
SFK are capable of tyrosine phosphorylation. In addition, Unc80 also function as an intermediate protein for G-
protein dependent inhibition of NALCN channel through activation of CaSR with extracellular Ca2+
. Similar to
neurons, NALCN channel can be activated in pancreatic beta-cells by Ach binding to M3R in a SFK-dependent
45
pathway. Co-expression of M3R and NALCN in HEK-293 cells and Xenopus oocytes indicated physical coupling of
receptor–channel complex, which were important in establishing ACh activation of NALCN current. Adopted from
(Lu and Feng, 2012). NALCN Na+ leak non-selective, TACR1 tachykinin receptor 1, SP substance P, SFK Src-family
kinase, APP adenosine diphosphate, P phosphate, CaSR, calcium-sensing receptor, Ach acetylcholine, M3R M3
muscarinic receptor
1.4.1.2 Homology
Currently, NALCN and NALCN-like genes have been identified in more than 20 different
species. Within vertebrates, NALCN is highly conserved with over 96% identity with its
orthologues. Human NALCN channel also shows 48% homology with both Caenorhabditis
elegans orthologues (NCA-1 and NCA-2) and 57% homology with the Drosophila melanogaster
orthologue (Dmα1U) (Table 1.1).
1.4.2 Biophysicology and pharmacology
1.4.2.1 Biophysical properties
Much of our current knowledge about the biophysical properties of the NALCN channel is
through overexpression studies in HEK cell-line, which do not endogenously express NALCN
channels. NALCN is a non-selective cationic channel with the permeability profile of:
Na+≈K
+>Cs
+>Ca
2+ (Lu et al., 2007). Current-voltage relation of NALCN whole-cell current in
overexpressed HEK cell-line indicates a reversal potential at approximately 0 mV. These
properties were shown to be contributed largely by the EEKE pore selectivity motif (Lu et al.,
2007). Unlike other voltage-gated Na+ and Ca
2+ channels, activation and inactivation of NALCN
does not dependent on voltage. The reduced number of positive amino acids on the S4 region of
NALCN was hypothesized to contribute to these properties (Lu et al., 2007). Given the persistent
activation of NALCN at rest, NALCN and NALCN-like channels could be a contributor to the
neuronal background Na+ current (Lu et al., 2007).
Regulation of NALCN activation/inactivation and localization requires many auxiliary subunits.
Currently, the most studied subunits of NALCN are UNC80 and UNC79. These are two highly
conserved proteins that initially demonstrated genetic interaction with NALCN orthologues in
46
Table 1.1 Homologous NALCN channel between different species. Adopted from (Lu and Feng, 2012).
invertebrates, identified by uncoordinated motor activities. Termed Unc-79 and Unc-80 in C.
elegans and D. melanogaster (Humphrey et al., 2007;Jospin et al., 2007;Yeh et al., 2008),
physical interactions with NALCN protein were later confirmed in mammals, where UNC79 and
UNC80 were identified based on their respective invertebrate orthologues (Lu et al., 2010;Wang
47
and Ren, 2009). NALCN interacts directly with UNC80 (Lu et al., 2010;Wang et al., 2010) and
indirectly with UNC79 (Lu et al., 2010). UNC79 has been shown to influence UNC80 membrane
expression (Lu et al., 2010). Although Na+-leak current has been recorded from the recombinant
NALCN pore forming subunit when expressed alone in HEK293 cells (Lu et al., 2007), Unc-80
and Unc-79 auxiliary subunits have been shown critical for the membrane localization NALCN
channels (Jospin et al., 2007;Yeh et al., 2008). Recent studies showed that membrane expression
of the functional NALCN channels requires NALCN-UNC80-UNC79 complex (Chen et al.,
2010;Lu et al., 2010). NALCN has also physically interacts with the M3 muscarinic receptor
(M3R) in pancreatic beta-cell line, Min6 (Swayne et al., 2009). Analyzing the leak conductance
of the NALCN in Min6 indicates a requirement M3 muscarinic receptor and Src activation,
though these is no evidence of NALCN physically interacting with the latter (Swayne et al.,
2009). Co-expression of M3R with NALCN was sufficient to reconstitute the NALCN current in
HEK293 cells and in Xenopus oocytes (Swayne et al., 2009).
1.4.2.2 Pharmacological properties
Currently, there is no specific blocker for the NALCN channel. NALCN shares many structural
similarities with voltage-gated Na+ and Ca
2+ channels; hence, sensitivities to blockers of these
channels were tested. NALCN is insensitive to NaV channel blocker, TTX, in overexpressed
HEK293 cells (Lu et al., 2007), Min6 cells (Swayne et al., 2009) and neurons (Lu et al., 2007;Lu
and Feng, 2011). Cs+, a non-specific Ih blocker also do not block neuronal NALCN current (Lu
et al., 2007). NALCN do not show sensitivity to various CaV channel blockers, such as
nifedipine, dialtizem, D-600, and mibefradil (Lu et al., 2007). In contrast, the current
conductance via recombinant (Lu et al., 2007) or native (Lu et al., 2007;Lu and Feng,
2011;Swayne et al., 2009) NALCN channels can be partially blocked by Gd3+
(IC50 1.4 µM),
Cd2+
(IC50 0.15 mM), Co2+
(IC50 0.26 mM) and verapamil (IC50 0.38 mM). Although Cav
channel blockers exhibit low efficacy on NALCN channel, Gd3+
can block NALCN with higher
sensitivity. Although Gd3+
can both transient receptor potential (TRP) and other ion channels,
NALCN block with Gd3+
is more sensitive (Biagi and Enyeart, 1990;Bleakman et al.,
1995;Caldwell et al., 1998;Elinder and Arhem, 1994;Mlinar and Enyeart, 1993;Tokimasa and
North, 1996;Yang and Sachs, 1989). The absence of a specific NALCN blocker is a barrier for
48
most conventional pharmacological studies. Therefore, developing specific pharmacological
tools for NALCN channels becomes essential in future physiology studies.
1.4.3 Channel regulation
NALCN conductance is modulated by various membrane receptors (Figure 1C). In neurons,
substance P and neurotensin has been shown to activate NALCN current (Lu et al., 2009).
Binding of substance P with tachykinin receptor 1 (TACR1) activates a G-protein independent
pathway and recruits Src family of tyrosine kinases that interact with UNC80 to activate NALCN
currents (Lu et al., 2009). A similar mechanism is found in the pacemaker cells of the
gastrointestinal cells. NALCN is also involved in a substance P-dependent depolarization of
pacemaker activity within the interstitial cells of Cajal (Kim et al., 2012). In β-cells, NALCN
current can also be modulated by acetylcholine through activation of M3R, and the Ach-sensitive
NALCN current is also Src-dependent (Swayne et al., 2009) (Figure 1C).
In addition to Src regulation, NALCN leak current is sensitive to low extracellular Ca2+
in mouse
hippocampal neurons (Lu et al., 2010) and Lymnaea pacemaker neurons (Lu and Feng, 2011). In
both systems, reduction of extracellular Ca2+
activates NALCN current that depolarizes
membrane potential. In hippocampal neurons, binding of Ca2+
to a Ca2+
sensing G-protein-
coupled receptor (CaSR) inactivates NALCN currents (Lu et al., 2010). As part of the NALCN
protein complex, UNC80 and UNC79 were identified as involved in the endogenous
extracellular Ca2+
sensitivity. UNC80 interacts directly with NALCN channel and is essential for
Ca2+
sensitivity, whereas UNC79 is indirectly involved (Lu et al., 2010). In HEK293 cells,
overexpressing NALCN with UNC80 and CaSR was sufficient to reconstitute extracellular Ca2+
sensitivity of NALCN current (Lu et al., 2010). The conserved mechanism represents a
fundament importance in regulating basal neuronal activity and excitability. In addition, it would
be of great interest to investigate how NALCN is involved in many pathophysiological
conditions involving large fluctuation of extracellular Ca2+
levels (Lu et al., 2010;Ren, 2011).
1.4.4 Expression and distribution
In humans, the mRNA of NALCN is widely expressed in brain, heart, and certain glandular
tissues (Lee et al., 1999;Lu et al., 2007;Swayne et al., 2009). Lee and colleagues (1999) first
cloned the NALCN channel and quantified the mRNA expression profile through northern blot
49
analysis of human brain regions as: amygdala = corpus callosum > caudate nucleus >
hippocampus > substantia nigra = subthalamic nucleus > thalamus. Further study by Swayne and
colleagues (2009) also confirmed high expression by using dot blot analysis of mRNA from
various brain regions as well as the spinal cord. In addition, moderate NALCN mRNA
expression was also identified in the heart, aorta, lymph node, pancrease, adrenal gland and
thyroid gland (Swayne et al., 2009). Similar high NALCN mRNA expression profile was also
identified in brain tissues of both rat through northern blot analysis (Lee et al., 1999), and mouse
through real-time PCR (Swayne et al., 2009) and in situ hybridization (Lu et al., 2007). Within
the pancreas, NALCN mRNA was almost exclusively expressed within endocrine tissues of the
islets rather than the exocrine tissues (Swayne et al., 2009). Kim and colleagues (2012) also
confirmed high NALCN mRNA and protein expression in murine interstitial cells of Cajal using
RT-PCR and western blotting, respectively (Kim et al., 2012). Currently, the cellular localization
pattern of NALCN has not been described in detail.
1.4.5 Physiological functions
1.4.5.1 Rhythmic activity
Physiological roles of NALCN were mostly derived from studies using genetic modulation.
Mutation in C. elegans of NCA-1 and NCA-2 resulted in impaired synaptic transmission
producing abnormalities in locomotion (Jospin et al., 2007;Yeh et al., 2008). In D. melanogaster,
mutation of Dmα1U shows a narrow abdomen phenotype (Lear et al., 2005;Nash et al., 2002)
and altered sensitivity to halothane (Krishnan and Nash, 1990;Leibovitch et al., 1995). Dmα1U
are highly expressed in the pacemaker cells responsible for the diurnal rhythm network (Lear et
al., 2005). Channel and pore selectivity motif mutation analyses indicate that Dmα1U functions
as an ion channel that is involved in regulating circadian rhythm (Lear et al., 2005). In mice,
targeted deletion of the NALCN exon resulted in a knockout strain with a lethal postnatal
phenotype characterized by abnormal rhythmic respiratory activity. C4 nerve root recording,
which indirectly measures respiratory network output, showed a substantial reduction in
spontaneous burst activities (Lu et al., 2007). However, although NALCN-dependent substance
P activation has been identified in the pacemaker of the gut, NALCN knockout did not
significantly alter resting inward current and basal pacemaker activity of interstitial cells of Cajal
(Kim et al., 2012). These different observations suggest possible regulated conductance in
50
membrane expressed NALCN channels or other similar type channels, indicating importance of
auxillary subunit interactions. Furthermore, although NALCN modified respiratory rhythm
output, it is unclear whether this effect was due to changes in intrinsic pacemakers of the
respiratory network, synaptic transmission, developmental abnormalities or signal transduction.
1.4.5.2 Resting membrane potential and cell excitability
Pharmacological and genetic evidence indicate NALCN permeable sodium leak is involved in
regulating resting membrane potential and neuronal excitability. Application of Gd3+
and
verapamil to cultured control hippocampal neurons resulted in over 75% reduction of action
potential frequency (Lu et al., 2007). In addition, Gd3+
hyperpolarized the baseline membrane
potential, which was recoverable by depolarizing current injection (Lu et al., 2007). NALCN
knockout or with Gd3+
block eliminated approximately 70% of INa Leak (Lu et al., 2007),
suggesting in neuronal hippocampal cells, NALCN constitute the major component of INa Leak (Lu
et al., 2007). Hippocampal neurons of NALCN knockout also show approximately 10 mV
hyperpolarization of resting membrane potential, similar to observed in pharmacological block
(Lu et al., 2007). Similarly, in the gastrointestinal pacemaker cells, NALCN knockout resulted in
more than 5 mV hyperpolarization in resting membrane potential (Kim et al., 2012).
Furthermore, mutation of NALCN or its associating subunits shows reduced sensitivity to
extracellular calcium induced inhibition (Lu et al., 2010). These observations suggest NALCN
conducting INa Leak is an important contributor to the baseline membrane potential of excitable
cells, specifically in neurons. Thus NALCN may function as a major participant in regulation of
resting membrane potential of pacemaker systems.
1.4.5.3 Synaptic regulation
Many rhythmic networks of the central nervous system depend on a combination of both
intrinsic and synaptic pacemaker rhythm. Some networks experience changes in intrinsic or
synaptic rhythm generation throughout development. For example, the pacemaker hypothesis of
respiratory network posits that the pre-Botzinger Complex is the sole respiratory rhythm
generator, as isolated brainstem and spinal cord of neonatal rat could generate fictive respiratory
rhythm (Smith et al., 1991), even with synaptic inhibition (Brockhaus and Ballanyi,
1998;Feldman and Del Negro, 2006). The group-pacemaker hypothesis suggests that periodic
51
activity of synaptic connection between bursting respiratory neurons recruits additional burst-
generating currents. NALCN as a pore-forming channel has already been demonstrated to
contribute toward regulating intrinsic membrane properties, but evidence also indicates that it is
also directly involved in synaptic transmission. In D. Melanogaster, NALCN orthologue
(Dmα1U) is highly expressed at the synaptic region compared to the cell bodies (Nash et al.,
2002), suggesting a functional role in mediating neurotransmitter release or postsynaptic
response. Disrupted circadian rhythms of Dmα1U mutants were also found to have decreased
release of neuropeptide, PDF (Lear et al., 2005). C. elegans orthologues of the NALCN channels
(NCA-1 and NCA-2) are critical in the conduction of depolarizing signal from the soma to the
axon (Yeh et al., 2008). Mutants of this channel showed reduce Ca2+
transient at the synaptic
terminal (Yeh et al., 2008). When NCA-1 and NCA-2 mutant were paired with Unc-80,
presynaptic release was impaired, possibly due to the involvement in synaptic vesicle recycling
mechanisms (Jospin et al., 2007). The highly conserved Unc-80 and Unc-79 proteins in C.
elegans are essential for proper NCA-1 and NCA-2 localization (Jospin et al., 2007;Yeh et al.,
2008).
1.4.5.4 Insulin release
Recent findings from the Monteil group have correlated NALCN with insulin release from the β-
cells of the pancreatic islets, as reviewed by Gilon and Rorsman (2009), and Swayne et al.
(2010). What is interesting to note is that NALCN interacts with M3R via a src-dependent
tyrosin kinase pathway that is G-protein independent, similar to the substance P sensitivity
observed in neurons (Swayne et al., 2009). Co-immuno precipitation shows NALCN interacts
with M3R protein. Reconstitution of Ach-sensitive NALCN current was successful in HEK293
cells where NALCN and M3R were coexpressed (Swayne et al., 2009), suggesting that NALCN
has different activation properties in different tissues by forming heteromeric receptor complex.
1.4.5.5 Osmoregulation
The Korstanje group recently demonstrated that NALCN is a potential player in osmoregulation
(Sinke et al., 2011). Halotype association mapping was used to identify and correlate various
mouse strains with differences in serum Na+ concentration. The NALCN gene was identified to
have the strongest correlation with Na+ concentrations. Analysis of heterozygous NALCN strains
52
identified hypernatremia suggesting NALCN is possibly involved in osmoregulation. The
detailed mechanism remains unclear, but Sinke and Deen (2011) proposed a model that
differential NALCN expression in osmoregulatory neurons alters osmolarity signals quantified
by changes in action potential frequency.
53
Chapter 2
Rationale, Hypothesis and Objectives
54
2 Rationale, Hypothesis and Objectives
2.1 Rationale
Much essential biological behaviour involves rhythms generated from complex combinations of
sub-cellular, cellular and network oscillations. The electrical activity of pacemaker cells, found
in many of these networks, is involved in generating and regulation of rhythmic output.
Membrane ion channels are a key determinant of pacemaker cell activities. Although
mechanisms perpetuating pacemaker activities varies between cells of different tissues,
fundamental mechanisms regulating pacemaker activities constitutes changes in resting
membrane potential. The resting membrane potential depends largely on the background
conductance of K+, Na
+ and Cl
-, whose membrane permeability is determined by various ion
channels and transporters. The depolarized basal membrane potential of pacemaker cells is
greatly permeable to Na+, indicating the likelihood of a Na
+ leak conductance. Recently, a new
category of non-selective cation channels, termed NALCN (Na+ leak channel), has been shown
to conduct voltage-independent and noninactivating current of Na+, K
+, and Ca
2+ (Lu et al.,
2007). These channels permeate an inward Na+ leak current at rest, which can regulate cell
electrical activity and vesicle release of both vertebrate (Kim et al., 2012;Lu et al., 2009;Lu et al.,
2007;Lu et al., 2010;Swayne et al., 2009) and invertebrate (Bouhours et al., 2011;Jospin et al.,
2007;Lear et al., 2005;Nash et al., 2002;Yeh et al., 2008) systems. High expression of NALCN
and NALCN-like channels has been identified in numerous excitable tissues, including tissues
with known pacemaker cells (Kim et al., 2012;Lear et al., 2005;Lee et al., 1999;Lu et al.,
2007;Swayne et al., 2009). Targeted deletion of NALCN exons in mice resulted in fatal postnatal
phenotypes characterized partially by an abnormal respiratory rhythm and absence of bursting
activities in C4 nerve root recording (Lu et al., 2007). The exact mechanism behind this rhythmic
defect is unknown given the high complexity of mammalian respiratory network. In addition,
whether NALCN-dependent Na+ leak could regulate pacemaker cell activity has yet to be
investigated.
2.2 General Hypothesis
The general hypothesis is that NALCN-dependent Na+ leak current regulates spontaneous
activities of pacemaker cells through regulation of resting membrane potential.
55
2.3 Objectives and approaches
Addressing the hypothesis involves three different objectives aimed to address three different
major questions.
2.3.1 Objective 1: Determine whether NALCN-dependent Na+ leak contributes to neuronal pacemaker activity
2.3.1.1 Major question
This objective is aimed to address the first fundamental question about whether NALCN is
involved in regulating pacemaker cell firing. Many lines of evidences direct at NALCN
involvement in regulating pacemaker activity (Lear et al., 2005;Nash et al., 2002) as well as
neuronal firing (Lu et al., 2009;Lu et al., 2007;Lu et al., 2010), but no conclusive evidence has
demonstrated a fundamental function of NALCN regulating the electrical activity of pacemaker
neurons.
2.3.1.2 Experimental approach
NALCN has been implicated to be involved in generation of respiratory rhythm following
measurement of the C4 nerve root recording, which partially represents respiratory network
output (Lu et al., 2007). However, investigation into whether NALCN contribute to respiratory
pacemaker neurons in mammal faces many challenges. Respiratory pacemaker neurons have not
been identified to drive fictive rhythm in adult systems. In neonatal preparations, pacemaker
neurons have been identified to regulate rhythmic output. However, there are at least two
different types of pacemaker neurons regulating different respiratory behaviors (i.e. eupnea,
gasping or sighing) (Del Negro et al., 2005;Pena et al., 2004;Tryba et al., 2006). In addition,
targeted genetic manipulation in the heterogenous respiratory central pattern generator region is
highly challeneging, albeit not impossible (Tan et al., 2008). Finally, investigating the intrinsic
properties in mammalian pacemaker neurons requires pharmacological isolation of synaptic
inputs, which might affect NALCN conductance; since it is has been report to contribute toward
synaptic currents (Lu et al., 2009;Swayne et al., 2009; Kim et al., 2012). Due to these reasons, I
opted to first investigate the fundamental role of NALCN channel in pacemaker cell using a
simplified, yet highly accessible respiratory model from Lymnaea stganalis. Using acute,
targeted RNAi gene silencing approach to manipulate gene expression and combined with
56
pharmacological interventions, I will characterize a NALCN-orthologue, U-type channel
(Spafford et al., 2003), in L. stagnalis and identify the following:
1. Determine whether U-type channel expression affects pacemaker neuron firing in an
isolated adult rCPG neuron, RPeD1.
2. Determine whether U-type channels conduct a Na+ leak current that regulates the
membrane potential of the pacemaker neuron.
3. Characterize the pharmacological properties of the U-type channel in relation to
mammalian NALCN channel.
4. Determine whether U-type channel expression is involved in maintaining respiratory
behaviour in L. stagnalis in vivo.
2.3.2 Objective 2: Identify the ionic mechanisms of Na+ leak regulation of spontaneous pacemaker activity
2.3.2.1 Major question
By establishing whether U-type channels regulate pacemaker neuron firing through conductance
of a Na+ leak, the next major question is to determine how Na
+ leak contributes to pacemaker
firing. Many neurons regulate leak and background current and the aim of this objective is to
quantitatively determine the differences between Na+ leak and another commonly attributed leak
component, K+ leak.
2.3.2.2 Experimental approach
In order to quantitatively describe the ionic constituents during action potential firing, I opted to
mathematically model the various ionic currents contributing to pacemaker neuron action
potential firing using a computation simulation of the RPeD1. Developing, quantifying and
running the computation model of RPeD1 will require the following:
1. Determine whether U-type channel expression affect action potential profile or major
voltage-dependent currents.
2. Characterize the major ionic currents necessary for generation of action potential in an
isolated adult pacemaker neuron, RPeD1.
57
3. Generate a single-compartment model to simulate the spontaneous action potential profile
of RPeD1 neuron.
4. Determine whether and how sodium leak can contribute to regulation of spike activity.
2.3.3 Objective 3: Determine whether NALCN contributes to Na+ leak regulation of pacemaker activity in isolated sinoatrial node cardiomyocytes
2.3.3.1 Major question
Finally, having established the major findings in the pacemaker neuron, my final objective is
aimed at addressing whether NALCN can also regulate non-neuronal pacemaker cell firing.
2.3.3.2 Experimental approach
Outside of the CNS, the highest expression of NALCN is in the heart. The pacemaker cells
located in the cardiac system is also well known and better characterized. Therefore, my final
objective will be addressed using the adult isolated cardiac pacemaker cells from the Mus
musculus sinoatrial node. Since functional NALCN has never been quantified in the cardiac
system, and given the lethal nature of the NALCN knockouts, NALCN-like conductance will be
probed using pharmacological identification and isolation from other known background Na+
current contributors. Completion of this objective requires the following:
1. Describe pacemaker activity of isolated SAN cardiomyocytes.
2. Characterize Na+ leak current necessary in establishing pacemaker activity.
3. Test whether Na+ leak current is sensitive to known NALCN blockers in combination
with blockers of other known background Na+ channels.
Profile NALCN and its subunits expression between pacemaker and non-pacemaker regions of
the heart.
58
Chapter 3
General Methodologies
Part of the work presented in this chapter corresponds to the following publication:
Lu TZ, Feng Z-P (2011) PLoS ONE 6(4): e18745.
59
3 General Methodologies
3.1 Electrophysiology
3.1.1 Solutions
3.1.1.1 Internal pipette and saline bath solutions
For various electrophysiological recordings, corresponding standard pipette and bath solutions
were used for each individual animal model (Table 3.1). Notably, the L. stagnalis osmolarity is
much lower than that of the mammalian cells. The average osmolarity of internal solution for L.
stagnalis is ~100 mOsm where as mammalian cells are at ~330-350 mOsm. Another difference
between snail and mammalian models is the internal [Ca2+
]. In L. stagnalis internal solution, the
buffered free [Ca2+
] is calculated at approximately 1 x 10 -8
M. L. stagnalis neurons are known to
hold intracellular [Ca2+
] to even below the internal solution concentration, provided that
concentration of free Ca2+
in the internal solution is lower than 1 x 10 -6
M (Byerly and Moody,
1984).
However, an important similarity shared between snail and mammalian model is the conserved
Nernst potential for the different ionic species. Using the experimental internal and bath
solutions the appropriate Nernst potential for various ionic species could be calculated using
equation 3.1. For potassium ions, Nernst potential calculated for cardiomyocyte is -83 mV. Snail
(equation 3.1)
potassium Nernst potential is calculated at -72 mV. For sodium ions, Nernst potential of sodium
ion for cardiomyocyte and snail neurons is 62 mV and 59 mV, respectively. Nernst potential of
calcium ion for cadiomyocyte and snail neurons is 252 mV and 260 mV, respectively. Chloride
Nernst potential is different between snail and cardiomyocytes, with snail neuron at -13 mV and
cardiomyocytes at -2 mV. Under physiological recordings, permeability of multiple ionic species
determines the observed reversal potentials, as determined by the Goldman-Hodgkin-Katz
equation (equation 1.1).
60
Table 3.1 Internal and external solutions used in different model systems for electrophysiology recordings.
61
3.1.1.2 Na+ free bath solutions
In order to investigate how Na+ contributes to regulation of membrane potential and leak
currents, bath substitution of Na+ with large positive charge carriers were used. In both snails and
mammalian cells, equimolar NMDG+ was used to replace extracellular Na
+. N-methyl-D-
glucamine (NMDG) is a large linear organic molecule. It consists of a charged methylamine
group at one end and a linear carbohydrate shaft and tail, making it highly hydrophilic. The large
structure of the molecule makes it approximately 6.4 Å wide and 12 Å long, with approximate
average diameter of 7.3 Å (Villarroel et al., 1995). Given the large diameter of the ion, most
selectivity pores do not permeate NMDG ions. However, slight NMDG permeability is observed
in a few ion channels such as the ATP-gated P2X channels (Khakh et al., 1999;Li et al.,
2005a;Virginio et al., 1999), epithelial Ca2+
channels ECaC (Nilius et al., 2000), voltage-gated
potassium channels (Wang et al., 2009), glutamate receptor channels (Ciani et al., 1997) and a
few mechanosensitive channels (Lawonn et al., 2003;Li et al., 2005b;Shiga and Wangemann,
1995;Zhang and Bourque, 2006). Under physiological conditions, NMDG permeability to most
ion channels is either very low or non-existent. This makes NMDG a suitable charge carrier in
many ion replacement studies.
3.2 Pharmacology and blockers
3.2.1 Pharmacological blockers
3.2.1.1 Tetrodotoxin (TTX)
As described in section 1.3.1.1., tetrodotoxin (TTX) is a specific blocker of the voltage-gated
sodium channel. NALCN has been identified as the TTX-insensitive Na+ leak current that is
contributing to the frequently observed TTX-insensitive background Na+ current in many
neurons (section 1.4). In order to identify a channel with NALCN like properties, TTX need to
be applied at significant concentration to inactivate Nav channels to determine whether it affects
a Na+ dependent background leak current. However, snails are inherently less sensitive to many
of the commonly used voltage-dependent ion channel blockers, often requires discovery of novel
blockers with higher sensitivity (Kits et al., 1996). Transient inward current in snails has been
reported to be inactivated at 10-4
M concentration range of TTX, hence a high concentration of
TTX was necessary to ensure sufficient block of voltage-dependent transient inward current. In
62
this thesis, IC50 of transient inward current to TTX is at 23.7 ± 2.1 µM (Appendix A1). At ~4
times the IC50 value, 90% of the transient inward current is inhibited. Therefore, a concentration
of 100 μM was used to investigate sensitivity of leak current to TTX.
3.2.1.2 Tetraethylammonium (TEA)
Tetraethylammonium is a quanternary ammonium ion commonly used blocker than binds
strongly to voltage-gated potassium channels. Experimental recordings has identified two major
binding sites in potassium channels, both are located near the pore at the intracellular and
extracellular regions (Choi et al., 1993;Heginbotham and MacKinnon, 1992;Kavanaugh et al.,
1991;Luzhkov and Aqvist, 2001;Newland et al., 1992). In addition, structural analysis also
confirmed the binding site near the selectivity filter of potassium channels at the external and
internal entrances (Luzhkov and Aqvist, 2001). Mutation combined with crystal structure
analysis identified a series of tyrosine residue that stabilizes TEA interaction with the entrance
site of potassium channel, forming a cage-like structure (Luzhkov and Aqvist, 2001). In this
thesis, TEA was used to block delay-rectifier current in snail neurons.
3.2.1.3 4-aminopyridine (4-AP)
4-aminopyridine (4-AP) is a potent blocker of the transient outward potassium current. Although
the exact mechanism of 4-AP block is not completely understood, electrophysiological
recordings identified a multistate blocking mode (Thompson, 1982). 4-AP block both opened
and closed channel configuration, with preferential block of open configuration (Kiss et al.,
2002). Open channel block is hypothesized by the use-dependent effect and possibly achieve by
a trapping mechanism of the inactivation gate (N-type inactivation) in closed state (Kirsch et al.,
1986;Kiss et al., 2002;Wagoner and Oxford, 1990). Closed channel block is possibly through
promotion of C-type inactivation (Baukrowitz and Yellen, 1996;Kiss et al., 2002). 4-AP has also
been observed to decrease P/Q type Ca2+
current densities, which formed the possible therapeutic
mechanisms for using 4-AP in clinical treatment of episodic ataxia type 2 (Strupp et al., 2008).
4-AP was used in this thesis to identify the A-type transient outward potassium current in snail
neurons.
63
3.2.1.4 Spermine
In this thesis, spermine is used as a pharmacological blocker of TRPM4 current. Intracellular
application of spermine has been shown to block TRPM4 currents (Nilius et al., 2004). This
sensitivity of TRMP4 to spermine depends on the high affinity binding near the pore entrance
region of the intracellular opening. This was identified by mutation of the Glu981
, resulting in a
functional channel that is poorly sensitive to spermine (Nilius et al., 2005a). IC50 of spermine to
TRPM4 is 61 ± 15 µM (Nilius et al., 2004) while other reported as 35 ± 11 μM (Ullrich et al.,
2005). Spermine also block TRPM5 channel with IC50 of similar value,
approximately 37 ± 12 μM (Ullrich et al., 2005). Spermine also blocks voltage-gated Na+
channels with IC50 of 17 ± 3 µM (Huang and Moczydlowski, 2001). KIR channel can also be
blocked by spermine with high sensitivity and complex blocking mechanisms (Guo and Lu,
2000). At voltages above 0, spermine completely inactivates at nM concentrations. At
hyperpolarizing voltages, higher spermine concentration is required for block (Guo and Lu,
2000). Extracellular application of spermine can block TRPM7 currents with high sensitivity
(Kerschbaum et al., 2003). Although spermine is a non-selective blocker for TRPM4, for the
purpose of this thesis, spermine is used as a potent blocker of possible candidate of background
Na+-dependent leak current.
3.2.1.5 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288)
The drug, ZD7288 is a commonly used bradycardiac agent which is produced due to its selective
block of If in heart (BoSmith et al., 1993). Outside of the cardiac system, Ih currents can also be
blocked by ZD7288 (Gasparini and DiFrancesco, 1997;Harris et al., 1994;Harris and Constanti,
1995;Satoh and Yamada, 2000). Kinetic of ZD7288 inhibition of If or Ih is slow (taking minutes).
In whole-cell mode, findings identified ZD7288 can block channels both in opened and closed
states (Harris and Constanti, 1995;Satoh and Yamada, 2000), however more accurate recording
using inside-out patch identified requirement of opened state (Shin et al., 2001). ZD7288 block is
also voltage-dependent, as hyperpolarization can release channel from a blocked state (BoSmith
et al., 1993;Gasparini and DiFrancesco, 1997;Harris and Constanti, 1995). This observation is
not well understood given the poor understanding of the gating mechanisms of Ih. Subunit
recombinant study found binding of ZD7288 to the pore site of the open channel, effectively
64
trapping the channel in a closed state (Shin et al., 2001). The stability of the binding depends on
three amino acids found on the S6 region of the channel, as indicated by mutation analysis. In
this study, ZD7288 is used as a preferential If blocker for isolated murine pacemaker cells. Stable
If block was achieved at a concentration that was sufficient to block more than 80% of If
(BoSmith et al., 1993). Although dose-response curve for ZD7288 in murine pacemaker cells has
not been reported; 0.1 µM, 0.3 µM and 1 µM were able to block 44%, 65%, and 78% of If in
isolated guinea pig SAN cells. Since ZD7288 acts primarily via open state block, it is assumed
that dose-response curve act through a sigmoidal shape suggesting 1 to 10 µM is capable of
blocking almost all If conductance. This thesis used 10 µM of ZD7288 applied for 5 minutes to
inactivate If in recorded cell.
3.2.2 Multivalent ion blockers
3.2.2.1 Gadolinium (Gd3+)
In this thesis, gadolinium was applied to investigate functional roles of NALCN. In addition,
Gd3+
is also a potent blocker of numerous ion channels. These include various stretch-activated
channels of the TRP families (Drew et al., 2002;Zhang et al., 2000b), voltage-gated L-type
calcium current (Lacampagne et al., 1994;Malasics et al., 2010) and delay-rectifier potassium
currents (Hongo et al., 1997). All of these conductances were identified in both cardiomyocytes
and neurons. IC50 of gadolinium to NALCN is reported at 1.4 µM (Lu et al., 2007), for L-type
calcium channel it is at 1.6 μM (Malasics et al., 2010), for delay-rectifier potassium current it is
at 24 μM (Hongo et al., 1997) and for mechanosensitive TRP currents it is at approximately 8
μM for neurons (Drew et al., 2002) and ~50 μM in cardiomyocytes (Zhang et al., 2000b). Since
Gd3+
sensitivity to NALCN current is much higher, a relatively low concentration of 10 µM Gd3+
was used to record Gd3+
sensitive component largely contributed by NALCN.
3.2.2.2 Cobalt (Co2+)
Cobalt was used in this thesis as an additional blocker of NALCN current. NALCN is partially
sensitive to cobalt block with an IC50 of 0.26 mM (Lu et al., 2007). A concentration of 1 mM
Co2+
was sufficient to block more than 80% of NALCN current. However, cobalt is known non-
specific Ca2+
channel blocker (Tsien et al., 1987), which inhibits Ca2+
and Ca2+
-dependent
currents. In order to analyze contribution of NALCN to leak current, proper protocols (described
65
below) would be necessary to stimulate and observe activity below threshold of voltage-gated
Ca2+
channel activation. This should limit contribution of Ca2+
and Ca2+
-dependent currents.
3.2.3 Whole-cell patch clamp recordings
Whole-cell ruptured patch clamp records was performed throughout the thesis in order to
measure the whole-cell current under various recording protocols. The ruptured recording
technique was performed similar to that previously reported for RPeD1 neurons (Feng et al.,
2002;Hui et al., 2007), SAN pacemaker cardiomyocytes (Cifelli et al., 2008;Mangoni and
Nargeot, 2001;Rose et al., 2007) and tsA201 cells (Barbara et al., 2009). In general, the
microelectrode pipettes were filled with appropriate internal solutions and suction was applied
following membrane contact. Whole-cell rupture was achieved through a combination of slight
negative pressure and a brief (0.1 ms) 1 V zap. Alternating bath solutions were achieved by
focally perfusing onto the cells with a gravity-driven perfusion system. The setup was assembled
using tubing, valve controls and a manifold purchased from Warner Instruments (Harvard
Apparatus Canada, St. Laurent Quebec). Signals were recorded and amplified with a personal
computer equipped with pClampex 9.2 (Axon Instruments) and MultiClamp 700A connected to
a Digidata 1322 digitizer, respectively. Data were filtered at 1 kHz (−3 dB) using a 4-pole Bessel
filter and digitized at a sampling frequency of 2 kHz. Leak currents were measured by
subtracting from the total current traces the non-linear voltage-dependent currents (Figure 3.1).
The representative recordings in Figure 3.1 show that the inward hyperpolarizing current (total
current) recorded from RPeD1 contained two components; a non-linear and a linear leak current.
The non-linear hyperpolarizing current was recorded at the identical voltage steps with an online
leak-subtraction protocol. P/4 subtraction protocol using pClamp 9.2 involved 4 hyperpolarizing
pulses of ¼ amplitude of the stimulating voltage to subtract the passive currents. The linear leak
current (ILeak) component was obtained by subtracting the non-linear current from the total
current. The current density-voltage (I-V) relations for the total, non-linear and linear currents
are shown in Figure 3.1 B. Data were analyzed with Clampfit 9.2 (Axon Instrument) and plotted
with Origin Pro v8 (Origin Lab Co., Northhampton, MA, USA). Curve fitting was performed
with Origin Pro v8. All recordings were performed at room temperature (~22C).
In general, leak current throughout this thesis is defined by the passive, non-voltage dependent,
sustained currents. Distinction is made between leak currents and background current; where
66
background conductance is a summation of all sustained conductances identified at a specific
holding voltage. These would include multitude of currents, which includes voltage-dependent
components. Leak currents are a collective group of voltage-independent, non-inactivating
conductance. These currents are especially prominent through measurement of the sustained
component following the voltage-recording protocol as indicated above.
3.2.4 Current clamp recordings
Current clamp recording varied between snail neuron and mammalian cells. The goal during
current clamp is to obtain accurate measurement of membrane voltage changes with minimal
disturbances to the intracellular solution. Generally, the most accurate form of recording is
through sharp electrode recording. The high resistance tip limits dialyzation of the intracellular
compartment. However, the act of impalement causes membrane damage that need to be
mitigated during recording. This is further complicated in contacting cells, where physical
movement would damage cell membrane, causing cell death. In these cases, patch recording is
preferred compared to sharp electrode recording. Patch electrode is better able to clamp the
spontaneous activities due to the large diameter of the tips. However, whole-cell patch in current
clamp configuration create a dialyzed intracellular compartment, which does not reflect physical
cellular activities. In these conditions, perforated patch is used instead of whole-cell patch as
perforated patch preserves membrane integrity and limits dialyzation of intracellular
compartment. The limitation of perforated patch is the accuracy of the recorded currents, since
depending on the antibiotics used for perforation, different currents could be conducted.
In the snail studies, conventional sharp electrode recordings were performed to monitor the
spontaneous bursting firing activity of RPeD1 cells. Sharp electrodes were filled with saturated
K2SO4 solution (70 - 80 MΩ). K2SO4 was preferred over KCl in order to limit increasing
intracellular chloride concentration, which can occur under prolonged recording protocol. In
mammals, sharp electrode recording was only performed on cardiomyocytes in situ from intact
SAN and RA tissue. 3M KCl was used as internal solution. Equipment setup identical to whole-
cell current recording was used for all current clamp recordings. Initial impalement resulted in
excessive activity due to damage to the cell membrane. A hyperpolarizing step was applied to
clam spontaneous activity and allow cell membrane to reseal. After 5 minutes, the membrane
potential is brought back to spontaneous level through a series of step voltages. Measurements
67
Figure 3.1. Whole-cell current in individual RPeD1 neurons isolated from naïve control animals. (A) Representative
hyperpolarizing inward current density traces of the total current, non-linear voltage-dependent current and inward
leak currents from one individual RPeD1 neuron. (B) Average current density-voltage (I-V) relation of the total
current (n = 7), the non-linear current (n = 7) and the leak current (n = 7) recorded from seven RPeD1 neurons. All
data were represented as mean ± S.E.M..
were then taken once the cell established stable activity or quiescence at 0 pA membrane
potential. All measurements were made at room temperature and in standard corresponding bath
salines.
For isolated cardiomyocytes, current clamp voltage oscillations measurements were performed
using perforated patch clamp recordings. Amphotericin B was used as the pore-forming
antibiotics as it allowed for rapid and low-resistance access to the cell membrane compared to
nystatin (Rae et al., 1991). These are polyene compounds that form pores in the membrane that
are generally permeable to very small molecules, with the upper limit of permeability to
approximately the size of a glucose molecule (Cohen, 1986;de and Demel, 1974). Application of
68
amphotericin B to lipid bilayer creates high selectivity for univalent ions, and largely
impermeable to multivalent ions (de et al., 1974). Application of amphotericin B to both sides of
the lipid bilayer results in higher selectivity for univalent anions, where as single sided
application produce higher selectivity for univalent cations (Borisova et al., 1986). 50 μL of 20
mg/mL Amphotericin B dissolved in DMSO stock is added to 5mL of cardiomyocyte internal
solution to a final concentration of 200 μg/mL. This solution was kept in the dark and on ice for
2 hours, leftovers were discarded and fresh solution was made again. In order to achieve a proper
seal followed by perforated-patch formation, approximately 500 μm of pipette tip was pre-filled
with standard cardiomyocyte internal solution as described in Rae et al., (1991). Successful
access were achieved between 10-15 minutes, with access resistance of <15 MΩ.
Evaluation resting membrane potential of spontaneously active cells was done by observing the
mid-point of the subthreshold depolarizing slope. The depolarizing slope is taken between
voltage range post-afterhyperpolarization and pre-threshold potential. The mid-point between the
two voltage ranges is then taken as one measurement of resting membrane potential. Final
resting membrane potential value takes an averaged value from 10 measurements gathered
within 1 minute measuring window.
3.3 RNAi designs and principles
3.3.1 RNAi gene silencing and nonspecific effects
RNAi is a process found in many eukaryotic cells were small RNA molecules regulate gene
expression by modulating mRNA translation. RNAi was initially identified in C. elegans (Fire et
al., 1998), to which both Andrew Fire and Craig Mello both received the Nobel Prize for their
pioneering work. The pathway functions as a parasitic defense mechanism, where long dsRNA is
cleaved by the enzyme, dicer, into short siRNAs. The complimentary single-stranded RNAs are
then incorporate into RNA-inducing silencing complexes where they regulate protein level via
mRNA translation/degradation. For this reason, RNAi has become an increasing popular
mechanism to specifically regulate endogenous gene expression and to study its corresponding
effects. However, there are many limitations to this technique, which lead to nonspecific effects
in applications.
69
3.3.1.1 Innate immunity
The first nonspecific effects of using RNAi for gene silencing is innate immunity (reviewed by
Robbins et al., 2009). Excessive siRNA activates innate immune responses that can produce
inflammatory cytokines and interferons (Clemens and Elia, 1997;Stark et al., 1998). In addition,
the delivery vector themselves can produce undesirable immune responses. The innate immunity
response to siRNA represents one of the major challenges to application of siRNA to human
therapeutic procedures. On the contrary, snail application of naked siRNA in vivo is able to
produce significant gene knockdown without immune complications. This is largely due to the
lack of interferon found in mammalian cells.
3.3.1.2 Off-targetting
Off-targeting is another nonspecific effects that needs to be considered in RNAi studies (Fedorov
et al., 2006). This is where complimentary non-targeted mRNAs are downregulated due to
siRNA acting as endogenous miRNA, which produces downstream effects that is not dependent
on the targeted gene. A remedy to this situation requires careful design (Birmingham et al.,
2007). Several control experiments and multiple RNAi knockdowns could be used to duplicate
consistency in knockdown effects. Design siRNA to avoid commonly conserved region and
targeting unique regions as identified by the known gene databases could further limit the effect
of off-targeting. Identification of off-targeting has been attributed to matching of 6-7 base pairs
along position 2 matching of 3’-UTR with siRNA (Birmingham et al., 2006). Further
confirmation of off-targeting could be done using genomic screening following microarray.
3.3.2 RNAi for Lymnaea stagnalis
RNAi has been effectively used to reduce gene expression in numerous L. stagnalis studies. This
worked effectively both in vivo (Fei et al., 2007;Korneev et al., 2002) and in vitro (Hui et al.,
2007;Nejatbakhsh et al., 2011). In both delivery conditions, naked short (Fei et al., 2007;Hui et
al., 2007;Nejatbakhsh et al., 2011) and long dsRNA (Korneev et al., 2002) is taken up by an
endogenous mechanism. Thus the delivering, monitoring and confirmation of gene knockdown
following RNAi application in L. stagnalis is more convenient than mammalian counter parts. In
this thesis, both siRNA (defined by a 27-mer short double stranded RNA) and dsRNA (defined
70
by a 300 base pairs long double stranded RNA) were used to determine the specificity and
consistency of the observed outcomes.
3.3.3 RNAi synthesis
RNAi gene silencing was used in chapters 4 and 5. The double-strained RNA was synthesized
following RT-PCR protocol using T7 phage polymerase promoter whereas siRNA were designed
and commercially purchased. For synthesis of the 300 bp dsRNA, the primers for the U-type
channel (GenBank#, AF484085) were designed with T7 phage polymerase promoters at the 5’
end of the primers, and the primer sequences were shown in Table 3.1. The U-type channel gene
was amplified from a standard cDNA library obtained from a collection 3 naïve control snail
ganglia, using the 2x PCR master mix (Fermentas, USA) following conventional PCR (PTC-
100TM Programmable Thermal Controllor). The samples were amplified with the temperature
profile of 94°C/2 min, 94°C/30”, (Tm - 5°C)/30”, 72°C/30” and a final elongation of 72°C for 10
minutes. The amplified products were then purified using the PureLinkTM
PCR Purification Kit
(Invitrogen, USA) according to the manufacturer’s instructions. The control dsRNA was
synthesized from a linearlized pcDNA3 vector with a T7 and SP6 promoter sequence. The RNA
was transcribed using MEGAscript High Yield Transcription Kit following instructions provided
by the manufacturer (Ambion). The synthesized RNAs were then denatured in 85oC water bath
for 10 minutes and allowed to gradually anneal as bath cooled to room temperature.
The 27-mer siRNAs specific to the U-type channel genes were designed using SciTools RNAi
Design online software (IDT DNA), and the siRNA sequences are shown in Table 3.2. Two
specific U-type channel siRNA and a control siRNA were purchased from IDT DNA as
described previously (Guo et al., 2010;Hui et al., 2007). TriFECTa control was used as the
control siRNA.
3.4 Data analysis and statistics
Summary of data points are presented as mean ± S.E.M.. Statistical analysis was carried out
using OriginPro v8 (Origin Lab Co) or SigmaStat (3.0, Jandel Scientific). Difference between
experimental groups was evaluated using a Student’s t-test for two groups and one-way analysis
of variance (ANOVA) followed by Holm-Sidak post hoc test for multiple experimental groups.
Significance was defined by probability level lower than 0.05 (P < 0.05).
71
*T7 promoter sequence (5’-TAATACGACTCACTATAGGGA-3’ has been tagged on to the U-type gene specific sequence for the
purpose of transcribing RNA into dsRNA for synthesis
Table 3.1 Primer sequences of U-type channel and β-actin. U-type and β-actin primers were used specifically for
real-time PCR studies. U-type T7 primer were used for synthesis of dsRNA.
Table 3.2 Sequences of siRNAs used in the knockdown study. Two U-type siRNA constructs were made from the
two corresponding U-type gene fragments (AF484085 and AF484086). dN represents a deoxyribonucleotide,
remaining 27-mer represents ribonucleotides.
72
Chapter 4
A Sodium Leak Current Regulates Pacemaker Activity of Adult
Central Pattern Generator Neurons in Lymnaea stagnalis.
The work presented in this chapter corresponds to the following publication:
Lu TZ, Feng Z-P (2011) PLoS ONE 6(4): e18745.
Author contributions:
Dr. Feng assisted in proposing, developing and designing experiments for this chapter. She also
provided all the necessary materials, reagents, and analysis tools. My role was conducting all the
experiments and analyzing the data. In addition, I also drafted and revised the manuscript. Dr.
Feng assisted in the editing and revision process.
73
4 A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator Neurons in Lymnaea stagnalis.
4.1 Abstract
The resting membrane potential of the pacemaker neurons is one of the essential mechanisms
underlying rhythm generation. In this study, I described the biophysical properties of theU-type
channel and investigated the role of the channel in the rhythmic activity of a respiratory
pacemaker neuron and the respiratory behaviour in adult freshwater snail, Lymnaea stagnalis.
My results show that the channel conducts an inward leak current carried by Na+ (INa Leak). The
INa Leak contributed to the resting membrane potential and was required for maintaining rhythmic
action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown
of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These
findings identified the Na+ leak conductance via the U-type channel, likely a NALCN-like
channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons
and respiratory behaviour in adult animals.
4.2 Introduction and Rationale
The rhythmic activities of the central pattern generator neurons (CPG) are essential for numerous
biological functions, including brain development (Sipila et al., 2006;Zheng et al., 2006),
locomotion (Harris-Warrick, 2002), energy balance (van den et al., 2004) and respiration (Pena
and Ramirez, 2004;Tryba and Ramirez, 2004). The true CPG pacemaker neurons are capable of
generating intrinsic bursting rhythms in dependent of synaptic input. One conserved mechanism
that is a prerequisite for spontaneous rhythmic activity of pacemaker neurons is regulation of the
resting membrane potential (RMP). K+ leak has been the classical mechanism to describe
regulation of the RMP (Hodgkin and Huxley, 1947); however the highly depolarized membrane
potential of many pacemaker neurons suggests additional current components (Khaliq and Bean,
2010;Tazerart et al., 2008;Tryba and Ramirez, 2004;van den et al., 2004). The principles of
rhythm generation and its modulation are conserved across species (Dickinson, 2006;Taylor and
Lukowiak, 2000).
74
Table 4.1. Protein sequence alignments of the U-type pore and S4 regions with the NALCNs. Regional protein
sequences of the U-type channel from Lymnaea stagnalis (GenBank AAO85435 and AAO84496) were aligned with
NALCN channel from Homo sapien (GenBank NP_443099) and Mus musculus (GenBank NP_796367), NCA-1
(GenBank NP_741413) and NCA-2 (GenBank NP_498054) from Caenorhabditis elegans, and α1U from
Drosophila melanogaster (GenBank AY160083). Positive residues of the S4 region are highly conserved across
different species. Pore forming sequence shows high degree of homology with a notable switch between the
transmembrane domain II and III.
75
The great pond snail, Lymnaea stagnalis (L. stagnalis), is a bimodal breather (JONES, 1961) and
its aerial respiratory activity can be easily described by measuring frequency and opening
duration of the respiratory gas-exchange orifice (pneumostome). The aerial respiration of L.
stagnalis is controlled by a simple well-described rCPG network consisting of three large
identified neurons (Syed and Winlow, 1991), including one intrinsic pacemaker neuron, the right
pedal dorsal 1 (RPeD1), that initiates rCPG rhythmic activity (Syed and Winlow, 1991;Winlow
and Syed, 1992). The pacemaker neuron RPeD1 exhibits rhythmic activity characterized by
intermittent action potential bursts (Syed et al., 1990;Taylor and Lukowiak, 2000). L. stagnalis
thus has been used as an animal model to study rCPG properties and regulation (Spencer et al.,
1999;Syed et al., 1990;Taylor and Lukowiak, 2000).
A putative ion channel (GenBank accession numbers, AF484086 and AF484085) has been
partially cloned from L. stagnalis and named the U-type channel, which stands for a functionally
unknown voltage-gated cation channel (Spafford et al., 2003). My protein sequence alignment
showed that the pore region of this uncharacterized putative ion channel has a 55% identity with,
NALCN (Sodium Leak Channel Non-selective) (Lee et al., 1999;Lu et al., 2007) of mouse
(GenBank: NP_796367) and human (GenBank: NP_443099), and 56% or 45% identity with the
NALCN orthologue of D. melanogaster (GenBank: AAN77520), and C. elegans isoforms NCA-
1 (GenBank: NP_741413) and NCA-2 (GenBank: NP_498054), respectively (Lu et al., 2007).
Specifically, this U-type channel has high homology in the pore and S4 region to its orthologues
(Table 4.1).
4.3 Hypothesis
I hypothesize that the U-type channel exhibits similar biophysical properties to its orthologues,
NALCN channels, and regulates the resting membrane potential.
4.4 Specific Aims
1. Determine whether U-type channel expression is required for pacemaker activity in
isolated rCPG neuron, RPeD1, from adult L. stagnalis in vitro.
2. Characterize the biophysical and pharmacological properties of U-type channel in L.
stagnalis rCPG pacemaker neuron, RPeD1.
76
3. Determine whether U-type channel expression is involved in maintaining respiratory
behaviour in L. stagnalis in vivo.
4.5 Materials and Methods
In this study, I investigated the biophysical properties and involvement of the U-type channel in
the rhythmic activity of the rCPG pacemaker RPeD1 neuron, and in the aerial respiratory
behaviour of the snail, using an RNAi gene silencing approach combined with
electrophysiological recordings.
4.5.1 Animals and aerial respiratory behavior observation
Freshwater pond snails, L. stagnalis, were obtained from an inbred culture at the Free University
in Amsterdam, and raised and maintained in 18 - 20°C aquaria on a 12 hr light / 12 hr dark cycle
aquaria at the University of Toronto (Fei et al., 2007;Guo et al., 2010). Six-week old snails were
used in all experiments. To study the aerial breathing behaviour of the snails, individually
labelled snails were placed in a 1000 mL beaker filled with 500mL of water. Snails were allowed
10 minutes to acclimatize to the new environment. Aerial respiratory behaviour was monitored
by observing the physical opening and closing of the gas exchange orifice, pneumostome, at the
water-air interphase. The duration and number of each pneumostome opening and closing event
were recorded for 1 hour.
4.5.2 Ganglionic RNA preparation and cDNA analysis
The central ring ganglia were excised from anaesthetized snails (in 10% v/v Listerine for 5 min).
Two excised ganglionic rings were used for each total RNA extraction following a modified
Trizol method (Invitrogen) as described preciously (Guo et al., 2010). First strand synthesis of
cDNA was conducted using SuperScript III reverse transcriptase (Invitrogen) with random
hexamer primer (Fermentas) in total volume of 20 µl for 1 µg of total RNA.
4.5.3 RNAi synthesis and delivery
The double-strained RNA and siRNA were synthesized as described in section 3.3.1.4. For
dsRNA/siRNA delivery, 10% Listerine were used to anaesthetize the snails for 7 minutes, a fine
forcep was used to gently lower the head of the snails to expose the mantle cavity below shell
77
and mantle skirt. The injection site was identified at an area caudal to the buccal mass and dorsal
to the central ring ganglia (Fei et al., 2007;Hui et al., 2007). A dead volume free needle micro-
syringe (MICROLITRE #7105KH, Hamilton Company, Reno, Nevada) was used to inject 2µl of
dsRNA (3µg/µl) or siRNA (20 µM). Following injection, the animal was transferred to a
recovery tank containing fresh lettuce and properly aerated water. The snails generally resume
normal locomotor behavior within 2 hours post-injection. Aerial respiratory behavior was then
observed on the third day after injection. Real-time quantitative polymerase chain reaction
(qPCR)
Real-time qPCR was performed using Platinum SYBR Green qPCR SuperMix (Invitrogen). 5 l
of the mix was added to 1 l of 2.5 M primers (Table 3.1) and 0.1 l cDNA, and topped off
with 0.5% diethyl pyrocarbonate-treated water to a final volume of 10 l. Individual cDNA
samples were run in identical triplicates. The reactions were performed in 384-well dishes and
run in a Real-Time PCR System (7900HT, Applied Biosystems, ABI) controlled by SDS2.2.1
software, with the cycling parameters of 50°C for 5 min and 95°C for 10 min, followed by 40
cycles of 95°C for 30 seconds and 55°C for 30 seconds followed by a melting curve protocol.
The peak of the first-derivative in the melting curve and the shape of the amplification curve
were used to assess the quality of the PCR. Ratiometric target (U-type)/control (β-actin)
transcript levels were analyzed using the ΔΔCt method (Pfaffl, 2001). The data were normalized
to corresponding naïve control, a reference group, (Ratio = (eff target gene)ΔCTtarget (control-treated)
/(eff
reference gene)ΔCTreference (control-treated)
). A value of one represents no change in the relative mRNA
expression levels, with values greater than one representing an increase and values less than one
representing a decrease in the relative mRNA expression.
4.5.4 Primary cell culture
Right pedal dorsal 1 (RPeD1) cells, the pacemaker neurons exhibiting spontaneous firing
properties and initiates the respiratory rhythm, were isolated and maintained in culture as
previously described (Feng et al., 1997;Syed et al., 1990). Snails were anaesthetized in 10%
(v/v) Listerine for 5 min prior to dissection of the central ring ganglia in HEPES buffered snail
saline. Digestion was performed by incubating ganglia with 3mg/mL trypsin (type III: Sigma-
Aldrich, Ontario, Canada) for 25min followed by additional 20 min of enzyme inactivation with
78
3 mg/mL of trypsin inhibitor (type III – soyabean: Signma-Aldrich). Ganglia were then washed
and pinned dorsal side up. Connective tissues surrounding the neurons were carefully removed
using a fine forceps. Identified RPeD1neurons were individually removed using a fire-polished
suction pipette coated with Sigmacote (Sigma-Aldrich). The isolated RPeD1 neurons were plated
in ploy-L-lysine-coated culture dishes and maintained in cultured medium (CM) for 12-24 hours
prior to electrophysiology recordings. For gene silencing experiments, whole animals were first
injected with 2 µl of dsRNA (3µg/µl) or siRNA (20 µM) 3 days prior to cell isolation. Isolated
cells were plated and maintained in culturing media containing 2 µg/ml of dsRNA or 20 nM of
siRNA.
4.5.5 Electrophysiology
Whole-cell patch clamp recordings (ruptured) were performed on cultured RPeD1 neurons, as
described in Chapter 3. Briefly, the microelectrode pipettes were filled with intracellular
solution, and while the bath solution consist of standard snail saline. Perfusates were focally
perfused onto the cells with a gravity-driven perfusion system. Na+-free solution contained (in
mM) 51.3 N-methyl D-glucamine (NMDG) substituted for Na+. Under voltage-clamp mode, leak
currents were measured by subtracting the total current traces with the non-linear voltage-
dependent currents recorded with a P/4 subtraction protocol using pClampex 9.2 (Figure 3.1).
Data were analyzed with Clampfit 9.2 (Axon Instrument) and plotted with Origin Pro v8 (Origin
Lab Co., Northhampton, MA, USA). Curve fitting was performed with Origin Pro v8. All the
recordings were performed at room temperature (~22C).
Under current-clamp mode, conventional sharp electrode recordings were performed to monitor
the spontaneous bursting firing activity of RPeD1 cells. Resting membrane potential was
measured within 2 minutes after impaling RPeD1. Input resistance was calculated by injecting
hyperpolarizing current steps and using the corresponding resulting membrane potentials ranging
from -70 to -120 mV to calculate resistance following Ohm’s law. In low extracellular Ca2+
experiements, membrane potentials were measured 10 s after change of solution.
Membrane potential was evaluated as an average of voltage readings from a 10 s measurement
window. At the end of each experiment resting membrane potential and electrode resistance were
again measured. The spontaneous action potential (AP) frequency and inter-spike intervals were
analyzed with Clampfit 9.2 (Axon Instrument). Logarithmic histograms of the inter-spike
79
intervals at bin size 20 were plotted with Clampfit 9.2 to describe the bursting firing pattern. AP
amplitude, rise time, decay time, and half width duration were measured Mini Analysis Program
ver. 6.01 (Synaptosoft, Decatur, GA, USA).
To test the sensitivity of leak current to tetrodotoxin (TTX) block, TTX was added in the bath
solution and perfused onto RPeD1 cells. In addition, snail saline containing either Gd3+
(10 μM)
or low [Ca]o (0.5 mM) was perfused onto the RPeD1 cells to study their effects on the leak
current activities and membrane potentials.
4.5.6 Data analysis
General analysis of data follows description outlined in section 3.4. Specifically for
electrophysiology, all recordings were considered during the analysis. Certain whole-cell patch
recordings were excluded from the final values due to poor access resistance and large
capacitance (>700 pF). Whole-cell voltage-dependent currents were also measured and averaged,
cells with inward and outward currents densities that fell outside of twice the standard deviation
were excluded. For current clamp recordings all voltage traces were considered for the analysis.
Within naïve control, approximately 14 cells were recorded; 4 out of the 14 did not exhibit
spontaneous activitiy or could not sustain continuous activity. Similar observations were made in
the control dsRNA/siRNA treated groups. For U-type knockdown, no spontaneous activity was
observed in all recordings.
4.6 Results
4.6.1 U-type channel regulates the resting membrane potential and is a prerequisite for RPeD1 pacemaker activity.
To determine whether U-type channels are involved in regulating the RMP, I took advantage of
the siRNA gene silencing approach to reduce the expression level of U-type channels, as
described previously (Fei et al., 2007;Hui et al., 2007;van Diepen et al., 2005). I first determined
the efficiency of the acute U-type channel knockdown by measurement of mRNA expression
using real-time PCR analysis. Results (Figure 4.1 A) showed that ganglionic expression level of
U-type mRNA transcripts was reduced by ~50% when either dsRNA or either siRNA specific to
the U-type gene was applied in vivo for 3 – 4 days. I then isolated the respiratory pacemaker
80
neuron, RPeD1, from whole animals that were injected with control dsRNA/siRNA or U-type
dsRNA/siRNA for 3 days, and cultured these neurons in respective RNAi treatments overnight.
Intracellular sharp electrode recordings from these treated cells show that the RMP, recorded
with zero current injection (Figure 4.1 B) was more hyperpolarized and input resistance (Figure
4.1 C) increased in the U-type dsRNA group relative to those of the controls. These data suggest
that the U-type channel may conduct current that is required to maintain the RMP at the more
depolarized potential.
To test indeed that the U-type channel regulates the pacemaker activities, I compared firing
patterns of the spontaneous action potentials (APs) in control and U-type knockdown
preparations. The distribution of the durations between action potentials (intra-spike interval)
over 20 minutes of recordings were analyzed to characterize the burst firing pattern. Naïve
control and control RNA groups (Figure 4.1 D2) had similar spike patterns and the inter-spike
intervals in the isolated RPeD1 cells showed two populations of shorter intervals (0.40s, and
0.79s) in the control groups. No spontaneous activity was observed at resting membrane
potential in all the U-type dsRNA treated cells (Figure 4.1 D1). To test whether these activity
patterns differed between groups was due to the more hyperpolarized membrane potential of
RPeD1 neurons in the U-type dsRNA group, I injected a compensatory current to depolarize the
membrane potential to -45 mV, thereby approximating the RMP of the control cells (Figure 4.1
B). The U-type pre-treated neurons resumed rhythmic firing (Figure 4.1 D1), albeit with a
different rhythmic distribution (Figure 4.1 D2). The results demonstrated that U-type expression
within the pacemaker neuron, RPeD1, may affected spontaneous rhythmic activity.
4.6.2 U-type channel conducts an inward Na+ leak current at hyperpolarizing voltages.
A major determinant of the RMP is the leak conductance. To investigate whether U-type
channels conduct a leak current, I first established a two-step recording protocol in a whole-cell
configuration to segregate a linear leak current from the voltage-dependent hyperpolarizing
current, as described in Chapter 3. The total hyperpolarizing current was initially recorded at
hyperpolarizing step voltages in order to limit activation of major voltage-gated channels
(Figure 3.1 A). The slope conductance ILeak was 12.21 ± 0.92 pS/pF (n=7). The two components
of the total hyperpolarizing current are clearly separated by the recording protocols (Figure 3.1).
81
Figure 4.1. Effects of the U-type dsRNA on rhythmic firing and intrinsic membrane properties in RPeD1 neurons.
(A) U-type specific dsRNA/siRNA in vivo knockdown was confirmed with real-time qPCR analysis. Expression
ratio of the U-type channel to β-actin mRNAs in different experimental groups was normalized to naïve control
ratio: control dsRNA (n = 7), U-type dsRNA (n = 7), control siRNA (n = 12), U-type siRNA 1 (n = 8), and U-type
siRNA 2 (n = 7). * indicate significant difference (P < 0.05) to the corresponding control dsRNA or control siRNA
treatment. Isolated individual RPeD1 neurons were maintained in culture in conditioned media (CM), CM + control
dsRNA, or CM + U-type dsRNA, and recording was conduced within 24 hours following isolation. (B) Average
resting membrane potentials of naïve control (n = 14), control dsRNA (n = 7), and U-type dsRNA (n = 9) treated
neurons recorded 2 min after impaling cells. (C) Average input resistance of naïve control (n = 20), control dsRNA
(n = 8), and U-type dsRNA (n = 13) treated neurons. (D1) Representative action potential traces of naïve control,
control dsRNA, and U-type dsRNA pre-treated neurons recorded at resting membrane potentials, and U-type
dsRNA pre-treated neuron depolarized to -45 mV. (D2) Distribution curves of inter spike durations for naïve control
neurons (n = 10), control dsRNA neurons (n = 4), U-type dsRNA neurons (n = 5), and U-type dsRNA neurons (n =
5) depolarized to -45 mV. Total inter-spike count is 940 in naïve control, 346 in control dsRNA, 0 in U-type
dsRNA, and 304 in U-type dsRNA depolarized to -45 mV. Distributions were best fitted with 4 terms Gaussian
curve. All significant difference (P < 0.05) between naïve control and U-type dsRNA treatment is denoted by *. All
significant different (P < 0.05) between control dsRNA and U-type dsRNA treatment is denoted by †.
82
I then used the established protocol to determine whether ILeak is conducted by U-type channels
in RPeD1 neurons. As shown in the representative recordings of Figure 4.2 A1, the leak current
was reduced in the RPeD1 cells pre-treated with U-type channel dsRNA/siRNAs. The I-V
relation of the ILeak conductance of U-type dsRNA treated RPeD1 neurons was significantly
reduced at all the tested voltage-steps (P < 0.05), relative to both the untreated naïve controls and
the control dsRNA treated neurons (Figure 4.2 A2). Similar results were observed in U-type
siRNA treated groups. In contrast, the non-linear hyperpolarizing current in RPeD1 neurons was
not affected by RNAi treatment (Figure 4.2 B1 and B2). These findings indicate that the U-type
channel contributes in part to the ILeak component of the RPeD1 cells at hyperpolarizing voltages.
The reversal potential of my observed leak current was not at -70mV (approximate reversal
potential of K+), suggesting additional contributing components. Therefore, I next tested whether
Na+ is conducted through U-type channel using an ion substitution approach. I first determined
whether U-type channels conduct a Na+ current that contributes to membrane potential
regulation, by measuring changes in the membrane potential in a Na+-free bath solution. As
shown in Figure 4.3 A1, the membrane potential became more hyperpolarized when Na+ was
substituted with equimolar NMDG+ in all the control groups, but no significant change was
observed in the U-type dsRNA or siRNA groups. The data are summarized in Figure 4.3 A2. A
~10 mV difference in the membrane potential was observed from the control cells in Na+-free
condition which was in agreement with the change in the resting membrane potential after
dsRNA or siRNA treatment (Figure 4.1 B). I then used whole-cell (ruptured) recording to
identify whether Na+ current through U-type channel contributes to the ILeak. As shown in
representative current recordings of Figure 4.3 B1 and I-V relation curves of Figure 4.3 B2, the
ILeak recorded at the hyperpolarizing voltages decreased in the naïve control, control dsRNA, and
control siRNA treated RPeD1 neurons, when Na+ in the bath solution was substituted with
equimolar NMDG+. In contrast, the current recorded with the same hyperpolarizing protocol in
the U-type channel dsRNA/siRNA groups was not affected by the Na+-free solution. The data
are summarized in Figure 4.3 B3 indicate that the leak conductance was significantly reduced in
Na+-free solution (P < 0.05) only in the control groups, but not in the U-type channel knockdown
groups. I compared the Na+ current components by subtracting the current recording in Na
+-free
condition from the total Na+ current in saline between the controls and the U-type knockdown
groups.
83
84
Figure 4.2. U-type RNAi knockdown reduces inward hyperpolarizing leak currents in RPeD1 neurons. U-type
RNAi treatment reduces inward hyperpolarizing Ileak. (A1) Representative ILeak, and (A2) average current density-
voltage (I-V) relation of the hyperpolarizing inward ILeak in RPeD1 cells from naïve control (n = 8), control dsRNA
(n = 5), U-type dsRNA (n = 7), control siRNA (n = 3), U-type siRNA 1 (n = 4), and U-type siRNA 2 (n = 4) treated
RPeD1 neuron. ILeak at all hyperpolarizing voltages reduced significantly in the U-type dsRNA/siRNAs groups as
compared to the control groups (P < 0.05). (C) Non-linear leak component was not significantly affected by the U-
type RNAi treatment. (B1) Representative non-linear component, and (B2) average current density-voltage relations
of the non-linear component for naïve control (n = 15), control dsRNA (n = 7), U-type dsRNA (n = 5), control
siRNA (n = 4), U-type siRNA 1 (n = 4), and U-type siRNA 2 (n = 4).
To test whether the Na+ leak current is sensitive to tetrodotoxin (TTX), a selective blocker of
voltage-gated sodium channel, I first studied the IC50 value of the voltage-gated sodium channels
in RPeD1 neurons to TTX. An IC50 value of ~25 µM TTX was observed (Appendix A1), which
was in a good agreement with that observed in the cerebral giant cells of L. stagnalis (Staras et
al., 2002), indicating the snail channels are intrinsically less sensitive to TTX block as compared
with mammalian channels. To ensure sufficient TTX concentration, 100 µM TTX (~4 fold of the
IC50 value) was perfused; however, no change was observed in the ILeak (Figure 4.3 C). These
findings indicate that the U-type channel carries a TTX-insensitive leak Na+- current (ILeak-Na) in
RPeD1 cells, which directly regulates the RMP.
4.6.3 U-type channel conductance is pharmacologically similar to reported NALCN channel conductance.
INa Leak is conducted by NALCN channels at rest in some neurons (Lu et al., 2007). These
channels are non-sensitive to TTX (Lu et al., 2007), can be partially blocked with Gd3+
(Lu et al.,
2007) and activated by low [Ca2+
]o (Lu et al., 2010). I thus asked whether my observed TTX-
insensitive ILeak has similar pharmacological properties to NALCN channels. I first tested
whether Gd3+
applications would affect ILeak. As shown the representative recordings in Figure
4.4 A1 and I-V curves in Figure 4.4 A2, 10 µM Gd3+
suppressed the ILeak in the control groups,
but with minimal effect in U-type dsRNA/siRNA treated groups. Gd3+
significantly reduced the
normalized leak current in the groups (P<0.05), but slightly enhanced the current in the U-type
RNAi groups (summarised in Figure 4.4 A3). The Gd3+
-blocked current component in the
control groups was smaller (Figure 4.4 A3) than the Na-dependent leak current component
85
86
Figure 4.3. ILeak conducted by the U-type channel in RPeD1 cells is carried by Na+ ions. (A1) Representative
voltage traces of naïve control, control dsRNA/siRNA, and U-type dsRNA/siRNA 1 & 2 with and without Na+ free
saline perfusion. (A2) Average voltage difference between Na+ free perfusion and saline perfusion for naïve control
(n = 9), control dsRNA (n = 6), U-type dsRNA (n = 6), control siRNA (n = 6), U-type siRNA 1 (n = 6), U-type
siRNA 2 (n = 4). (B1) Representative ILeak in RPeD1 cells from naïve control, control dsRNA/siRNA, and U-type
dsRNA/siRNA 1&2, at hyperpolarizing voltages in saline or Na+ free solution (B2) Representative ILeak density-
voltage (I-V) relations of naïve control, control dsRNA/siRNA, and U-type dsRNA/siRNA 1&2 in saline or Na+ free
solution. (B3) comparison of the average normalized ILeak in Na+ free solution over saline at -160 mV, -110 mV, and
-60 mV for naïve control (n = 8), control dsRNA (n = 6), U-type dsRNA (n = 7), control siRNA (n = 4), U-type
siRNA 1 (n = 4), and U-type siRNA 2 (n = 4). A significant reduction in normalized ILeak under Na+ free condition
was observed in RPeD1 cells from the naïve control and control dsRNA/siRNA groups, but not from U-type
dsRNA/siRNA groups. All significant difference (P < 0.05) between Na+ free saline current normalized to saline is
denoted by *. (C) Application of 100 µM TTX did not significantly affect Ileak. (C1) Representative leak current
traces of naive control neurons in saline, 100 µM TTX, and saline wash under a hyperpolarizing step voltage
protocol. (C2) Representative current-voltage relation for the leak current observed in saline, 100 µM TTX, and
saline wash. Line represents a linear fit. (C3) Summary of relative current density of 100 µM TTX normalized to
that in saline at various hyperpolarizing voltages: -160 mV (n = 5), -110 mV (n = 5) and -60 mV (n = 5). No
significant difference was observed in the normalized currents among all the groups.
(Figure 4.3 B3); therefore, my findings indicate that Gd3+
only partially blocks the INa Leak
conducted by U-type channel. To further confirm that a partial blockade by Gd3+
affects the
functional properties of U-type channel, I measured the membrane potential of RPeD1 in saline
containing 10 µM Gd3+
. The membrane potential in the control cells was reduced by ~5 mV in
Gd3+
condition (Figure 4.4 A) but was not affected by Gd3+
in the U-type knockdown group,
consistent with my voltage-clamp data showing that Gd3+
partially blocked the leak current of
RPeD1.
I then tested whether the U-type channel properties are affected by low [Ca2+
]o. As shown the
representative recordings in Figure 4.5 A1 and I-V curves in Figure 4.5 A2, reducing [Ca2+
]o
from 4.1 mM to 0.5 mM resulted in a depolarization of the membrane potential in the control
groups, but with a minimal effect in U-type dsRNA/siRNA treated groups. The low [Ca2+
]o also
significantly enhanced the normalized leak current in the control groups (P<0.05), but with no
significant effect on the current in the U-type RNAi groups (Figure 4.5 B). Taken together, my
data suggest similar pharmacological properties between U-type channels and NALCN channels.
87
Figure 4.4 Gd3+
partially blocked ILeak via the U-type channels in RPeD1 neurons. (A) ILeak conducted by the U-
type channel is partially blocked by Gd3+
. Representative ILeak (A1) and ILeak - voltage (I-V) relations (A2) of naïve
control, control dsRNA/siRNA, and U-type dsRNA/siRNA 1&2 in saline with or without 10 µM Gd3+
. (B3)
Average normalized ILeak in 10 µM Gd3+
over saline at -160 mV, -110 mV, and -60 mV for naïve control (n = 4),
control dsRNA (n = 4), U-type dsRNA (n = 3), control siRNA (n = 4), U-type siRNA 1 (n = 4), and U-type siRNA 2
88
(n = 4). The dashed line represents the current activity in saline. 10 µM Gd3+
reduced significantly the ILeak in the
naïve control and control dsRNA/siRNA groups, but not in U-type dsRNA/siRNA groups. Data are presented as
mean ± SEM. Significant difference (P < 0.05) between Na+ free saline current normalized to saline is denoted by *.
(B) 10 µM Gd3+
hyperpolarized the membrane potential. (B1) Representative records of the membrane potentials of
RPeD1 from naïve control, control dsRNA/siRNA, and U-type dsRNA/siRNA group. 1 & 2 indicate perfusion with
and without 10 µM Gd3+
, respectively. (B2) Average difference of the membrane potential by 10 µM Gd3+
in naïve
control (n = 4), control dsRNA (n = 3), U-type dsRNA (n = 4), control siRNA (n = 3), U-type siRNA 1 (n = 4), U-
type siRNA 2 (n = 3) groups. * and † indicate the significant difference (P < 0.05) between naïve control and U-type
RNAi treatment and between control RNAi and U-type RNAi treatment, respectively.
89
Figure 4.5 Low extracellular Ca2+
depolarizes the membrane potential by enhancing U-type channel activity in
RPeD1 neurons. (A) U-type channel knockdown prevents low extracellular Ca2+
-induced membrane depolarization.
(A1) Representative voltage traces of naive control, control siRNA, and U-type siRNA treated RPeD1 neurons
perfused with saline, Na+ free or low [Ca
2+]o saline. Partial U-type knockdown reduces membrane hyperpolarization
by Na+ free solution or depolarization by 0.5 mM [Ca
2+]o. (A2) Average voltage differences of naive control (n = 4),
control siRNA (n = 5), and U-type siRNA (n = 4) treated RPeD1 under Na+ free or 0.5 mM [Ca
2+]o perfusion. (B)
Low extracellular Ca2+
increased Ileak is reduced in U-type knockdown. (B1) Representative Ileak traces of naive
control, control siRNA, and U-type siRNA treated RPeD1 in saline, Na+ free, and 0.5 mM [Ca
2+]o solution. (B2)
Average ILeak normalized to saline of naive control (n = 5), control siRNA (n = 4), and U-type siRNA (n = 4) treated
RPeD1 in Na+ free, and 0.5 mM Ca
2+ at -100 mV. The dashed line represents the current activity in saline. Data are
presented as mean ± SEM. * and † indicate significant difference (P < 0.05) between naïve control and U-type RNAi
treatment and control RNAi and U-type RNAi treatment, respectively.
4.6.4 Partial U-type knockdown reduces the aerial respiratory behavior in adult animal in vivo.
Having established that U-type channel knockdown slows firing rhythmic properties of isolated
RPeD1 neurons (Figure 4.1), I next investigated whether the U-type channel plays a role in
regulation of respiratory behaviour the adult animal in vivo. I measured aerial respiratory activity
of the snails from the naïve control, control dsRNA or U-type dsRNA treated groups. Figure 4.6
A shows that the total duration of pneumostome opening of the U-type dsRNA group (55.8±7.1
s, n=17, p<0.05) during the 1 hour observation period was significantly reduced relative to the
naïve control (134.60±17.76 s, n=24) and control dsRNA (123.8 ±15.9 s, n=13) groups.
Interestingly, the average duration of opening event (Figure 4.6 B) was only slightly decreased
in U-type dsRNA group (21.09±1.46 s, n=17, p>0.05), whereas the number of opening events
(Figure 4.6 C) was significantly reduced (2.68±0.31, n=17, p<0.05). Acute U-type channel gene
knockdown was confirmed using real-time qPCR (Figure 4.6 D); U-type specific dsRNA treated
animals exhibiting a reduced total breathing time showed significantly low U-type mRNA
expression level ratio (0.81±0.15, n=7, p<0.05) as compared to the naive control (1.545±0.40,
n=8) and control dsRNA group (1.77±0.15, n=7). These observations support the hypothesis that
U-type channel plays an important role in maintaining the respiratory activity in adult animal via
regulation of rCPG rhythmic activity by regulating the resting membrane potential of the
pacemaker neurons.
90
Figure 4.6 Acute U-type dsRNA knockdown suppresses aerial respiratory behaviour in adult L. stagnalis in vivo.
Summary of the (A) total breathing time, (B) duration per opening event, and (C) number of opening events for
naïve control (n = 24), control dsRNA (n = 13), and U-type dsRNA injected snails (n = 17). U-type dsRNA injected
snails only showed significant reduction in total breathing time (55.82 ± 7.13s) and number of opening events (2.65
± 0.31) when compared to naive control (total breathing time of 134.60 ± 17.76s and 5.42 ± 0.36 opening events)
and control dsRNA (total breathing time of 123.77 ± 15.82s and 4.94 ± 0.45 opening events). Average opening
duration for U-type dsRNA (21.09 ± 1.46s) injected group is not significantly different from the naive control (24.85
± 1.51s) and control dsRNA (25.05 ± 2.86s) injected snails. (D) Summary of real-time PCR performed following
behavior experiments. Ganglia from U-type dsRNA injected snails that showed decrease in aerial respiratory
behavior were used to confirm U-type channel knockdown with real-time qPCR. The result was then compared with
naive control and control dsRNA results. Βeta-actin control gene was used for ratiometric analysis of U-type mRNA
expression level. * and † indicate significant difference (P < 0.05) from the naïve control and control dsRNA
treatment, respectively.
4.7 Discussion
In this study, I have demonstrated that an inward Na+-conductance leak current component at
rest regulates the rhythmic activity of a respiratory pacemaker neuron, RPeD1. Acute
knockdown of the channel gene decreased the functional channel expression and suppressed the
rhythmic firing of the pacemaker neuron, and inhibited breathing activity. The U-type channel
protein sequence shares ~50% homology with the pore region of the NALCN channel and
exhibits similar biophysical and pharmacological properties, thus the U-type channel is likely a
91
Lymnaea orthologue of NALCN-like channel. The INa Leak conducted though the U-type channels
are critical in modulating the rCPG rhythmic activity. This is the first study providing direct
evidence of a NALCN-like channel regulating the activity of pacemaker neurons, and its
functional significance in breathing activity is determined by the pacemaker neuron regulating
respiration at the whole animal level.
Many ion channels have been identified as contributors to pacemaker activity in vertebrates.
These include: persistent Na+ currents (INaP) (Del Negro et al., 2002;Do and Bean, 2003;Koizumi
and Smith, 2008;Puopolo et al., 2007;Raman et al., 2000), leak K+ current (IK Leak) (Koizumi and
Smith, 2008), Ca2+
-activated voltage-insensitive non-specific cation current (ICAN) (Del Negro et
al., 2005), hyperpolarizing activated current (Ih) (Bennett et al., 2000;Chan et al., 2004;Forti et
al., 2006;Thoby-Brisson and Ramirez, 2000), small conductance Ca2+
-activated K+ current (ISK)
(Bennett et al., 2000;Hallworth et al., 2003;Wolfart et al., 2001), and subthreshold Ca2+
currents
(ICaT) (Onimaru et al., 1997;Puopolo et al., 2007). In invertebrates, similar currents have also
been identified to modulate various CPG network models (Dickinson, 2006;MacLean et al.,
2005;Staras et al., 2002). In my study, Na+ leak through U-type channels (IU) represents a major
component in subthreshold current densities. Background Na+ current has been previously
identified in respiratory pacemaker neurons in rodent (Tryba and Ramirez, 2004), by stabilizing
bursting activities. Given the relative depolarized nature of the RMP in rodent rCPG pacemaker
neurons (Tryba and Ramirez, 2004) and other pacemaker neurons (Tazerart et al., 2008;van den
et al., 2004), it is likely that the INa Leak current via NALCN channel regulates the resting
membrane potential in the similar manner of U-type channel in the snail. The voltage-dependent
component of inward hyperpolarizing current (Figure 4.2) is likely an Ih conductance. Although
previous studies have not identified Ih component from other L. stagnalis neurons (Staras et al.,
2002), the presence of the Ih in RPeD1 cells has not been excluded. Pacemaker bursting activity
in pacemaker neurons is also mediated by INaP and IK Leak in rodents (Koizumi and Smith,
2008;Smith et al., 1991) and L. stagnalis (Staras et al., 2002). INa Leak via the NALCN or
NALCN-like channels could be another conserved component that is essential for bursting
activity across species.
The RMP is one of the essential prerequisites that determines pacemaker activity (Kandel, 1977),
and is determined by the ionic permeability of the cell membrane (Hodgkin and Huxley,
92
1947;Keynes, 1951) at rest. Membrane permeability to K+ through K leak channels has been
considered the key conductance to determine the RMP (Hodgkin and Huxley, 1947). However,
the RMP of most pacemaker neurons are more depolarized than the equilibrium potential of K+,
suggesting additional outward current component (Tazerart et al., 2008;van den et al., 2004).
Resting Na+ conductance may be derived from numerous sources, including window current
from voltage-gated Na+ channels, hyperpolarization-activated channels, persistent Na
+ channels
and Na+-coupled transporters. NALCN channels have been considered to be the major
contributor to background Na+ leak current in hippocampal neurons (Lu et al., 2007;Lu et al.,
2010). The NALCN channel was first cloned from rat brain (Lee et al., 1999). The leak current
conductance of the channel, however, remains inconclusive because of a lack of the background
Na+ leak conductance following overexpression of the NALCN channel on HEK293 cells
(Swayne et al., 2009). My results in RPeD1 neurons support the notion that NALCN-like
channels conduct leak Na+ current at rest.
The in-vivo knockdown through RNAi gene silencing has been widely used to study protein
function (Coumoul and Deng, 2006), including L. stagnalis (Fei et al., 2007;Hui et al.,
2007;Korneev et al., 2002). Both long dsRNA and short siRNA have been used to induce gene
silencing in L. stagnalis (Fei et al., 2007;Hui et al., 2007;Korneev et al., 2002). One of the
shortcomings of the RNAi approach is the possibility of its off-targeting effect on non-specific
genes. I attempted to remedy this possibility by employing three different dsRNA sequences
consisting of both long dsRNA and short siRNAs. The two siRNA were synthesized from two
different fragments of U-type mRNA sequence (AF484085 and AF484086). The selection
criteria consist of selecting for moderate to low G/C content, biasing toward the 3’-terminus and
purposely avoiding sequences encoding the transmembrane domain (Reynolds et al., 2004). The
resulting siRNA sequence was BLAST searched against known genes sequences in all available
databases to avoid complementary paring with any known ion channel sequences. All three
RNAi sequences resulted in a >40% knockdown in U-type mRNA level, a reduction of the INa
Leak, a hyperpolarized resting membrane potential, a decrease in AP firing activity of the
pacemaker RPeD1 neuron, and suppressed respiratory behaviour. My findings are consistent and
thus suggest that the U-type channel RNAi in L. stagnalis is specific.
93
It should be noted that this study observed ~50% knockdown of U-type mRNA level, which
translated to a large describe in function U-type channel. Given that U-type knockdown group
resulted in almost complete abolition of spontaneous activity, Na+-dependent regulation of
membrane potential, and Gd3+
sensitivity; it suggest that higher functional membrane expressed
U-type channel reduced. It should be noted that gene expression does not directly translate to
changes in physiology. In mammals, although knockout NALCN resulted in lethal pups (Lu et
al., 2007), NALCN heterozygotes are viable with mild abnormalities to serum sodium
concentration (Sinke et al., 2010). The discrepancies could be attributed to membrane expression
of the NALCN and NALCN-like channels, and possible regulatory mechanisms that controls
NALCN gating.NALCN channel may affect synaptic transmission. C. elegans orthologues of the
NALCN channels are critical in the conduction of depolarizing signal from the soma to the axon
(Yeh et al., 2008) and mutation of this channel impairs presynaptic release (Jospin et al., 2007).
NALCN orthologue is highly expressed at synapses in D. melanogaster (Nash et al., 2002), but
perisynaptically in C. elegans (Yeh et al., 2008). In rodents, the NALCN is modulated by
substance P and neurotensin through G-protein independent and Src family of tyrosine kinases-
dependent pathway (Kim et al., 2012;Lu et al., 2009). The NALCN channel conductance in the
pancreatic -cells is activated by M3 muscarinic receptors (Swayne et al., 2009). The rCPG
network in adult L. stagnalis consists of three neurons (Syed et al., 1990). RPeD1 forms mutual
inhibitory synapses with VD4 neuron, which innervates motor neurons that close pneumostome.
RPeD1 forms an excitatory synapse with IP3I which leads to pneumostome opening. Reduction
of U-type channel expression decreases the number of openings, but has less effect on closing of
the pneumostome (Figure 4.6), indicating the rCPG network is differentially regulated by INa Leak
conductance. Shifting the RMP to the hyperpolarizing direction may reduce the excitatory
synaptic input of RPeD1 to IP3I and relieve the inhibitory input on VD4; thus, the rCPG output
favours the decrease in pneumostone openings rather than duration of the opening. This study
leads to new avenues to further our understanding of rhythm regulation of CPG network.
4.8 Acknowledgements
The work was supported by an operating grant to ZPF from National Sciences and Engineering
Research Council of Canada (NSERC-249962-09). TZL is a recipient of a Canadian Graduate
94
Studentship (CGS-MSc) and Postgraduate Scholarship (PGS-D3) of NSERC. ZPF holds a New
Investigator Award from the Heart and Stroke Foundation of Canada.
95
Chapter 5
Robust Regulation of Spontaneous Activity Depends On A Sodium
Leak Current Involving U-type Channel In A Mollusca Pacemaker
Neuron Simulation
The work presented in this chapter corresponds to the following manuscript:
Lu TZ, Kostelecki W, Pérez Velázquez JL, Feng Z-P. In preparation for submission.
Author contributions:
Dr. Feng and I both worked in conceiving, developing and design the experiments. I worked to
perform all the electrophysiology recording and analysis. Mr. Kostelecki, a 4th
year PhD student,
collaborated with me in contributed to the development, optimization and analysis of the isolated
RPeD1 computation model. Dr. Feng provided the materials/reagents/analysis tools for
electrophysiology analysis and Dr. Pérez Velázquez provided the computation
developing/analysis tools.
96
5 Robust regulation of spontaneous activity depends on a sodium leak current by U-type channel in a molluscan pacemaker neuron simulation.
5.1 Abstract
Pacemaker activity is characterized by the intrinsic ability of generating spontaneous electrical
oscillations without external stimulations. Different cells utilizes different mechanisms for
pacemaker generation, however the fundamental principle of pacemaker generation depends on
the background currents regulating voltage-gated activations. Traditionally, background current
has been considered to be largely regulated by K+, with a smaller Na
+-dependent component.
Recent characterization of a sodium leak, non-selective channel (NALCN) has identified it as a
major candidate for background Na+ current in neurons. NALCN knockouts and NALCN-like
knockdowns produced hyperpolarized resting membrane potential and reduced neuronal
activities in vitro. However, it is unclear the functional significance of background Na+ leak
regulating spike activity. In this study, I have investigated the role of Na+ leak current partially
through NALCN-like, U-type, channel in generating action potentials of a pacemaker neuron in a
simplified molluscan model. Using a conductance-based computation model that was generated
from experimental recordings, I was able to accurately simulate the action potential profile and
spontaneous activity of the pacemaker neuron. By dividing the leak component to Na+ and K
+
leak, I was able to demonstrate the higher sensitivity of spiking rate with Na+ leak compared to
K+ leak. The results provide strong indication of the robust function for Na
+ leak in regulating
spontaneous neuronal activities.
5.2 Introduction and rationale
Pacemaker cells are identified by their intrinsic ability to generate spontaneous activity. Under
experimental conditions, true pacemaker cells are capable of electrical activity following
physical or pharmacological isolation. Regulating these activities depends on the different
membrane expressed ion channels that drives electrical oscillations and shape the action potential
profile. In neurons, generation and perpetuation of pacemaker rhythm depends on a myriad of
voltage-gated and ligand-gated currents (Bean, 2007). Many computation models of pacemaker
neurons have investigation of the underlying ionic mechanisms involved in generation of
97
pacemaker activity. Generating these models stemmed from works by Hodgkin and Huxley
(1952), who described action potential firing of squid giant axon using the first conductance-
based computation simulation. Since then, many conductance-based models have accurately
simulated and describe ionic conductance during neuronal firing (Amini et al., 1999;Amini et al.,
2005;Butera, Jr. et al., 1999;Rose and Hindmarsh, 1985). Fundamental to all model simulation is
a component of leak conductance. The importance of leak is well recognized; however, the
functional contributions of non-voltage gated currents are relatively less understood.
Traditionally, voltage-independent leak current has been hypothesized as functional component
of background conduction necessary to establish resting membrane potential (Hodgkin and
Huxley, 1952;Hodgkin and Huxley, 1947). Leak currents have been identified to regulate
network oscillation (Koizumi and Smith, 2008;Pang et al., 2009;Zhao et al., 2010), cell
excitability, (Brickley et al., 2007b;Rekling et al., 2000) and pacemaker regulation (Farkas et al.,
1996;Hagiwara et al., 1992;Jackson et al., 2004;Liu et al., 2002;McCormick and Huguenard,
1992;Pena and Ramirez, 2004;Ptak et al., 2009;Shen and North, 1992b;Shen and North, 1992a).
Characterization of the leak conductance in neurons has identified major K+ and a smaller Na
+
component. Recently, a novel sodium leak channel (NALCN) has been identified to be the major
contributing ion channel to the background Na+ leak current. Knockout of NALCN in neurons of
mice results in hyperpolarized resting membrane potential and reduced excitability (Lu et al.,
2007). In Chapter 4, I have described how NALCN-like (U-type channel) conductance in a L.
stagnalis pacemaker neuron model contributes to rhythmic generation as well as spontaneous
activities. Results suggested that Na+ leak through U-type channel represent an essential
component to pacemaker activity. However it is unclear the significance of regulating a small
Na+ leak current in relation to much larger K
+ leak in modulating rhythmic activity of spike
activities.
5.3 Hypothesis
Na+ leak current conducted by U-type channel contributes to the major subthreshold current
necessary in regulating action potential firing rate of RPeD1.
98
5.4 Specific Aims
1. Determine and characterize how U-type channel knockdown affects action potential
profile.
2. Characterize ionic currents necessary for rhythm generation in the isolated adult
pacemaker neuron, RPeD1.
3. Generation of a conductance-based model to simulate the spontaneous action potential
profile of RPeD1 neuron.
4. Describe quantitatively how Na+ leak regulates action potential frequency and action
potential profile.
5.5 Materials and Methods
5.5.1 Experimental animals
Freshwater pond snails, Lymnaea stagnalis, obtained from an inbred culture at the Free
University in Amsterdam were maintained in a 12 h light/dark cycle under 18-20°C water at
University of Toronto. Adult snails of 2 month old (approximate shell lengths of 20mm – 25mm)
were used. All experiments were performed according to the guidelines of the Animal Care
Committee of the University of Toronto.
5.5.2 Primary cell cultures, cell isolation and RNAi gene silencing
Detailed descriptions of the isolation procedure for right pedal dorsal 1(RPeD1) neurons have
been previously made in section 4.5.5. Similar RNAi synthesis (Chapter 3) and silencing
protocol (Chapter 4) was performed in this study to induce acute U-type channel knockdown in
culture.
5.5.3 Bath solutions and chemicals
In order to characterize various ionic current we substituted bath and pipette solutions with
blockers or large charge carriers to isolate different ionic currents of the RPeD1 neuron. Bath
solutions were focally perfused onto cells using a gravity-driven perfusion setup. The DM
consisted of serum-free 50% (v/v) Liebowitz L-15 medium (without salts or L-glutamine; Gibco,
99
Grand Island, NY, USA), with the addition of (in mM): 51.3 NaCl, 1.7 KCl, 4.1 CaCl2, 1.5
MgCl2, 10 glucose, 1 L-glutamine, 2 HEPES, 20 mg/mL gentamycin, and with pH adjusted to
7.9 with 1M NaOH. Sodium free saline contained (in mM) 51.3 N-methyl D-glucamine in
replacement of sodium ions. Low calcium saline contained 0.5 mM CaCl2 in replace of 4.1 mM
found in control saline. For studying of calcium and blocking of potassium currents, we used
saline consisted of (in mM) 51.3 mM tetraethylammonium chloride (TEA-Cl), 1.7 KCl, 4.1
CaCl2, 1.5 MgCl2, 2 HEPES, and pH was adjusted to 7.9 with 1M TEA-OH. One potassium
blocking experiment also included 4 mM of 4-aminopyridine (4-AP). Pipette solution for
potassium and hyperpolarizing activated current experiments contained (in mM): 29 KCl, 2.3
CaCl2, 2 MgATP, 10 HEPES, 11 EGTA, 0.1 GTPTris and pH was adjusted to 7.6 with 1 M
KOH. Pipette solution for sodium and calcium experiments contained (in mM) 29 CsCl, 2.3
CaCl2, 2 MgATP, 10 HEPES, 11 EGTA, 0.1 GTPTris and pH was adjusted to 7.6 with 1M
CsOH.
5.5.4 Electrophysiology
In order to describe the ionic currents involved in generation of spontaneous action potentials of
acute isolated RPeD1 neurons, I performed whole-cell (ruptured) voltage-clamp recordings as
described in section 3.2.3. I also characterized the action potential frequency and profiles using
conventional sharp electrode recordings as described in section 3.2.4. The spontaneous action
potential (AP) frequency was analyzed with Clampfit 9.2 (Axon Instrument). AP amplitude, rise
time, decay time, and half width duration were measured Mini Analysis Program ver. 6.01
(Synaptosoft, Decatur, GA, USA). Curve fittings were carried out using OriginPro v8 (Origin
Lab Co). Current-voltage relations of calcium currents were fitted to the modified Boltzmann
equation: (1/(1 +exp(–(V − Vh)/S)))*(V − Vrev)*G, where V is the applied voltage, Vh is the half-
activation voltage, S is the slope factor, Vrev is the reversal potential and G is the slope
conductance. All the recordings were performed at room temperature (~22°C).
5.5.5 Data analysis
As described in section 3.4. All current traces were included in the analysis. Cells with
capacitance larger than 700 pF were excluded due largely to the inability to maintain a proper
seal.
100
5.5.6 Computer simulation
Using the RPeD1 model described by Bungay and Campbell (2009) as a basis, this study builds
on previous work to develop an RPeD1 model of spontaneous rhythmic activity that is more
experimentally accurate. This study uses a similar combination of ionic currents with the
exception that the persistent sodium current described by Bungay and Campbell (2009) was
excluded. Equations describing the kinetics of ionic currents (equations equation 4.2) were
reproduced identically with differences only in the values of model parameters shown in Table
5.1. Simulations were performed using a single neuron Hodgkin-Huxley parallel conductance
model that incorporated voltage-gated sodium ( ), potassium ( and ), calcium ( ),
hyperpolarizing-activated (h) currents and a leak current ( ) consisting of sodium and potassium
leak components. Changes in simulated membrane potential, , were described by the equation
(equation 4.1)
where a membrane capacitance of was used (Bungay and Campbell,
2009;McComb et al., 2003). For parameter estimation, was used to simulate patch clamp
conditions but excluded from all other simulations. Individual currents were calculated from the
equations
(equation 4.2)
(equation 4.3)
(equation 4.4)
(equation 4.5)
(equation 4.6)
(equation 4.7)
where is the conductance for ion channel , and is its reversal potential. The activation and
inactivation parameters ( , , , , , , and ) were updated according to the equation
101
(equation 4.8)
where the steady-state activation and inactivation proportions were calculated as
(equation 4.9)
and was either treated as a constant or dependent on membrane potential according to the
equation
(equation 4.10)
(see Table 5.1). Numerical integration of simulations was performed in MATLAB using the
ode15s solver (Shampine and Reichelt, 1997) with a maximum step size of 5 ms, and absolute
and relative tolerances of . Simulations were performed with an initial membrane potential
of -48 mV, and initial activation and inactivation parameters set to (equation 4.9).
5.5.7 Parameters estimation and tuning
Voltage-gated channel parameters were estimated using patch clamp data for joint and
currents, and individual ,
, and currents. Patch clamp simulations were performed
using manually set conductances and initial parameters settings provided by Bungay and
Campbell (2009). After simulating each patch clamp protocol, the squared error was calculated
between simulated currents and representative recordings over the portions of the patch clamp
procedure during the voltage step. Parameters were updated by calculating numerical gradients
of the error function and performing gradient descent to minimize error. Initially, parameters for
,
, and were estimated individually. Results for
were incorporated into a
simulation that included voltage-gated sodium channels and the procedure was repeated to
estimate parameters and while refining parameters.
After parameter estimation, several manual adjustments were made to produce action potential
102
Table 5.1 Parameters of used in establishing the simulation of isolated RPeD1. Values were determined through use
of both literature and data. Fine tuning of the parameters were accomplished to replicate the action potential profile
from recordings.
profiles that matched the observed data more closely. Most notably, was reduced by
approximately a factor of 4 to more closely approximate the upward stroke of experimental
action potentials. The conductance of each population of ion channels was then adjusted to
produce the desired extent of depolarization and hyperpolarization. At this stage, minor
adjustments were made to voltage of half-activation and slope factor parameters to refine the
simulated action potential shape. To finalize the model, the leak current conductance (for a fixed
leak equilibrium potential of – 52 mV) was adjusted to reach the experimentally observed firing
rate of approximately 1 Hz.
103
Final parameter settings are displayed in Table 5.1 and were used for all simulations unless
noted otherwise.
5.5.8 Model evaluation
For each setting of parameters, a simulation of 20 sec duration was generated, from which the
final 15 seconds of the simulation was used to calculate the spike rate and the final spike-centred
window entirely within the simulation was extracted for illustrative and comparative purposes in
Figure 5.6 and Figure 5.7. When necessary, windows of data were resampled using linear
interpolation to match the sampling rate of the recordings. To determine the level of variability in
individual spikes, recordings of 223 spikes during rhythmic RPeD1 activity were obtained and 1
second time windows centred on the peak of the spike were used to calculate the root-mean-
square-error (RMSE) between all combinations of recordings according to the equation
√
∑(
)
(Equation 4.11)
where
is the membrane potential of spike at time and describes the root mean
squared error between spikes and . The resulting distribution of RMSE is shown in Figure 5.7
D as a histogram. This was repeated for one representative simulated spike, , and the histogram
for the distribution is shown in Figure 5.7 E.
5.5.9 Variation of leak sodium/potassium currents
The leak current was separated into two component potassium and sodium leak currents
described by
(
)
(equation 4.12)
where the corresponding conductances and equilibrium potentials are shown in Table 5.1.
Simulations were performed for different settings of the sodium leak and potassium leak
conductances and the resulting rhythmic behaviour and spiking frequency is shown in Figure
5.7.
104
5.6 Results
5.6.1 U-type channel knockdown does not significantly alter spike profile and voltage-gated currents
Having shown in my previous chapter that U-type conductance constitute a major component at
sub-threshold voltages, I first looked at how U-type knockdown affect voltage-dependent
conductance by comparing action potential profiles. Using a similar U-type knockdown approach
as that in Chapter 4, I measured voltage-oscillation using sharp electrode intracellular recording
of action potentials at -45 mV in naïve control, control dsRNA and U-type dsRNA treated
groups. Quantification of action potential profile was made by comparison along 4 different
measurements: action potential amplitude, rise time constant, decay time constant and half-width
at 50% amplitude. In comparison to naïve control and control dsRNA treated groups, U-type
knockdown did not significantly affect all 4 parameters (Figure 5.1 A). This suggests U-type
conductance is not a significantly contributing toward shaping action potential, which is largely
regulated by voltage-dependent channels. To further indicate U-type conductance is not
contributing to action potential shape, I compared voltage-dependent currents between those of
controls and U-type knockdown (Figure 5.1 B). These currents were recorded under P/4 leak
subtraction at depolarizing protocol from a holding potential of -50mV, which only records
voltage-dependent conductance. Analysis of the amplitude for peak transient inward current and
the sustained outward current showed no significant difference between the U-type knockdowns
versus the controls (Figure 5.1 B2), suggesting U-type conductance is not a significant
contributing current at depolarizing voltages. Taken together, these findings indicate that U-type
conductance does not significantly alter major voltage-dependent conductance that shape action
potential profile.
5.6.2 RPeD1 expresses major voltage-gated Na+, Ca2+, K+, and hyperpolarizing-activated currents
In my previous chapter, U-type channel contribute to a significant portion of INa Leak. In order to
determine the functional significance of INa Leak toward action potential firing, I proposed to
generate a conductance-based model based on the Hudgkin-Huxley model in order to simulate
RPeD1 action potential firing. This requires identification and quantification of major Na+-, K
+-
and Ca2+
-dependent currents expressed in RPeD1.
105
Figure 5.1 U-type knockdown does not significantly affect voltage-dependent current or action potential profile.
(A1) Representative traces of control, control dsRNA, and U-type dsRNA action potential recorded from isolated
RPeD1 neurons. (A2) Quantification of amplitude, rise and decay time constant, and half-width duration showed no
significant differences between controls and U-type knockdown group. (B1) Representative voltage-dependent
current traces of naïve control, control dsRNA, and U-type dsRNA treated group. (B2) Current-density versus
voltage relation for transient inward current and sustained out-ward rectifying current. No significant differences
were observed between all three groups.
106
Voltage-dependent Na+ conductance has been reported in other L. stagnalis pacemaker neurons
as well as RPeD1 (Gyori et al., 2000;Onizuka et al., 2005;Staras et al., 2002). Recording under
low extracellular calcium solution yielded a large transient inward current in the isolated RPeD1
neurons (Figure 5.2 A1). This large current was reversibly abolished under Na+-free bath
perfusion (Figure 5.2 B1), indicating the dependence on extracellular Na+. This current was also
sensitive to Na+-channel-specific blocker, TTX (IC50 23.7 ± 2.1 µM; Appendix A1). In addition,
threshold of activation for this current was the range of -40 mV to -30 mV (Figure 5.2 A2) with
the peak current at 1.8 ± 5.7 mV (n=7). These observations are consistent with a voltage-gated
Na+ channel.
In addition to the transient inward current, recording of outward potassium currents under Na+
free bath solution revealed two voltage-dependent currents; a delayed-outward rectifier and a
transient outward current. These are similar to the previously reported IKv and IA current reported
in RPeD1 (Sakakibara et al., 2005). In order to confirm their identity and voltage-dependent
properties, IKv was blocked by 51 mM of extracellular tetraethylammonium (TEA). 70 ± 16 % (n
=5; Figure 5.3 B2) of the sustained outward current is blocked compared to the control. The
residual component includes the transient outward current and TEA-insensitive IKv. Current-
voltage (I-V) relation indicates the activation range at -25 to -20 mV (Figure 5.3 B1). TEA-
sensitive IKv is the major component of the sustained outward current between -20 to 10 mV. The
transient outward current is characteristic of the A-type current (IA), which is prominently
observed at a more hyperpolarized holding voltage (Sakakibara et al., 2005;Staras et al., 2002).
IA is highly sensitive to 4-AP potassium channel blocker. Bath application of 4 mM 4-AP
resulted in a 93 ± 12 % (n=5; Figure 5.3 D2). The I-V relation indicates current activation at
approximately -20 mV (Figure 5.3 D1). During depolarization, IA contributes to over 85% of the
fast outward voltage-dependent component (Figure 5.3 D1). These observations suggest that
both IKv and IA represent major components of the voltage-dependent potassium current during
RPeD1 action potential firing. Calcium conductance is one of the major inward current in RPeD1
neurons. My previous recordings under Na+ free bath solution indicate a residual transient
inward current (Appendix A2), suggesting a voltage-dependent calcium component. In order to
isolated calcium currents, recordings were carried out in Na+-free bath solution with 51 mM
TEA and 4 mM 4-AP to block major voltage-dependent potassium currents. Figure 5.4 A1
107
Figure 5.2 Characterization of Na+ currents in the RPeD1 neuron. (A) Typical traces of the transient inward current
(INa(T)). Voltage step protocol was elicited at -50 mV holding voltage (resting membrane potential) (A2) Current-
voltage relation of transient inward Na+ current. (B) INa(T) generated from -50 mV holding to +10 mV is largely
eliminated in Na+ free perfusion.
108
109
Figure 5.3 Characterization of K+ currents in the RPeD1 neuron. (A) Representative current traces recorded from an
isolated RPeD1 neuron under Vh=-60 mV followed by 5mV steps from -80mV to 25mV. (A1) Control recording
indicate a slow activated outward rectifying current that is then blocked by 51 mM of TEA (A2). The subtracted
current shows the blocked slow-activated K+ current (IKv) (A3). (B) Voltage-current relation of the stead-state
current from the control (A1), TEA-insensitive current (A2), and TEA sensitive IKv (A3). (B2) Percentage block of
sustained outward current at 10 mV. (C) Representative current traces recorded under Vh=-100mV followed by
5mV steps from -80mV to 35mV. (C1) Control recorded at -100 mV indicate a fast-activated outward transient
current that is senstive to 4 mM of 4-AP (C2). The Subtracted current indicate an fast-activated transient outward
component that is similar to A current (C3). (D) Voltage-current relation of the transient outward current showing
the control (C1), 4-AP insensitive (C2), and 4-AP sensitive IA type K+ current (C3). (D2) Percentage block of
transient outward current at 10 mV.
showed a large inward current at holding voltage of -100 mV. This current is activated between -
70 mV to -60 mV and peaks at 14.9 ± 5.3 mV (n=7). Recording at holding voltage of -50 mV
revealed a small reduction in amplitude with the activation between -45 mV to -40 mV with a
peak amplitude at 16.3 ± 10 mV (n =7). Peak voltage between -100 mV and -50 mV holding
potential was not significantly different (p>0.05), suggesting the major contributor to the peak
calcium conductance is the high voltage-activated calcium channels. Subtraction between the -
100 mV and -50 mV holding voltage revealed a small low voltage-activated current, with peak
amplitude of -10.0 ± 3.1 mV (Figure 5.4 A2). These findings were similar to calcium currents
recorded by in RPeD1 neuron (Onizuka et al., 2005).
In the previous chapter, I identified a voltage-dependent hyperpolarizing activated current (Ih) in
isolated RPeD1 (Figure 4.2). Voltage of activation for an inward rectifying Ih is between -55 mV
to -60 mV. The functional importance of this conductance to action potential firing is unclear as
it is largely inactive at resting membrane potential. Further evidence was identified in current-
clamp recording with hyperpolarizing step current injections (Figure 5.5 B). Holding membrane
potential at -45 mV, hyperpolarization to -70 mV, -80 mV and -90 mV did not produce
noticeable depolarization “sag”, suggesting Ih does not contribute to membrane potential
depolarization at these voltages. Hyperpolarizing membrane potential from -45 mV to -105 mV
produce a large depolarizing “sag”, resulting in a membrane depolarization of ~8 mV. These
findings suggest that although voltage-dependent activation of Ih is a component identified in
110
RPeD1, it may not be a major contributor current at physiological pacemaker membrane
potential range.
Figure 5.4 Characterization of Ca2+
currents in the RPeD1 neuron. (A1) Typical voltage-gated Ca2+_
current
recorded from RPeD1 at various holding potential. Step voltage protocol is applied at 10mV interval from -80mV to
70mV. Holding voltage at -100mV would activate both high voltage activated (HVA) currents and low voltage
activated (LVA) currents. Holding voltage at -50mV would inactivate the LVA component leaving only the HVA
current. Subtracting current from Vh at -50mV from -100mV would provide the LVA Ca2+
current. Future
recordings will be made with HVA and LVA specific channel blockers. (A2) voltage-current density relation of
RPeD1 Ca2+
currents.
5.6.3 Simulated RPeD1 action potential profile correctly represents ones from recording
Having established the major ionic currents of RPeD1 neuron, I then worked in collaboration
with Kostelecki W. to develop a simulation of RPeD1 action potential activity, based on the
Hodgkin-Huxley model. Previously, the Bungay and Campbell (2009) model has been generated
to simulate RPeD1 activity. We have further modified ion current parameters using our
experimental observed parameters to better reflect the action potential profile from our current-
clamp recordings. Parameters for the voltage-dependent Na+, Ca
2+, hyperpolarizing-activated,
111
Figure 5.5 Characterization of hyperpolarization-activated current in the RPeD1. (A) A typical inward rectifying
current recorded from RPeD1 following the voltage-step P/4 protocol under naïve control condition. (B) Presence of
a ‘depolarizing sag’ suggesting a Ih current. Four hyperpolarizing step current pulses on a current clamped isolated
L. stagnalis RPeD1. Under 0 pA resting potential is at -45 mV. With larger increasing negative current, the
membrane potential became more hyperpolarized.
delay rectifier and transient K+ currents were all input into the model from experimental. The
parameters used to generate our simulated RPeD1 are presented in Table 5.1. The respective
current profiles during action potential stimulation are shown in Figure 5.6 B and C. In order to
112
quantify the natural variations of spontaneously generated action potential, we determined the
mean of the root-mean-square difference between all 1 second windows of recorded spikes
(center on the spike peak) was 2.63 mV based on 223 different spontaneous action potential
spikes (Figure 5.6 D). Experimental spontaneous spike frequency was 1.17 Hz. When a
frequency-matched simulation was produced, simulated spikes exhibited little error when
compared to real data rhythmic oscillations (Figure 5.6 A). Analysis of the mean RMSE for 75
simulated spikes showed a value of 2.48 mV (Figure 5.6 E). The tight distribution between one
simulated spike and all real spikes suggests a low enough error that a simulated spike is more
similar to all real spikes than the two most dissimilar spikes.
Figure 5.7 Simulated action potential fitted to natural variation of the spontaneous action potential recorded in
isolated RPeD1 neuron. (A) Mean recorded action potential with standard deviation at each point in time (black)
with overlaid simulated action potential (red). (B) Voltage gated currents corresponding to the simulated action
potential in (A). (C) Leak currents and hyperpolarizing activated current corresponding to the simulated action
113
potential in (A). (D) Histogram of root-mean-square-error (RMSE(I,j)
) between all combinations of recorded action potentials. (E) Histogram of RMSE
(I,j) between all recorded action potentials and the simulated action potential from
(A). Mean RMSE(I,j)
is in (D) and (E) is indicated with a vertical dotted line.
5.6.4 Spiking activity is more sensitive to Na+ leak current compared to K+ leak current.
Having established that our model accurately reflect experimental RPeD1 action potential
profile. We then investigated how variation of Na+ leak and K
+ leak affect spontaneous action
potential activity. The leak current was separated into its component current of Na+ and K
+.
Simulations were generated from variation of Na+ leak against constant K
+ leak or variation of
K+ leak against constant Na
+ leak. Relation of spike rate versus Na
+ and K
+ leak conductance is
presented in Figure 5.7 C and D, respectively. Simulation indicates partial background Na+ leak
is necessary for spontaneous activity. A Na+ leak of 0.24 nS was necessary for spontaneous
action potential firing. Beyond the threshold of Na+ leak conductance, spontaneous firing rate
increased drastically with a continuing increase in Na+ leak conductance. Maximum firing rate of
the simulated neurons was approximately 10 Hz at which point, simulations fail to exhibit the
expected spontaneous rhythmic spikes. In contrast, no K+ leak produced firing frequencies of 5.8
Hz. Increasing K+ resulted in a gradual reduction in spike frequency, untilreaching 1.7 nS at
which point no action potential firing was observed. Summary of spike rate sensitivity to change
in gNaL (Figure 5.7 C) and gKL (Figure 5.7 D) indicate a higher sensitivity (Hz/nS) to Na+ leak
than K+ leak. Relation of gNaL and gKL to spike rate is summarized in Figure 5.7 E, which
indicate the high spike rate sensitivity to gNaL versus gKL is linearly conserved. This suggests
high sensitivity of Na+ is observable, even at low gKL.
5.7 Discussion
The results from this chapter indicate the importance Na+ leak current contributing toward the
generation of spontaneous activity of an rCPG pacemaker neuron. Although U-type knockdown
affects a large component of the sub-threshold current (Chapter 4), action potential profile and
voltage-dependent current were not significantly affected. This suggests that primary function of
U-type channel is to regulate sub-threshold conductance and modulate pacemaker cell activity at
114
Figure 5.7 Rhythmic spiking during RPeD1 simulations using different settings for gLNa (A) and gLK (B).
Corresponding spike rates for the range of simulated conductances are shown in C, D (blue), and E. Spike rate
sensitivity to changes in conductance are shown in C and D (green).
or near threshold. I was also able to record major voltage-dependent Na+, Ca
2+, K
+ and
hyperpolarizing-activated currents. Using measurements gathered from experimental voltage-
clamp recordings, Kostelecki W and I were able to generate and fine-tune a computer simulated
RPeD1 model that accurate reflect experimental observations. Investigation of the Na+ leak and
K+ leak component of the RPeD1 model revealed higher sensitivity of RPeD1 spike frequency to
Na+ leak than K
+ leak conductance, further supporting the importance of Na
+ leak through U-
type channel regulation pacemaker activity.
My recording of leak current from isolated RPeD1 indicated at sub-threshold voltage (below -40
mV), linear current represent the majority of background conductance (Figure 3.1). At -60 mV,
background Na+ current amplitude represents approximately 32% of total background current
(Appendix A3). Slope conductance of the Na+ leak from control is 3.88 ± 0.17 pS/pF (n=8,
115
Appendix A3), which is less than 1/3 of the total leak current (12.21 ± 0.92 pS/pF, n=7; Figure
3.1 B). This suggests the major linear conductance is carried by K+ component. The finding that
higher sensitivity of Na+ leak regulating action potential frequency successfully explains my
results from Chapter 4, which indicates that knockdown U-type channel reduced Na+ leak current
resulted in complete absence of spontaneous activity (Figure 4.1 A). This higher sensitivity is
largely dependent on the electrochemical gradient of Na+, which has a reversal potential in our
simulation at +50 mV. Under physiological conditions, intracellular often [Na+] fluctuates during
high frequency simulations (Turner and Stuenkel, 1998;Zhong et al., 2001). Under pathological
conditions, intracellular [Na+] increases has been reported in neurons during hypoxia and anoxia,
resulting in anoxia-dependent depolarization (Banasiak et al., 2004;Friedman and Haddad,
1994;Fung et al., 1999). Intracellular [Na+] also fluctuates during traumatic insults (Bauer et al.,
1999;Schwartz and Fehlings, 2002). Many of these conditions are characterized by abnormal
changes in excitability contributing to spiking activity irregularities, and eventually cell death.
The high sensitivity of Na+ leak to spike rate indicates that it represents a highly robust
mechanism of regulating neuronal firing.
This model of RPeD1 is currently the most accurate simulation of the pacemaker neuron of
RPeD1, due largely to the conductance-based approach and fine tuning based on experimental
observations. Compared with the Bungay and Campbell (2009) model of the RPeD1 neuron, this
model accurately generated high frequency action potential trains with a shape closely reflecting
the spontaneous spikes from isolated neurons. In addition, results from this model sufficiently
explain findings from my previous chapter, providing insight on the importance of Na+ leak
toward spiking activity. Comparison with other pacemaker neuron simulations (Toporikova and
Butera, 2011), this is the first model to dissect out the leak component and present strong
evidence of the functional effect of Na+ leak, despite its smaller proportional conductance to K
+
leak. Traditionally, modeling of leak current has been largely attributed K+ permeability with
smaller role given to Na+. Koizumi and Smith (2008) model of the respiratory rCPG network
indicate bursting generation depends on subthreshold conductance of INaP and ILeak. Component
of leak conductance includes a large IK and small INa, found largely from the -65 mV reversal
potential of ILeak (ELeak). Other models also describe large IK Leak in simulation of IL component
(Toporikova and Butera, 2011). Based on evidence observed from this study, it would be great
116
interest to re-examine spontaneous rhythmic activity of other pacemaker models with separate
INa Leak component of IL.
Results from performing U-type knockdown indicate U-type channel does not affect action
potential profile. It is not clear whether NALCN knockouts in mammals change action potential
profile. NALCN conducts a regulated leak current whose permeable ions includes Na+, K
+ and
Ca2+
(Lu et al., 2007;Lu et al., 2010). Although gating of endogenously expressed NALCN
channels is currently unknown, partial permeability of NALCN to other cations suggest possible
role in regulating intracellular signaling event as well as activation of Ca2+
-dependent ion
channels. Ca2+
permeability in non-selective cationic TRP channels have been implication in
regulating cell depolarization and activation of Ca2+
-dependent mechanisms (Launay et al.,
2002). It is possible NALCN has a similar importance in biological system. Knockouts of K2P
channels in mammals often produce abnormal action potential shapes. TASK-3 knockouts
produced action potential of broad shape and reduced overshoot in cerebellar granule neurons
(Brickley et al., 2007a). TASK-1 knockouts did not produce noticeable changes in action
potential profile; however it was attributed to the compensation with upregulation of TASK-3
(Aller et al., 2005). TRESK mutant dorsal root ganglia neurons showed large after-
hyperpolarization amplitude and decreased duration of action potential (Dobler et al., 2007).
Based on these studies, it would not be surprising if NALCN regulate action potential shape in
mammalian system. NALCN and U-type channel form an important component to the sub-
threshold conductance. Although not examined in this study, the importance of U-type channel
would possibly suggest roles in regulating after-hyperpolarization profile and how it regulates
sub-threshold activation of action potential. Future studies could directly address these questions.
5.8 Acknowledgement
The work has been supported by an NSERC Discovery Grant to Z.P.F. NSERC – PGS D3
scholarship awarded to T.Z.L.
117
Chapter 6
Identification of a Novel Background Sodium Current Contributing
to Pacemaker Generation in Adult Mouse Sinoatrial Node
Cardiomyocytes.
Author contributions:
Dr. Feng and I both worked in conceptualizing, developing and designing the experiments. I
performed all the electrophysiology recordings and analyses. As well I performed all qPCR
experiments as well as analysis for western blot. Dr. Feng provided the
materials/reagents/analysis tools for electrophysiology analysis.
118
6 Identification of a Novel Background Sodium Current Contributing to Pacemaker Generation in Adult Mouse Sinoatrial Node Cardiomyocytes.
6.1 Abstract
Pacemaker activity of the sino-atrial node (SAN) requires complex and dynamic ionic currents
that are carried through diverse groups of ion channels. Unlike non-pacemaking cells of the right
atrium, SAN cardiomyocytes require high background sodium current to establish the highly
depolarized resting membrane potential necessary for pacemaking generation. While HCN has
been considered as a major contributor to rhythmic activities, it does not fully explain the
relatively more depolarized resting membrane potential in SAN cells. Recent studies in the
nervous system have identified NALCN (Na+ leak current, non-selective) as a candidate
background Na+ conductance. My work in Lymnaea stagnalis showed that knockdown of a
NALCN orthologue reduced neuronal background Na+ current of a pacemaker cell and
abolished/altered spontaneous activity due to hyperpolarization of resting membrane potential.
Tissue expression profile indicates NALCN gene is highly expressed in the heart. In this study, I
hypothesizes that NALCN-like current is similarly involved in pacemaker generation in the SAN
cardiomyocytes. To test this hypothesis, I carried out patch-clamp recordings and used
pharmacology to isolate the NALCN-like current in isolated adult mouse SAN pacemaker cells.
The data suggests that a NALCN-like Na+ current contributes to the membrane potential of SAN
pacemaker cardiomyocytes. I further studied the properties of the channels using recombinant
DNA approach. My findings indicate that functional NALCN channels required coexpression of
auxiliary subunit in tsA201 cells. Taken together, my results provided the first identification of a
novel current involved in pacemaker generation of SAN cardiomyocytes.
6.2 Introduction and rationale
Adult heart beat largely originates from the spontaneously generated rhythms of the sinoatrial
node (SAN). In mammals, pacemaker activity of heart originates from a specialized group of
cardiomyocytes found in the SAN region that are capable of frequent burst of electrical
oscillations. The intrinsic ability of pacemaking has been correlated to the dynamic interplay of
membrane ion channels (Mangoni and Nargeot, 2008). The mechanisms that perpetuate
119
pacemaker activity have continued to be a topic of intense investigation, however, much less is
known about the background currents that are necessary to establish pacemaker rhythm.
Fundamentally, the resting membrane potential is highly depolarized in SAN cardiomyocytes in
comparison to adjacent atrial myocytes. This was largely attributed to the differences in the
background currents, specifically the background Na+ current (Hagiwara et al., 1992). However,
the identity of the major ion channels contributing to the background Na+ current is currently
unclear (Mangoni and Nargeot, 2008).
Recently, a novel ion channel characterized and described in neuron, pancreas and GI tract has
been implicated to regulating cell excitability (Kim et al., 2012;Lu et al., 2007;Lu et al.,
2010;Swayne et al., 2009). Termed NALCN (Na+ leak, non-selective) channel, it is a newly
described non-selective cationic channel that is characterized by voltage-independent activation
and non-activating current profiles. NALCN contributes to the sodium leak current responsible
for regulating the resting membrane potential of neurons (Lu et al., 2007). Mice pups of NALCN
knockout do not survive past 24hrs postnatal, indicating the importance of NALCN for animal
viability. My earlier work has identified that NALCN orthologue in a molluscan model
pacemaker neuron is a major component of sub-threshold conductance contributing to the
background Na+ current, which is necessary to depolarize resting membrane potential to
pacemaking level (Chapter 4 and 5). Mammalian expression profile identified high NALCN
gene expression in cardiac tissues as well as numerous excitable tissues (Lee et al., 1999;Swayne
et al., 2009), however, the functional role of NALCN in the heart has yet to be investigated.
6.3 Hypothesis
I hypothesize that NALCN conductance is involved in establishing pacemaker activity and is
unique from other known background Na+ ion channels in the SAN cardiomyocytes.
6.4 Specific Aims
Due to a lack of appropriate genetic models to investigate NALCN function in the adult heart, I
approached this study through a pharmacological approach. Our objectives are as follows:
1. Describe pacemaker activity SAN cardiomyocytes.
120
2. Characterize Na+ leak current necessary in establishing pacemaker activity.
3. Test whether Na+ leak current is sensitive to known NALCN blockers in combination
with effective blockers of other known background Na+ channels.
4. Profile NALCN and its subunits expression between pacemaker and non-pacemaker
regions of the heart.
6.5 Materials and Methods
In this study, we investigated the presence of NALCN-like conductance in isolated SAN
cardiomyocytes through a pharmacological approach combined with electrophysiological
recordings. We also profiled the cardiac regional expression of NALCN and its major associating
subunits to correlate expression pattern with electrophysiology observations.
6.5.1 Solutions and Chemicals
Dissection Solution (in mM): 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 1.8 CaCl2, 1 MgCl2, 5.55 D-
glucose, 5 HEPES, pH to 7.4 with NaOH.
Low Ca2+
and Mg2+
free (in mM): 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 0.2 CaCl2, 50 taurine, 18.5
D-glucose, 5 HEPES, 1 mg/mL BSA, pH to 7.2 with NaOH.
Modified Kraftbrühe (KB) solution (in mM): 100 K-glutamate, 10 K-aspartate, 25 KCl, 10
KH2PO4, 2 MgSO4, 20 taurine, 5 creatine monohydrate, 0.5 EGTA, 20 D-glucose, 5 HEPES, 1
mg/mL of BSA, pH to 7.2 with KOH.
Na+ free solution for cardiomyocytes contained 140 mM N-methyl D-glucamine (NMDG)
substituting for sodium ions. Na+ free solution for tsA201 cell culture recordings contained either
150 mM NMDG+ or Tris
+ substituting for sodium ions. GdCl3 (10 µM) and CoCl2 (1 mM) were
used to block leak currents (Lu et al., 2007). 10 µM of If-specific blocker, ZD7288 were used.
300 µM of intracellular spermine, which is 5X concentration of TRPM4 IC50 (Nilius et al.,
2004), was used to block TRPM4 currents in isolated SAN cardiomyocytes.
121
6.5.2 SAN region identification
Adult SAN region is identified both based on location and electrophysiological characteristics of
spontaneous contracting cardiomyocytes. We identified SAN region based on regional
boundaries demarcated by the cristea terminalis, the superior and inferior vena cava and the
interatrial septum (Figure 6.1). We also performed sharp electrode recordings in situ to record
the spontaneous action potential firing of the intact SAN with RA. We compared the differences
in action potential profile with those known in the literature and confirmed that are regional
demarcations are in-line with current knowledge of SAN region.
Figure 6.1 Characterization of electrical properties of SAN and right atrial (RA) cardiomyocytes. (A1) Location of
SAN region in intact heart tissue. (A2) Intracellular sharp electrode recording of intact SAN and right atrial regions.
3 M KCl pipettes were used to impale cardiomyocytes and measure spontaneous action potential activities. SAN
region myocytes have slower upstroke and reduced amplitude compared to atrial region myocytes. CT: cristae
terminalis, IVC: infereior vena cava, SVC: superior vena cava, RA: right atrium.
122
6.5.3 Animals and cardiomyocytes isolation
All experimental animals were used in according accordance with guidelines approved by the
University of Toronto Animal Care Committee. Beating hearts were dissected out from pregnant
adult (11-13 weeks) female Swiss white mice and immediately submerged into pre-warmed
(37°C) dissection solution. SAN and RA regions were identified (Figure 6.1), excised and cut
into strips. The tissue strips were then soaked and rinsed with low Ca2+
and Mg2+
free solution,
which was then followed by 30 min enzymatic digestion at 37°C in solution containing: 226
U/mL collagenase type II (Worthington), 1.9 U/mL elastase (Worthington) and 0.6 U/mL
protease type XIV (Sigma) dissolved in low Ca2+
and Mg2+
free solution. Digested strips were
transferred to modified KB solution and manually agitated using a wide-boar pipette. Dissociated
cells were readapted to physiological Ca2+
concentration by incremental increases in Ca2+
concentration using Tyrode’s soltuion (with 1mg/mL of BSA). Cells were then plated onto a
35mm gelatin-precoated dish for recording.
6.5.4 Plasmid preparation
DNA plasmids pcDNA-RnNCA and pcDNA-Unc79-2 (Figure 6.2) were amplified and collected
for use in culture transfection. Both plasmids were a generous gift from Dr. Terrance P. Snutch
(University of British Columbia, Michael Smith Laboratories, Department of Psychiatry,
Neuroscience Division Zoology Biomedical Research Centre). DNAs were transformed with E.
coli cells (DH5α, Invitrogen) as per the manufacturer’s instructions. Briefly, 5 µL of plasmids as
well as pcDNA3 controls were added to 50 µL of DH5α cells in a 15 mL tube and gently mixed.
The solution was kept incubated on ice for 15 mins. The tubes were then placed on a heated
water bath at 42°C for 90 sec and immediately transferred on ice to be incubated for 2 mins. The
solution was then diluted by add 1 mL of room temperature LB medium (1 L solution contains:
10 g tryptone, 5 g yeast extract, 5 g NaCl, 1 mL 1M NaOH). The tubes were shaken at 37°C for
1 hr. Collection of the culture was done by spinning down the tube at 3,000 RPM for 3 mins and
the supernatant (~700 µL) was discarded. The remaining culture was spread onto LB plate
containing 50 µg/mL ampicillin and grown overnight at 37°C.
The next day, discrete colonies were grown due to uptake of ampicillin resistant pcDNA3.
Picked colonies were placed in a 200 mL LB medium containing 50 µg/mL of ampicillin and
123
Figure 6.2 Vector constructs map for pcDNA3-RnNCA and pcDNA-Unc79-2.
124
continued to incubate overnight at 37°C with shaking at 240 RPM. The next morning, maxipreps
were carried out as per manufacturer’s instructions (PureLink HiPure Plasmid DNA Purification
Kits; Invitrogen). DNA quantity and purity was confirmed with spectrophotometry.
6.5.5 Cell culture and plasmid transfection
Cultures of tsA201 cells were obtained from a frozen stock. Cells were maintained in DMEM
(GIBCO 11995) + 10% FBS (Sigma F1051) + 1% Penecilin (GIBCO 15070063) at 37°C and 5%
CO2. 6 hours prior to transfection, tsA201 cells were plated onto 35mm dish at 60% confluency
with 1.5 mL growth medium with antibiotics. Cells were divided into 5 groups of naïve control,
transfection control, eGFP control, rat NALCN only or NALCN+UNC79 transfection groups
(Table 6.1). Mock transfections were performed using Polyethylenimine (PEI, Sigma P3143)
only or with the addition of pcDNA3-eGFP plasmid. NALCN (pcDNA3-RnNCA) and
NALCN+UNC79 (pcDNA-Unc79-2 and pcDNA-RnNCA) plasmids were also introduced with
PEI following manufacturer suggested instructions.
Table 6.1 Transfection treatment conditions for tsA201 overexpression study.
125
6.5.6 Electrophysiology
Signals were recorded and amplified with a personal computer equipped with Clampex 9.2
(Axon Instruments) and MultiClamp 700A connected to Digidata 1322 digitizer, respectively.
Data were filtered at 1 kHz (−3 dB) using a 4-pole Bessel filter and digitized at a sampling
frequency of 2 kHz. All the recordings were performed at room temperature (~22C).
6.5.6.1 Voltage-clamp recordings
Whole-cell patch clamp recordings (ruptured) were performed on transfected tsA201 cultures or
isolated cardiomyocytes of SAN and RA as described previously (Mangoni and Nargeot, 2001).
Microelectrodes of ~5 MΩ filled with either tsA201 pipette or cardiomyocytes pipette solutions.
Bath solutions were perfused onto the cells using a gravity-driven setup.
6.5.6.2 Current-clamp recordings
For in situ recordings of intact SAN and RA tissue, we used sharp electrode intracellular
recordings. The electrodes were filled with 3 M KCl and either SAN region or RA region was
impaled. Recordings and measurements were taken approximately 5 minute following
impalement of the cell. All recordings were made in Tyrode’s solution at room temperature. For
in vitro recording of isolated SAN and RA cardiomyocytes, we used perforated patch clamp
recordings to monitor spontaneous voltage oscillations. 50 μL of 20 mg/mL Amphotericin B
dissolved in DMSO stock is added to 5mL of pipette solution to a final concentration of 200
μg/mL. Successful access were achieved between 10-15 minutes with access resistance of <15
MΩ.
6.5.7 Real-time quantitative PCR (qPCR)
SAN, RA and left ventricle (LV) tissues were dissected and total RNA extraction was performed
following a modified Trizol method (Invitrogen). First strand synthesis of cDNA was conducted
using SuperScript III reverse transcriptase (Invitrogen) with Oligo(dt)20 Primer (Invitrogen) in
total volume of 20 µL for 1 µg of total RNA. Serial dilutions of 1 µL, 0.333µL, 0.111 µL, 0.037
µL, and 0.012 µL cDNA using 5 µL of SYBR GreenER Reagent System (Invitrogen), 1 µL of
appropriate primer set (Table 6.2) and appropriate volume of DEPC-treated water to a reaction
volume of 10 µL. Individual cDNA samples were amplified using the following temperature
126
profile: 50°C for 5 min and 95°C for 10 min, followed by 40 cycles of 95°C for 30 seconds and
55°C for 30 seconds. Ratiometric target /GAPDH control transcript levels were analyzed by
performing Ct vs Ct plot analysis.
Table 6.2 Primer sequences of NALCN, GAPDH, UNC79 and UNC80.
6.5.8 Western blotting
Excised tissues from SAN, RA and LV were lysed with lysis buffer containing (50mM Tris HCl
pH 8.0; 150mM NaCl; 1% NP-40). 15 µg of solubilized proteins were loaded and separated on a
5-8-12% Tris-glycine gel. Proteins were transferred to a nitrocellulose membrane that was
washed and incubated with either 1:200 anti-NALCN (NeuroMab, clone N185/7) or 1:10000
anti-GAPDH (Sigma, G8795-25UL) at 4°C overnight. Secondary blot with Gt×Ms
IgG(H+L)HRP (Millipore AP124P). Blot was developed using Western Lightning Plus-ECL
(PerkinElmer NEL105001EA).
127
6.5.9 Data analysis
As described in section 3.4.
6.6 Results
6.6.1 Background Na+ leak current in isolated SAN cardiomyocytes depolarizes resting membrane potential and is essential for spontaneous pacemaker firing.
When cardiomyocytes of the SAN and RA were compared in isolation, SAN cardiomyocytes
were smaller, spiral-shaped, deficient in myofilaments, and spontaneously contract when
immersed in physiological Tyrode’s solution (Figure 6.3 A). The spontaneous contraction of the
SAN cardiomyocytes correspond to the spontaneous action potential activities observed
following current-clamp recording using perforated patch (Figure 6.3 B). Under similar
recording condition of RA cardiomyocytes, spontaneous activity was not observed. The lack of
spontaneous activity was also characterized by a more hyperpolarized resting membrane
potential compared to SAN cardiomyocytes. The resting membrane potential (RMP) of SAN
pacemaker cardiomyocytes (-45.1±2.2 mV, n=7,p<0.05) was significantly more depolarized than
RA cardiomyocytes (-73.3±0.9 mV, n=5;Figure 6.3 C). Spontaneous action potential firing in
RA cardiomyocytes would be induced following a DC current injection (Figure 6.3 B),
indicating that these cells were viable.
Background Na+ conductance has been described as the major contributing background current
of pacemaker activity in both SAN cardiomyocyte and neurons (Khaliq and Bean, 2010;Kim et
al., 2012). My next approach was to determine whether the Na+ background conductance
contributes toward the significantly depolarized RMP of SAN cardiomyocytes. I replaced Na+
with equimolar of a large, positive charge carrier, NMDG+. Under current-clamp recording, Na
+
free Tyrode’s solution reversibly abolished spontaneous action potential activity of SAN
cardiomyocytes (Figure 6.4 A1) and significantly hyperpolarized the membrane potential with
an average change of -10.7±2.6 mV (n=7,p<0.05;Figure 6.4 A2). In RA cardiomyocytes, Na+
free solution slightly depolarized the membrane potential with an average change of 2.3±0.8 mV
(n=5). I then measured the leak current of both SAN and RA in order to determine whether Na+
was a major ion contributing to the background conductance. Using the recording protocol
128
Figure 6.3 Morphological and electrophysiological properties of SAN and right atrial cardiomyocytes in isolation.
(A) Representative images of isolated SAN myocytes (i and ii) are narrow, spindle-shaped or elongate-shaped cells
that contain reduced myofilaments as compared to larger and myofilament enriched (iii) atiral myocytes. (B) SAN
cardiomyocytes also exhibit spontaneous contraction in isolation, which can be recorded as action potentials. This
spontaneous activity is largely absent in atrial myocytes (0 pA), but can be induced through depolarizing currents (+
20 pA). Dotted lines represent 0 mV. (A3) RMP of SAN pacemaker cells and right atrial cardiomyocytes. Data are
presented as mean ± SEM. * denotes significant difference (p<0.05) from the right atrium.
129
established in Chapter 3, I recorded ILeak between -100 mV to 0 mV in both SAN and RA under
saline and Na+ free saline. A large Na
+ leak component was recording in SAN cardiomyocytes,
characterized by a significant reduction in slope conductance Na+ free saline (14.3±2.1 pS/pF,
n=5,p<0.05) versus that of the control (33.4±4.2 pS/pF,n=5) and shift in reversal potential from -
25.4±4.8 mV (n=5) in saline to -63.0±8.0 mV (n=5,p<0.05) in Na+ free saline (Figure 6.4 B2).
RA cardiomyocytes showed a significant positive shift in ILeak reversal potential between saline
(-112.3±10.6 mV, n=5) and Na+ free (-63.6±3.8 mV, n=5, p<0.05) solution, but slope
conductance (saline: 8.1±0.8 pS/pF, n=5; Na+ free: 6.6±0.5 pS/pF, n=5) was not significantly
altered (Figure 6.4 B2). These results demonstrate that Na+ leak current is a major component
contributing to depolarization of the RMP in isolated SAN cardiomyocytes.
6.6.2 Na+ leak current in isolated SAN cardiomyocytes is sensitive to Gd3+ and Co2+ block.
The molecular identity of the INa Leak is currently unclear, but numerous candidates have been
proposed though not directly investigated. In neurons, NALCN has been identified as a major ion
channel that contributes to the INa Leak (Chapter 4). However, due to a lack of effective adult
NALCN knockout model, targeted genetic investigation proved difficult. Therefore, I
approached this study using a pharmacological approach. NALCN current has been
demonstrated to be partially inhibited by Gd3+
and Co2+
both in over expressed cultures and
endogenously expressed cells (Lu et al., 2009;Lu et al., 2007;Lu et al., 2010;Swayne et al.,
2009). Therefore, I investigated whether application of Gd3+
and Co2+
could inhibit ILeak and its
regulation of RMP (Figure 6.5 A). Addition of 10 M Gd3+
or 1 mM Co2+
has been shown to
effectively block more than 80% of NALCN-dependent current in culture (Lu et al., 2007). In
my observation under current-clamp recordings with perforated patch, pacemaker cells that are
hyperpolarized by Na+ free solution were also significantly hyperpolarized by 10 μM Gd
3+ (-
4.1±1.5 mV, n=5, p<0.05) and 1 mM Co2+
(-4.5±1.1 mV, n=5, p<0.05) compared to RA (Figure
6.5 A2). This sensitivity of membrane potential to Gd3+
(-3.4±1.3 mV, n=5, p<0.05) and Co2+
(-
4.2±1.5 mV, n=5, p<0.05) was conserved even when SAN pacemaker cells were artificially
hyperpolarized to RA cardiomyocyte membrane potential of -70 mV. RA were not sensitive to
Gd3+
(-0.4±0.4 mV, n=5) and Co2+
(-0.3±0.6 mV, n=5) application (Figure 6.5 A2), indicating
an intrinsic difference in currents that regulates RMP between RA and SAN pacemaker cell.
130
Figure 6.4 Na+ leak current regulates resting membrane potential and pacemaker activity in SAN cardiomyocytes.
(A) Current-clamp recording of isolated SAN and RA cardiomyocytes indicates a background Na+ conductance
essential for regulating RMP and establishing pacemaker activity. (A1) Representative traces of perforated-patch
clamp recordings from SAN and atrial myocytes. Black bar indicates switching to Na+ free bath solution and dotted
line indicates 0 mV. (A2) Summary of changes in membrane potential observed following Na+ free perfusion. Data
are presented as mean ± SEM. * denotes significant differences (p<0.05) from the right atrium. (B) Whole-cell
voltage-camp recording of leak currents indicating a large Na+ leak in SAN but not RA myocytes. (B1)
Representative current density traces of SAN and atrial myocytes in both saline and Na+ free bath perfusates. Dotted
lines represent 0 pA/pF current. (B2) Average current-densitiy-voltage (I-V) relations of the leak current for SAN
and atrial myocytes under saline and Na+ free conditions. R
2 value determines the correlation of the linear fit.
131
I then measured the sensitive of Gd3+
and Co2+
application to leak current conductance (Figure
6.5 B). Recording of ILeak in pacemaker cell showed significant reduction in slope conductance of
both Gd3+
(10.7±3.1 pS/pF, n=5, p<0.05) and Co2+
(13.2±2.8 pS/pF, n=6, p<0.05) perfusion
versus those of the ILeak recorded in saline (Figure 6.5 B2). In addition, reversal potential of ILeak
was significantly hyperpolarized to -60.1±18.1 mV (n=5, p<0.05) for Gd3+
and -57.8±12.0 mV
(n=6, p<0.05) for Co2+
(Figure 6.5 B2). Gd3+
(8.6±0.7 pS/pF, n=5) and Co2+
(9.8±0.7 pS/pF,
n=5) did not significantly alter slope conductance of ILeak (Figure 6.5 B2), indicating that RA
cardiomyocytes were not sensitive to both Gd3+
and Co2+
application. These results suggest a
Gd3+
and Co2+
-sensitive current contributes to the depolarized RMP of SAN pacemaker cell,
which is not found in the RA cardiomyocytes.
6.6.3 Gd3+ and Co2+ sensitive Na+ leak current is unique from known background Na+ currents of SAN cardiomyocytes.
In neurons, numerous ionic conductances, including NALCN, contributes to the regulation of the
RMP. Likewise in the SAN pacemaker cells, many ionic currents have been hypothesized to
regulate depolarization of the RMP. Chiefly, the major candidates for the Na+ background
conductance has been the HCN channel conducting If (Mangoni and Nargeot, 2001) and the
TRPM4 channel carrying a non-selective cationic background current at hyperpolarizing
voltages (Demion et al., 2007). I investigated whether Gd3+
-and Co2+
-sensitive component are
unique from these two known components. Using a pharmacological approach, I applied Gd3+
or
Co2+
in combination with effective blockers of HCN (ZD7288) or TRPM4 (spermine)
conductance. Under current-clamp recording, application of ZD7288 at 10X the concentration
that inhibits approximately 44% of If in isolated cardiomyocytes (BoSmith et al., 1993),
produced slowing of pacemaker activity (Figure 6.6 A2) a small reduction in the RMP (-1.4±0.1
mV, n=4; Figure 6.6 A3). Indicating If is partially involved in depolarization and rhythmic
generation of pacemaker cells. When ZD7288 is applied with Gd3+
or Co2+
, spontaneous activity
is abolished (Figure 6.6 A2) and RMP is significantly more hyperpolarized (Gd3+
: -3.6±1.5 mV,
n=4, p<0.05; Co2+
: -2.9±0.6 mV, n=4, p<0.05; Figure 6.6 A3) compared to with only ZD7288
application. This result indicated that although If is involved in rhythmic regulation of pacemaker
activity, regulation of the depolarized RMP is contributed by a component that is independent of
If.
132
Figure 6.5 The membrane potential and leak current of isolated SAN myocytes are sensitive to Gd3+
and Co2+
. 10
µM and 1 mM Co2+
are capable of blocking more than 80% of NALCN conductance in overexpressed cultures (Lu
B et al., 2007). (A1) Representative traces show reversible hyperpolarization of resting membrane potential
following Gd3+
and Co2+
perfusion at resting membrane potential for SAN mycoytes at both resting membrane
potential and at -70 mV, but not RA myocytes. Dotted line indicates 0 mV. (A2) Summary graph of changes in
membrane potential during Gd3+
and Co2+
perfusion. Data are presented as mean ± SEM. * denotes significant
differences (p<0.05) from the right atrium. (B1) Representative leak current recordings of both SAN and atrial
myocytes during saline, Gd3+
and Co2+
perfusions. Dotted lines indicate 0 pA. (B2) Average current-density and
voltage (I-V) relations for both SAN and atrial myocytes during saline, Gd3+
and Co2+
perfusions. R2 value
determines the correlation of the linear fit.
133
TRPM4 has been identified in cardiac pacemaker cells of SAN region (Demion et al., 2007) and
it has been proposed to potentially contribute to background Na+ current (Guinamard et al.,
2010;Mangoni and Nargeot, 2008). In overexpression systems, TRPM4 current is sensitive to
intracellular block of spermine with IC50 of 61 μM (Nilius et al., 2004). In order to block
potential TRPM4 currents, SAN cardiomyocytes were dialyzed with patching pipette loaded with
300 μM spermine (approximately 5X concentration of TRPM4 IC50). Cells were allowed to
dialyze for 5 min prior to recording. Measurement of the ILeak from SAN cardiomyocytes with
spermine showed a small, but not significant reduction in slope conductance (31.0±1.8 pS/pF,
n=10) compared to without spermine controls (33.4±4.2 pS/pF, n=5; Figure 6.6 B2). Application
with Na+ free solution still indicate a significant reduction in ILeak (17.5±1.8 pS/pF, n=5, p<0.05),
suggesting additional component to TRPM4 is contributing to the Na+ leak current. Application
with Gd3+
(21.0±2.5 pS/pF, n=8, p<0.05) and Co2+
(23.5±3.2 pS/pF, n=8, p<0.05) with
intracellular spermine also showed significant reduction in ILeak. Taken together, these findings
indicate that Gd3+
- and Co2+
-sensitive INa Leak is a unique component that is different from known
conductance that contribute to background Na+ current.
6.6.4 NALCN is highly expressed in both SAN and RA, but NALCN subunits expressions are region specific.
The observed results share many similarities with the biophysical and pharmacological
properties of NALCN channel. In order to determine whether NALCN channel could have
contributed to the observations, I tested NALCN mRNA and protein expression of SAN and
compared it to two non-pacemaker regions of RA and left ventricle (LV). By comparing
normalized NALCN expression level with that of the GAPDH control gene, I found that
NALCN expression was significantly higher in both RA (mRNA: 0.0038±0.0008, n=8, p<0.05;
protein: 0.33±0.05, n=5, p<0.05) and SAN (mRNA: 0.0038±0.0005, n=4, p<0.05; protein:
0.22±0.04, n=5, p<0.05) compared to LV (mRNA: 0.0004±0.0002, n=5; protein: 0.042±0.038,
n=5;Figure 6.7 A and B). This suggests that NALCN only expression could not sufficiently
explain our electrophysiology recordings.
Many α pore-forming subunits require auxiliary protein expression for appropriate membrane
localization and functional membrane currents (Bichet et al., 2000;Chien et al., 1995;Pragnell et
al., 1994). Numerous studies have demonstrated that NALCN membrane localization depends on
134
135
Figure 6.6 Gd3+
and Co2+
sensitive background current is unique from known candidates of background current.
(A1) Representative voltage traces of SAN myocytes perfused with HCN blocker (ZD7288) and with either Gd3+
or
Co2+
. Dotted lines indicate 0 mV. ZD7288 reduces HCN current in SAN myocytes by 44% at 0.1 µM (BoSmith S et
al., 1993). (A2) Average action potential frequency of SAN pacemaker cells recorded under ZD7288 perfusion and
ZD7288 with Gd3+
or Co2+
. (A3) Average voltage difference between perfusion with ZD7288 and ZD7288 with
Gd3+
and Co2+
. Data are presented as mean ± SEM. * denotes significant (P<0.05) difference between ZD7288
perfusion only. (B1) Representative leak current traces of SAN in control versus intracellular dialyzed with 300 μM
spermine under saline, Na+ free, Gd
3+, and Co
2+ perfusions. Intracellular block of TRPM4 with spermine have an
IC50 of 61 µM (Nilius B et al., 2004). (B2) Average current density and voltage (I-V) relation comparing control
with 300uM spermine in Saline, Na+, and with Gd
3+ and Co
2+ perfusions. R
2 value determines the correlation of the
linear fit.
expression two highly conserved subunits, UNC79 and UNC80 (Chen et al., 2010;Lu et al.,
2010;Yeh et al., 2008). I then investigated whether UNC79 and UNC80 are differentially
expression between SAN and RA regions, which could potentially contribute to differential
regulation of NALCN membrane current (Figure 6.7 C). Using qPCR analysis for both UNC79
and UNC80 mRNA, I found that SAN expression of both subunits (UNC79: (27.99±9.88)x10-5
,
n=6, p<0.05; UNC80: (190.00±44.97)x10-5
, n=6, p<0.05) were significantly higher than the RA
(UNC79: (5.38±0.39)x10-5
, n=6; UNC80: (38.87±5.79)x10-5
, n=5) and LV (UNC79:
(12.88±1.61)x10-5
, n=5; UNC80: (98.24±20.43)x10-5
, n=6). These results suggest differential
regulation of NALCN subunits could contribute to functional membrane current expression.
6.6.5 Reconstituting NALCN-dependent Na+ leak requires UNC79 coexpression.
Having established the differential regulation of NALCN subunits, I then investigated whether
NALCN and its subunit are sufficient to reconstitute the INa Leak in a heterologous expression
system. NALCN and UNC79 were stably coexpressed in tsA201 cell line, which do not
endogenously expressed NALCN. In comparison (Figure 6.8 A), ILeak recorded from NALCN +
UNC79 coexpressed tsA201 culture is significantly larger (10.60±1.10 nS, n=9, p<0.05) those of
NALCN only (0.46±0.04 nS, n=6), eGFP vector control (0.86±0.35 nS, n=4), transfection
control group (0.31±0.11 nS, n=3) and naïve tsA201 cultures (0.78±0.10 nS, n=7). Furthermore,
Na+ substitution in NALCN + UNC79 with both NMDG
+ and Tris
+ showed a significant
reduction (NMDG+: 0.43±0.16, n=9, p<0.05; Tris
+: 0.25±0.22, n=9, p<0.05) in the normalized
136
Figure 6.7 NALCN is highly expressed in both SAN and RA, but NALCN subunits expression is region-specific.
(A1) Average NALCN mRNA expression in left ventricle (LV), right atrium (RA), and sinoatrial node (SAN)
normalized to GAPDH. (B1) Representative western blot stains of NALCN protein (blue box) and GAPDH
expression for LV, RA, and SAN regions. (B2) Average NALCN protein expression normalized to GAPDH for LV,
RA, and SAN regions. (C1) Average Unc-80 mRNA and (C2) Unc-79 expression in LV, RA, and SAN regions.
Data are presented as mean ± SEM. * and † indicate significant difference (P < 0.05) between expression level of
right atirum and left ventricle, respectively.
leak current at -80 mV compared to NALCN only (NMDG+: 1.02±0.19, n=5; Tris
+: 1.02±0.30,
n=5) and eGFP controls (NMDG+: 0.93±0.09, n=4; Tris
+: 1.02±0.20, n=4; Figure 6.8 B1). This
suggests that the NALCN, when expressed with UNC79, carries a large Na+-dependent ILeak.
These results are similar to the observation of a Na+-dependent leak currentin the pacemaker
SAN cardiomyocytes, suggesting the possibility that Na+ leak conductance regulating the resting
137
Figure 6.8 NALCN-dependent Na+ leak current reconstituted in tsA201 overexpressed with NALCN and UNC79.
(A1) Average current-voltage (IV) relations and (A2) representative leak currents recorded from either control and
transfection control tsA201 cells or overexpressed with eGFP, NALCN only, and NALCN + Unc-79. (B1)
Summary of normalized currents under various Na+ free perfusion at -80 mV. (B2) Representative leak currents for
eGFP, NALCN, and NALCN + Unc-79 under saline and Na+ free bath perfusions. Data are presented as mean ±
SEM. * and † indicates significant (P<0.05) difference between eGFP control and NALCN only, respectively.
138
membrane potential of SAN cardiomyocytes could be contributed by functional NALCN
conductance that is dependent on the regional expression of NALCN subunits.
6.7 Discussion
My results show that Na+ leak current is an essential component of the background conductance
that contributes to the highly depolarized resting membrane potential of SAN pacemaker
cardiomyocytes. Large component of the background leak current is sensitive to Gd3+
and Co2+
block, which is a unique component from currently known candidates of the background Na+
current. These biophysical and pharmacological properties are reminiscent to NALCN
conductance, which both mRNA and protein analysis found to be highly expressed in both SAN
and RA. However, high regional distribution of UNC80 and UNC79 in only the SAN region
suggest functional NALCN conductance requires its auxiliary subunit. Functional NALCN-
dependent Na+ leak current required UNC79 coexpession in tsA201 cell line. This is the first
study to investigate the major molecular identity for the background Na+ conductance, which is
essential for regulation of pacemaker activity of SAN cardiomyocytes.
The background Na+ conductance has been previously well characterized in SAN
cardiomyocytes. Early description indicates it as an essential component to pacemaker
generation. In SAN pacemaker cells, Na+ contributes to more than 33% of the background
conductance (Seyama, 1977). Background Na+ current in isolated rabbit SAN pacemaker cells
was reported as 0.73±0.21 pA/pF at -50 mV (Hagiwara et al., 1992), which is similar to my
observed result (0.98±0.49 pA/pF, n=5; Figure 6.4 B2). The exact molecular identity of this
conductance is unknown. Many ion channels and transporters have been proposed to contribute
to the regulation of background Na+ current, these includes the HCN, TRPM4, Nav, Ist channels
and Na+/Ca
2+ transporters (NCX). All of these channels are voltage-gated and INCX is heavily
dependent on intracellular [Ca2+
]. HCN and TRPM4 are not fully inactivated, thus they permeate
a background leak conductance at hyperpolarizing voltages, which were thought to contribute to
background Na+ current. This study demonstrates that HCN and TRPM4 current is insufficient to
account for the background Na+ conductance, as demonstrated by a novel INa Leak that is sensitive
to Gd3+
and Co2+
block.
139
The observed conductance is highly similar to the reported NALCN conductance found in many
neurons (Lu et al., 2009;Lu et al., 2007;Lu et al., 2010), including pacemaker neurons (Chapter
4). A TTX-insensitive Na+ component that regulates membrane potential has been identified in
isolated SAN cells (Sanders et al., 2006b). My findings further support the candidacy of NALCN
as a possible membrane channel that contribute to the background Na+ leak current. However, it
is important to be mindful of the limitations of our pharmacological approach. Gd3+
has been
extensively used in cardiomyocytes to investigate stretch activated cationic conductance (likely
activation of TRP channels) (Caldwell et al., 1998). Besides blocking TRP channels, Gd3+
also
non-specifically block L-type calcium current (Lacampagne et al., 1994) and delay-rectifier
potassium current (Hongo et al., 1997). Co2+
has been extensively used as a non-specific Ca2+
channel blocker, which blocks Ca2+
and Ca2+
-dependent currents (Tsien et al., 1987). TRPM4
has been attributed to Ca2+
-activated monovalent cationic conductance in SAN pacemaker cells
(Demion et al., 2007;Nilius et al., 2003). In this study, 300 µM intracellular spermine was used
to block TRMP4 current, however it is possible that the Gd3+
and Co2+
-sensitive component of
leak current could be contributed by residual TRP channels (Ju et al., 2007). Therefore, direct
evidence of whether NALCN channel is involved in Na+ leak conductance requires further
investigation with targeted immune ion channel block with NALCN-specific antibodies, genetic
knockdown with NALCN-targeted siRNAs or inducible NALCN knockout models.
Regional NALCN expression in cardiac tissue has not been fully described. NALCN expression
in heart was first described by Lee and colleagues (1999). Using northern blot analysis, NALCN
mRNA was found to be highly expressed in cardiac tissues of both rat and human (Lee et al.,
1999). Further analyses by Swayne and colleagues (2009) confirmed high NALCN mRNA
expression in heart using dot blot analysis. In addition, high NALCN mRNA expression was
identified in aorta, right atrium and left atrium. NALCN was not highly expressed in the
ventricles, the ventricular septum and apex of the heart (Swayne et al., 2009). This study
provides further characterization within cardiac SAN expression of NALCN mRNA and protein
and for the first time, NALCN subunit mRNA expression.
Currently, a number of studies indicate that functional NALCN current in non-neuronal cells do
not contribute to background Na+ conductance (Kim et al., 2012;Swayne et al., 2009). In
gastrointestinal pacemaker cells, NALCN contributes to substance P-dependent depolarization of
140
the membrane potential, but current clamp recordings of NALCN knockout animals do not show
significant change in basal membrane potential (Kim et al., 2012). NALCN knockout also did
not significantly alter background current (Kim et al., 2012). Similarly Swayne and colleagues
(2009) were not able to observe the background conductance of endogenous NALCN expression
in Min6 pancreatic-beta cells or overexpressed HEK293 cells. My results provide a possible
explanation, where NALCN-dependent Na+ leak requires both the pore-forming α subunit and
sufficient auxiliary subunits expression. One possibility for this could be that proper NALCN
membrane localization requires its auxiliary subunits. In both invertebrate (Chen et al., 2010;Yeh
et al., 2008) and vertebrate (Lu et al., 2009;Lu et al., 2010) systems, UNC79 and UNC80 have
been demonstrated to be essential for functional NALCN conductance. In overexpression
systems, NALCN conductance also requires UNC79 and UNC80 (Chen et al., 2010;Lu et al.,
2010). Mice with UNC79 knockout exhibit some similar phenotype as NALCN knockout,
suggesting UNC79 is linked to functional NALCN expression (Speca et al., 2010). It is possible
that the UNC79 and UNC80 is involved in membrane trafficking of the pore-forming α subunit,
similar to the functional roles of the cytosolic β subunits of voltage-gated Ca2+
channels (Bichet
et al., 2000;Chien et al., 1995;Pragnell et al., 1994).
Another possibility is the interaction between auxiliary subunit regulating NALCN gating
properties. In neurons, NALCN gating is modulated by UNC80 that forms a scaffold for Src
kinases (Lu et al., 2009;Wang and Ren, 2009). NALCN gating modulation is also regulated by
association with other membrane receptors such as the M3R in pancreatic-beta cells (Swayne et
al., 2009). Differential NALCN association with various auxiliary subunits in cardiomyocytes of
pacemaker and non-pacemaker cells could modulate gating behavior of NALCN conductance.
Currently, the exact gating mechanism and gating properties of the pore-forming subunit is
unclear. Therefore, future single-channel recordings could elucidate mechanisms of NALCN
gating properties.
Lastly, different tissues could exhibit alternative splice variants that produce functionally
different NALCN channels and its associating subunits (Table 6.3). In C. elegans, genomic
NCA-1 (NC_003282) produces numerous splice variants of NCA-1 mRNAs (NM_171352.2,
NM_171353.2, NM_068525.2 and NM_068526.2). Similarly, genomic sequences of NCA-2
(NC_003281) of C. elegans and na of D. melanogaster (NC_004354) also produce multiple
141
splice variants (NCA-2: NM_065653 and NM_001136363; na: NM_001103511 and
NM_167401). Currently, splice variants of NALCN has not been identified in human, mouse or
rat, predicted NALCN mRNA variants were identified in other vertebrate systems (B. taurus).
Splice variants of auxiliary subunits have also been identified. In invertebrate models, both Unc-
79 and Unc-80 exhibit multiple slice variants. In human, two mRNA slice variants of UNC80
have been identified: homologue isoform 1 (NM_032504) and isoform 2 (NM_182587).
Different splice variants of NALCN and its subunits could produce functionally different
proteins complex. These could be regulated differently between different tissues. How different
isoforms are regulated and how they function in NALCN permeability is a subject for future
investigation.
6.8 Acknowledgement
The work has been supported by an NSERC Discovery Grant to Z.P.F. NSERC – PGS D3
scholarship awarded to T.Z.L.
142
Table 6.3 Different splice variant of NALCN, Unc79 and Unc80 subunits in C. elegans, D. melanogaster, M.
musculus, R. norvegicus and H. sapiens.
143
Chapter 7
Results Summary and General Discussion
\
Part of the text presented in this chapter is reproduced with permission from the following
publication:
Lu TZ, Feng Z-P (2012) Molecular Neurobiology 45(3): 415-423.
144
7 Results Summary and General Discussion
7.1 Summary of results
In this thesis, I have identified a molecular constituent that partially contributes to the
background Na+ current of pacemaking cells. In Chapter 4, I identified a novel functionally
expressed ion channel (U-type channel) within the pacemaker neuron of the rCPG pacemaker
neuron of adult L. stagnalis. Using a genetic knockdown approach, I was able to demonstrate
that the U-type channel is a major component that contributes to the background Na+ leak
current, resulting in a depolarized resting membrane potential. Furthermore, pharmacological
and biophysical characterizations of the U-type channel suggest it is a L. stagnalis homolog of
mammalian NALCN channel. Acute knockdown of U-type channel reduced aerial respiratory
behavior, similar to that observed in the NALCN knockout mouse at postnatal level. I then
quantified the importance of Na+ leak in regulating spontaneous spike activity of the rCPG
pacemaker neuron. U-type knockdown indicate insignificant changes to action potential profile,
suggesting it is functionally more important at the subthreshold voltage range. Working in
collaboration with W Kostelecki, we were able to develop an accurate simulation of RPeD1
action potential profile based on parameter inputs from various voltage-dependent currents
recordings. By segregating the leak current into its respective Na+ and K
+ components, I was able
to demonstrate that spike rate is highly sensitive to background Na+ conductance compared to K
+
conductance. These results indicate that despite a smaller ratio of Na+ conductance in many
pacemaking cells, it potentially represents a significant regulator of spike activity.
In my last chapter, I extended my finding of NALCN-like conductance from a pacemaker neuron
and applying them to an establish pacemaker model within the heart. I was able to identify a
novel conductance that is critical for generation of pacemaker activity in isolated adult SAN
cardiomyocytes. This current which is sensitive to both Gd3+
and Co2+
was distinct from
currently known molecular channels for background Na+ current. Expression profile identified
that NALCN was highly expressed, but regional specificity could be regulated by the auxiliary
subunits. Further analysis of the NALCN conductance in overexpressed tsA201 cell cultures
revealed that NALCN-dependent Na+ leak requires UNC79 coexpression. These results suggest a
novel molecular constituent that potentially regulate the depolarized resting membrane potential
of SAN pacemaker cell.
145
7.2 General discussion
7.2.1 NALCN regulation of respiratory rhythm and rCPG neuron activities
In mice, targeted deletion of the NALCN exon resulted in a knockout strain with a lethal
postnatal phenotype characterized by abnormal rhythmic respiratory activity. C4 nerve root
recording, which indirectly measures respiratory network output, showed a substantial reduction
in spontaneous burst activities (Lu et al., 2007). My knockdown study of a NALCN-like U-type
channel in L. stagnalis demonstrated an ohmic NALCN-like conductance is necessary for
spontaneous pacemaker activity. First, knockdown U-type channels using a gene silencing
approach led to a more hyperpolarized RMP, which was sufficient to eliminate the spontaneous
activity in isolated pacemaker neurons of the L. stagnalis respiratory central pattern generator.
Secondly, depolarizing current injection in the U-type knockdown group restored rhythmic
activity to the wild-type, suggesting the U-type channel is involved in regulating intrinsic
membrane properties necessary for spontaneous bursting. Thirdly, biophysical and
pharmacological analysis of the U-type channel conductance showed it conducts a TTX-
insensitive Na+ leak current that can be partially blocked with Gd
3+. This Na
+ leak current is also
sensitive to extracellular Ca2+
. Sequence alignment of U-type channel shows high level of
homology with mammalian NALCN channel. This evidence suggests that U-type channel is a
molluscan NALCN orthologue. Finally, in vivo knockdown of U-type channel resulted in
reduced respiratory output (Figure 7.1). This study was the first to directly test, identify and
characterize NALCN-like current as an essential component to pacemaker neuron activity.
Detailed analysis of the isolated respiratory pacemaker interneuron identified NALCN channel
as an essential component to pacemaker generation, which directly affects respiratory rhythm
output.
How U-type channel affect in vivo respiratory rhythm generation is a contentious question. Since
U-type knockdown used in this study is a global knockdown in whole animal, it is difficult to
gauge which neuron is affecting the decrease in respiratory behavior. Specifically, in situ
recording using semi-intact preparations demonstrated that under normal physiological
conditions, most RPeD1 do not exhibit spontaneous pacemaker activity. This is due to inhibitory
input from the network and from peripherial connections that prevents RPeD1 firing (Bell et al.,
146
2007;Inoue et al., 2001;Inoue et al., 1996). RPeD1 only then fires in bursts following
pneumostome opening, which is a result of combined disinhibition and network feedback.
Pacemaker function is important during the respiratory gas exchanges where activation of the
rCPG is essential in maintain penumostome opening. Dispite RPeD1 firing in burst mode during
pacemaking, high expression of U-type channel facilitates bursting by depolarizing the
membrane potential. Differential U-type channel expression in different rCPG neurons could
also facilitate different rhythmic feedback mechanisms that ultimately maintain respiratory motor
outputs.
Whether NALCN involves regulating pacemaker activity in the mammalian respiratory network
largely remains unknown. However, many studies provide evidence that suggest possible
NALCN-like involvement. In rodent studies, a TTX-insensitive background Na+ current has
been identified in respiratory pacemaker neurons (Tryba and Ramirez, 2004). Furthermore, the
TTX-insensitive current is activated by substance P via a neurokinin 1 receptor (NK1R) in the
preBotC neurons (Pena and Ramirez, 2004). The substance P-activated current is also sensitive
to low-extracellular calcium (Pena and Ramirez, 2004). A portion of the NK1R and somatostatin
expressing neurons in preBotC, have also been shown to be responsible for rCPG rhythmic
activity (Gray et al., 1999c;Tan et al., 2008). These results are similar to the many biophysical
and pharmacological properties of the neuronal NALCN channel (Lu et al., 2009;Lu et al.,
2007;Lu et al., 2010). Although there is evidence to suggest that TRPC channel conducting ICAN
might be the molecular constituent responsible for the substance P-activated conductance (Ben-
Mabrouk and Tryba, 2010), evidence also suggest additional component of substance-P
dependent slow depolarization that are insensitive to ICAN blocker (Pena and Ramirez, 2004).
Given the relative depolarized nature of the RMP in rodent rCPG neurons (from -60 mV to -40
mV), it is plausible that additional Na+ leak serves as an additional mechanism to establish the
bursting activity of the neurons. Bursting in preBotC pacemaker neurons is conditional
depending on the baseline membrane potential (Feldman and Del Negro, 2006). Regulating ILeak
has been identified to be an important cellular mechanism to control baseline membrane
potential, which contributes to rhythm generation in preBotC (Koizumi and Smith, 2008). Since
respiratory pacemaker neurons serve a more important role in driving emergent respiratory
network rhythm in neonatal animals (Brockhaus and Ballanyi, 1998;Gray et al., 1999a;Onimaru
147
Figure 7.1 Working model of U-type conductance contributing to rCPG rhythmic output and respiratory behavior.
U-type channel, NALCN orthologue in L. stagnalis, conducts an inward leak current which combined with
potassium leak channels, regulates the resting membrane potential. Blue cell body represents low activity and red
cell body represents high activity. U-type knockdown hyperpolarizes basal membrane potential of right pedal dorsal
1 (RPeD1), rCPG pacemaker neuron which reduces its intrinsic activity. This results in reduction of excitation to
input 3 interneuron (IP3) and reduction of inhibitory input to visceral dorsal 4 (VD4). This results in a shift in rCPG
network rhythmic output toward pneumostome (respiratory orifice) closing resulting in reduced respiratory behavior
in adult animal. rCPG network model adopted from (Syed et al., 1990;Winlow and Syed, 1992).
148
et al., 1990;Smith et al., 1991), NALCN conductance could be responsible for abnormal rhythms
of respiratory pacemaker neurons observed in the neonatal knockouts (Lu et al., 2007).
7.2.2 NALCN and other subthreshold currents
As demonstrated in this thesis, NALCN contributes to a major component of the subthreshold
conductance. In the pacemaker neuron of the L. stagnalis, I have identified two major
components at subthreshold membrane potentials. These include a voltage-independent leak
current and a hyperpolarizing-activated inward current. At resting membrane potential of
RPeD1, linear current contributes to a larger proportion of background conductance than non-
linear current. Knockdown of U-type channel produced approximately 40% reduction in the
linear current amplitude, suggesting U-type channel contributes to a significant portion of the
background conductance. Current-clamp recordings indicate that U-type knockdown resulted in
approximately 15 mV hyperpolarization of the resting membrane potential, which brought
RPeD1 neurons out of the spontaneous spiking range, resulting in an with absence of
spontaneous activity, without DC current injection. Similar findings were observed in the acute
hippocampal neuron cultures of neonatal NALCN knockouts (Lu et al., 2007). Also, my
observations of the isolated SAN cardiomyocytes, application of Gd3+
and Co2+
, which are
capable of blocking NALCN conductance, were able to abolish pacemaker rhythm. These results
suggest NALCN is an essential component of subthreshold conductance that maintains
pacemaker cells at a membrane potential to allow activation of voltage-gated ion channels
(Figure 7.2).
It is important to note that persistent activation of certain voltage-dependent ion channels can
also contribute to the background conductances. In my U-type knockdown study, partial
reduction of U-type channel expression resulted in approximately 80% reduction of Na+-
dependent leak conductance, with only 40% reduction in the total leak current. This suggests
other currents contribute to the residual conductance observed in my recordings. In mammalian
systems, Ih has very slow inactivation kinetics; thus in neurons expressing high Ih, the current
contributes to a portion of the background conductance necessary to establish resting membrane
potential (Bal and McCormick, 1997;Harris and Constanti, 1995;Maccaferri and McBain, 1996).
In addition, many pacemaking neurons express subthreshold voltage-gated currents that produces
resurgent inward conductance that depolarizes the membrane potential during tonic or bursting
149
activity. These includes the persistent sodium current (Del Negro et al., 2002;Del Negro et al.,
2005;Do and Bean, 2003;Pace et al., 2007), calcium-activated inward non-selective cationic
current (ICAN) (Caeser et al., 1993;Crepel et al., 1994;Del Negro et al., 2005;Lee and Tepper,
2007;Mrejeru et al., 2011;Partridge and Valenzuela, 2000) and subthreshold Ca2+
currents (ICaT)
(Kim et al., 2001;Onimaru et al., 1997). NALCN is hypothesized to be a major contributor of
background Na+ conductance (Lu et al., 2007;Ren, 2011), but other mechanisms exist in neurons
and other excitable cells. NALCN expression changes Na+ leak conductance and sodium balance
(Sinke et al., 2010), which have implication to sodium dependent mechanisms that regulates
membrane potential. For example, Na+-dependent K
+ currents could be greatly affected. This
class of current is poorly understood, but has been identified in numerous tissues. It has been
proposed to regulate many different cellular functions, including hyperpolarization following
burst activities (Franceschetti et al., 2003;Safronov and Vogel, 1996), ischemia (Bhattacharjee et
al., 2003;Dryer, 1994;Dryer, 2003) and locomotion (Dale, 1993). In addition, the significance of
NALCN conductance could vary from neuron to neuron. Knockout studies of potassium leak
channels (TASK-1) indicate that certain cells are able to compensate in order to maintain
background conductance (Aller et al., 2005). Therefore it is important to be mindful that multiple
mechanisms maintain the background conductance and resting membrane potential.
Furthermore, the mechanism of pacemaking is different from cell to cell. Pacemaking drive from
the oscillation of subthreshold currents depends on a combination of voltage-dependent and
voltage-independent currents. In thalamaic pacemaker neurons, Ih and ICaT form the fundamental
conductance that drives pacemaker activity (McCormick and Bal, 1997). In dopaminergic
midbrain neurons, selective coupling of ICaT and ISK channels help generate spontaneous intrinsic
bursting activity and regulate temporal integration of synaptic inputs (Wolfart and Roeper,
2002). In some respiratory pacemaking neurons, INaP and ICaT are important for bursting activity
and enhancement of rhythmogenesis (Pace et al., 2007). Others adopt INaP and IK Leak as a
principle mechanism for pacemaking generation (Koizumi and Smith, 2008). In heart,
pharmacological studies combined with using genetic knockouts of HCN, Cav 1.3 and 3.1 all
suggest a complex interplay of ionic conductance that generates cardiac pacemaker rhythm
(Mangoni and Nargeot, 2008). In adrenal chromaffin cells, tight coupling of Cav 1.3 and BK
channels was found to affect pacemaker firing and action potential profile (Marcantoni et al.,
2010;Vandael et al., 2010). The complex relationship of ion channels that drive spiking is
150
highlighted by the vast array of action potential waveforms produced from excitable cells of
different regions and tissues (Bean, 2007). Therefore, few mechanisms are conserved in
pacemaker cells of various systems. My thesis in combination with other studies, indicate
NALCN and NALCN-like conductance is possibly involved maintaining resting membrane
potential of neuronal and cardiac pacemaker cells; whereas other studies in pancreatic β (Swayne
et al., 2009) and gastrointestinal pacemaker cells (Kim et al., 2012) suggest NALCN is not
involved in regulating background Na+ leak current. These results suggest NALCN function in a
complex mechanism that varies from tissue to tissue.
7.3 Clinical significances
7.3.1 Na+ leak fluctuations
Ion homeostasis is often perturbed under pathological conditions. In pacemaker cells, this change
in ionic concentration can affect spontaneous activity through alteration of firing rate. Na+ is one
of the major ions that regulate neuronal excitability and cardiac contractility. Findings from my
thesis suggest that some pacemaker cells are highly sensitive to background Na+ conductance.
Given that this conductance is largely dependent on the Na+ gradient, altering this can have
significant effect on firing activity of pacemaker cells. As already mentioned, intracellular [Na+]
of neurons is highly affected by oxygen concentration. An increase in intracellular [Na+] results
in depolarization (Banasiak et al., 2004;Friedman and Haddad, 1994;Fung et al., 1999), which is
linked to hyperexcitability and Ca2+
influx that eventually leads to cell damage and death. In
cardiac systems, regulation of intracellular [Na+] has been attributed to Na
+ channels, Na
+/Ca
2+
exchanger, Na/2Cl/K cotranspotrer, Na/HCO3 cotransporter, Na+/H
+ exchangers, stretch
activated channels, and Na+/K
+ pump. Under physiological conditions, high stimulation of
cardiomyocytes produces acute increase in intracellular [Na+] fluctuating by 4-5 mM at 2-6 Hz
of simulation in guinea pig (Wang et al., 1988), rat (Frampton et al., 1991) and rabbit (Cohen et
al., 1982). Under pathological conditions such as hypertrophy and heart failure, intracellular
[Na+] significantly increases in various animal models (Baartscheer et al., 2011;Despa et al.,
2002;Gray et al., 2001b;Jelicks and Gupta, 1994;Pieske et al., 2002;Verdonck et al., 2003).
Increases in intracellular [Na+] is highly linked to delayed afterdepolarizations, due largely to an
increase in sarcoplasmic reticulum (SR) Ca2+
release (Guo et al., 2007;Pogwizd et al., 2001).
This is often observed in forms of arrhythmia.
151
Figure 7.2 Proposed model of NALCN channel function in regulating pacemaker activity. (A) NALCN conductance
is essential in establishing resting membrane potential for spontaneous activity. (B) During action potential firing,
voltage-dependent persistent Na+ current (INaP) depolarizes membrane potential to threshold activating voltage-gated
Na+ current (INav). Rapid depolarization of membrane potential activates voltage-gated Ca
2+ currents(ICav), and
voltage-gated K+ current (IKv). After hyperpolarization is mediated by the Ca
2+ dependent K
+ current (IK Ca),
followed by hyperpolarizing-activated current (Ih) and T-type low voltage activated Ca2+
current (ICaT).
Na+ conductance through NALCN (INALCN) channel is a passive regulator of depolarizing phase of action potential,
which potentially regulates rhythmic spiking. Adopted from (Lu and Feng, 2012)
152
7.3.2 Ion channel regulation
Numerous sources could contribute to increases in intracellular [Na+] in the cardiac system.
These includes a decrease Na+/K
+ pump activity (Verdonck et al., 2003), altered Na
+/K
+ pump
isoforms (Verdonck et al., 2003), increased stretch channel activation due to an increase in cell
volume (Alvarez et al., 1999;Kent et al., 1989;Youm et al., 2005), altered activity of various Na+
exchangers (Chiappe de et al., 2001;Perez et al., 1995;Sandmann et al., 2001) and changes in
Na+ channel isoform expression to increase window current (Alvarez et al., 2000;Huang et al.,
2001;Undrovinas et al., 1999). If NALCN carries a significant portion of the background Na+
leak in cardiac myocytes, it is conceivable that NALCN expression could be significantly altered
under similar pathological conditions. This would raise many interesting questions and
possibilities of how NALCN is regulated; such as the possible functional significance of its
regulation, the molecular subunits that are possibility also regulated, and the regional expression
profile during pathological condition for NALCN and its associating subunits.
Current remodeling is commonly observed in many pathological conditions within the cardiac
system (see review by Nattel et al., 2007). These include chronic cardiac failure (see reviews by
Cha et al., 2004 and Janse, 2004), myocardiac infarctions (see review by Janse and Wit, 1989),
and atrial fibrillations (see reviews by Allessie et al., 2002 and Bosch et al., 1999). Changes to
rhythm generation include altered SAN pacemaking output (Opthof et al., 2000;Zicha et al.,
2005), early afterdepolarization (Li et al., 2004;Li et al., 2002;Nuss et al., 1999), delayed
afterdepolarization (de Bakker et al., 1988;Pogwizd et al., 2001;Vermeulen et al., 1994),
modified action potential duration/profile (Kaprielian et al., 2002;Li et al., 2002;Sah et al.,
2003;Undrovinas et al., 1999) and altered signal conduction throughout cardiac tissue (Gardner
et al., 1985;Spear et al., 1983a;Spear et al., 1983b). These changes have been attributed to
modification of the underlying ionic currents found in various cardiac tissues, including changes
to the quantities of membrane expressed ion channels (Borlak and Thum, 2003), different
membrane expressed isoforms ratios (Gidh-Jain et al., 1995;Gidh-Jain et al., 1998;Munch et al.,
2001;Yang et al., 2000), modifications of channel/transporter activities (Cao et al., 2003;Christ et
al., 2004;Tessier et al., 1999;Vest et al., 2005) and modified protein assembly into protein
complexes (Kurokawa et al., 2004;Maguy et al., 2006;Marks et al., 2002;Marx et al.,
2000;Thomas et al., 2003). Findings from my thesis implicate NALCN as an important regulator
153
of rhythmic activity. Furthermore, its conductance is a major component to neuronal and
possibly cardiac background Na+ current. Its functional properties are highly dependent on
auxiliary subunit expression. These ideas suggest a strong likelihood of NALCN-dependent
regulation under pathological conditions.
7.4 Limitations and future directions
In this thesis, many limitations need detailed consideration, which will direct future
investigations.
7.4.1 Specificity of genetic knockdown
As with most genetic knockdown approach, specificities of the dsRNA need to be considered at
gene, protein, and functional levels. In my study, I used two different 27-mer siRNAs and one
300 bp dsRNA to induce knockdown of endogenously expressed U-type channel. Both real-time
PCR analysis and functional electrophysiological recordings confirmed knockdown affect;
however protein data are noticeably missing. This was largely due to the lack of targeted
antibodies for U-type channel. I have been testing antibodies for the channel throughout the
experimental process. I have made two polyclonal antibodies against specific regions
(ENKGTALLTVDQRRC; TPTQSDGGTRSEKDC) of the U-type channel protein sequence
(Genscript). At the same time, I have also tested the commercially available NALCN antibody
used in the SAN cardiomyocyte study (UC Davis/NIH NeuroMab Facility, N185/7).
Unfortunately, all three antibodies failed to observe any clear signal at the range of the predicted
protein size (~200 kDa). Future approach to resolving the antibodies issue, would involve
production of a targeted antibodies to the U-type channel at a different region that is non-
homologous with other known voltage-dependent ion channels. In addition, other commercially
available NALCN antibodies could also be investigated (Santa Cruz, sc-84518, sc-134937,sc-
84520; Sigma, HPA031958; Abnova, PAB22599, PAB22787, PAB22788; Novcus Biologicals,
NBP1-70397, NBP1-70396).
7.4.2 Pharmacological specificities
In this thesis, Gd3+
and Co2+
were applied to investigate functional roles of NALCN. Both
present their own limitations in specificity. As already mentioned (section 3.3.2.1), Gd3+
is a
154
potent blocker of numerous ion channels. In order to remedy this, I first reduced the scope of
pharmacological investigation to only hyperpolarizing voltage ranges; this limited the
contributing effect of voltage-dependent calcium and potassium currents. In addition, Gd3+
sensitivity to NALCN compared to mechano-sensitive TRP channels is higher in both neurons
and cardiomyocytes. Specifically in the cardiomyocyte study, I used a concentration of Gd3+
that
is below activation threshold of mechanosensitive currents. In previous findings with atrial
myocytes, 100 μM of Gd3+
resulted in a hyperpolarzation of the resting membrane potential of
atrial myocytes (which was not observed in my study), attributed to the inactivation of stretch-
activated currents (Zhang et al., 2000b). Furthermore, 10 μM of Gd3+
was found to be ineffective
in blocking stretch-activated increase in resting calcium, suggesting poor blockade of stretch-
activated channels at 10 μM Gd3+
(Hongo et al., 1997). In my thesis, by using low [Gd3+
] and
investigating leak conductance at hyperpolarizing voltages, I was able to mitigate known
limitations and non-specific properties with Gd3+
as a pharmacological blocker.
Using both Gd3+
and Co2+
, I was able to identify a conductance that has not been described in
SAN cardiomyocytes. However, it is important to be mindful that various TRP channels are also
sensitive to Gd3+
and intracellular Ca2+
, which can be affected by Co2+
block (Demion et al.,
2007;Nilius et al., 2003). As well Na+/Ca
2+ exchanger currents depend heavily on the influx of
Ca2+
, which can be partially sensitive to Co2+
. Although various means have been used to limit
contribution from other known ion channels, it is important to be aware of additional channel
contributing to the observation. Future investigations will employ an immunological approach
using targeted NALCN antibodies to inhibit NALCN-dependent conductance (Huang et al.,
2009), genetic approach using gene-silencing approach of performing acute NALCN knockdown
in the cardiac region (Fechner et al., 2008;Suckau et al., 2009), or conditional knockout approach
using cardiac-specific NALCN knockout in adult mice (Rosati et al., 2011).
ZD7288 is used as a specific pharmacological blocker of If current in isolated murine SAN cells.
However, future studies could investigate how much of the current is blocked by using
activators. HCN channels are particularily sensitive to activation by cAMP and cGMP (Biel et
al., 2009;Yanagihara and Irisawa, 1980). Identification of appropriate HCN channel block could
be confirmed through whole-cell voltage clamp recordings, and addition of cAMP analogue to
155
activate If and monitor how it is blocked. This when combined with Gd3+
and Co2+
could allow
for more detailed separation of currents.
7.4.3 Synaptic alterations
In my U-type knockdown study, global reduction of U-type channel resulted in reduced
respiratory behavior even though U-type knockdown eliminated spontaneous activity of RPeD1
neuron; this suggestion possible compensatory mechanisms within the network to maintain
respiratory output at whole-animal level. In mammalian systems, neuronal respiratory drive
shifts under conditions of oxygen deprevation (Baker and Mitchell, 2000;Blitz and Ramirez,
2002;Bureau et al., 1984;Harris et al., 2006;Neubauer et al., 1990). Network activities that
produce rhythmic output under eupnea, gasping or sighing are inherently different (Paton et al.,
2006;St-John and Paton, 2004;Tryba et al., 2006). Hypoxia induces a biphasic response in whole
animals, characterized by an augmentation of respiratory activity followed by depression
(Bureau et al., 1984;Neubauer et al., 1990;Richter et al., 1991). Continuous acute hypoxia results
in short-term depression whereas episodic intermittent hypoxia results in long-term facilitation
(Baker and Mitchell, 2000;Blitz and Ramirez, 2002). Long-term facilitation in respiratory
activity has been observed in numerous vertebrate models following intermittent hypoxia
treatment (Baker and Mitchell, 2000;Cao et al., 1992;Turner and Mitchell, 1997;Turner and
Sumners, 2002), including humans (Harris et al., 2006). These various respiratory outputs
depend on modulation of rCPG with modulators and network circuitry. My observations suggest
a possible compensatory mechanism exists at the network level in U-type knockdown group.
Two possibilities exist, either U-type channel is directly involved in regulating synaptic
transmission of the network activity, or U-type channel is indirectly involved through regulation
of intrinsic cell properties that alters synaptic output. NALCN and its homologous has been
directly implicated in modulating synaptic transmission through regulation of both presynaptic
release (Jospin et al., 2007;Lear et al., 2005) and post-synaptic responses (Kim et al., 2012;Lu et
al., 2009;Swayne et al., 2009). In addition, NALCN also regulates cell excitability (Lu et al.,
2007;Lu et al., 2010) and signal transduction (Yeh et al., 2008), suggesting it could also be
indirectly involved in synaptic transmission. How U-type channel contribute to synaptic
regulation and network remodeling requires further investigation that is beyond the scope of this
study. Additional experiments investigating U-type knockdown on known membrane-expressed
156
ligand-gated currents, such as GABA and glutamate, could provide evidence to U-type role on
direct post-synaptic responses.
In cardiac system, NALCN could also mediate post-syantpic responses. Current evidence
suggests NALCN conducts a substance P activated current in both neurons (Lu et al., 2009) and
gastrointestinal pacemaker cells (Kim et al., 2012). In pancreatic beta-cells, NALCN interacts
with M3R to conduct an acetylcholine activated inward current (Swane et al., 2009). Both have
been partially identified in the cardiac system. In particular, M3R subtypes have been
extensively investigated; its physiological and phathological importance has been well
documented (reviewed by Wang et al., 2007). In particular, M3R has been identified to activate a
K+ current which is found to be significantly enhanced under atrial fibrillation (Dobrev et al.,
2002). Future investigation could look into whether NALCN and M3R interact in a similar
manner with those found in pancreatic beta-cell. If identified, it could also further provide
evidence of a different gating mechanism as M3R interactin with NALCN prevents background
Na+ conductance (Swayne et al., 2009).
7.4.4 Membrane localization
Functional NALCN conductance requires membrane expression of the pore-forming subunits.
One possibility of lack of Na+ leak conductance in atrial myocytes is the reduced membrane
expressed ion channel of NALCN due to requirement of UNC79 and UNC80 subunit formation.
Numerous studies using endogenously expressed vertebrate (Lu et al., 2009;Lu et al., 2010;Speca
et al., 2010) and invertebrate (Yeh et al., 2008) NALCN suggest membrane localization requires
UNC79 and UNC80. Overexpression systems also suggest functional NALCN conductance
requires UNC79 and UNC80 (Chen et al., 2010;Lu et al., 2010). My results further confirm
NALCN only expression does not produce functional Na+ leak current, which is observed in
NALCN+UNC79 coexpressed system. Since UNC80 and UNC79 drives membrane expression
of NALCN, it is possible that atrial expression of NALCN protein is not localized to the
membrane to produce high Na+ leak current (Figure 7.3). However, mammalian membrane
expression pattern and localization information is currently unknown. In order to confirm
whether NALCN localization at the cellular level is responsible for the lack of Na+ leak current,
future investigation will use immunocytochemistry to determine NALCN localization in SAN
and atrial cardiomyocytes. In addition, confirmation of membrane localization in overexpression
157
Figure 7.3 Proposed working model of NALCN channel in SAN and atrial cardiomyocytes. In SAN pacemaker
cells, high expression of UNC80 and UNC79 drives membrane localization/stablization of NALCN channel. In
addition, interaction between UNC80 and UNC79 with NALCN produce a functional channel complex that conduct
background Na+ leak, resulting in a depolarized resting membrane potential. In atrial myocytes, low background
Na+ leak is observed. This could be explained by a number of reasons. First, low expression of UNC80 and UNC79
possibly reduce membrane expression of NALCN function. Membrane expressed NALCN channels in atrial
myocytes could also be non-conducting channels. Other possibility for low background Na+ leak is the expression of
a different NALCN isoform in the atrial cardiomyocyte.
158
system with NALCN only and NALCN+UNC79 will determine whether internalization of
NALCN protein is responsible for the lack of Na+ leak observed in my recordings.
7.4.5 NALCN conductance with UNC80
In my study, over expression of NALCN with UNC79 in tsA201 cell line was sufficient to
generate a large Na+-dependent leak current. Other studies in overexpression systems indicate
NALCN expression requires UNC80 (Chen et al., 2010) or UNC79 and UNC80 (Lu et al., 2010).
In my study, UNC80 mRNA was also highly expressed in the SAN region. Therefore, a
fundamental question remains as to how UNC80 and UNC79 coordinate NALCN conductance.
This could be addressed with future experiments by combining expression of UNC80 subunit
with NALCN, and UNC80 along with NALCN+UNC79. Additional analysis of the Na+ leak
current will be able to determine whether different subunits affect NALCN conductance.
Furthermore, immunocytochemistry will also help to establish whether UNC80 and UNC79 will
affect membrane expression and localization of NALCN channel.
7.4.6 General application of computation model findings
Taken together my finding using the computation modeling, I have identified a strong correlation
between background Na+ conductance and neuronal spiking rate. Although neuronal simulations
rarely distinguish between Na+ leak and K
+ leak, segregation of background Na
+, K
+ and Ca
2+
current has been reported in some of the earliest SAN models (Bristow and Clark, 1982;Noble et
al., 1989;Noble and Noble, 1984). Contrary to my findings, changing background Na+
conductance produces small changes in spike frequencies (Noble et al., 1992). There are various
reasons to account for the apparent discrepancies between my model and established cardiac
models. First, my model groups all Na+ and K
+ leak into its respective gNaL and gKL. Many
conductances that contribute to background Na+ current are separately described with different
parameter. The Noble-DiFrancesco-Denyer model (1989) used separate equations to describe
slow inward current (Isi), Na+/Ca
2+ exchanger current (INCX), hyperpolarizing-activated current
(If) and background Na+ current (INa,b) (Noble et al., 1989). Noble and colleagues (1992)
adopted this model to vary background Na+ to indicate spike frequency change from 100%
background Na+ at 4 Hz to 0% background Na
+ at just less than 3 Hz. However, as more data
became available and newer models are developed, a better illustration of the pacemaker model
159
indicate a strong dependence on the background Na+ current. Dokos-Celler-Lovell (1996) SAN
model used different proportion of background Na+ with other ion voltage-dependent channel
and ion exchanger current produced SAN model that is higher sensitivity to background Na+
conductance, where absence of INa,b resulted in no spontaneous activity and hyperpolarized
membrane potential at approximately -65 mV. A major differences between the Dokos et al.
(1996) and Noble et al. (1992) models are the action potential waveform where peak
depolarizing and hyperpolarizing voltage varies by ~30 mV. This suggests proper fitting of
computation model to experimental recordings are critical to produce physiologically relevant
results. Although the model produce in this thesis fits the experimental RPeD1 model, it is still
important to consider other ionic conductance that can contribute to background Na+ leak. An
important current to consider in neuronal model is persistent Na+ current (INaP). Future revision
of RPeD1 model will include experimental quantificatied INaP. Comparisons will be made to
determine whether inclusion of INaP will affect spike rate sensitivity to background Na+ leak.
7.5 Concluding remarks
The findings from this thesis provide a new perspective on the molecular constituent that
contributes to the major background Na+. In my studies as well as many others, significant
background Na+ conductance is an essential intrinsic property that forms the prerequisite for
spontaneous activity in many pacemaker cells. It remains to be seen how extensive NALCN
regulation of the depolarized resting membrane potential is with other pacemaker cells; but this
study offered compelling evidence to suggest NALCN plays a fundamentally important role in
regulating pacemaker cell excitability. There is still much to be learned about the biophysical,
molecular and electrophysiological function of NALCN channel. Lack of appropriate
pharmacological tool for NALCN channel makes it a difficult channel to study in the biological
system. However, as demonstrated by this thesis, appropriate planning and utilizing strengths
from various animal models to bypass the known limitation, can provide a great wealth of
knowledge.
160
Reference List
Adams,B.A., Tanabe,T., Mikami,A., Numa,S., and Beam,K.G. (1990). Intramembrane charge
movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs.
Nature 346, 569-572.
Alboni,P., Baggioni,G.F., Scarfo,S., Cappato,R., Percoco,G.F., Paparella,N., and Antonioli,G.E.
(1991). Role of sinus node artery disease in sick sinus syndrome in inferior wall acute
myocardial infarction. Am. J. Cardiol. 67, 1180-1184.
Alig,J., Marger,L., Mesirca,P., Ehmke,H., Mangoni,M.E., and Isbrandt,D. (2009). Control of
heart rate by cAMP sensitivity of HCN channels. Proc. Natl. Acad. Sci. U. S. A 106, 12189-
12194.
Aller,M.I., Veale,E.L., Linden,A.M., Sandu,C., Schwaninger,M., Evans,L.J., Korpi,E.R.,
Mathie,A., Wisden,W., and Brickley,S.G. (2005). Modifying the subunit composition of TASK
channels alters the modulation of a leak conductance in cerebellar granule neurons. J. Neurosci.
25, 11455-11467.
Allessie,M., Ausma,J., and Schotten,U. (2002). Electrical, contractile and structural remodeling
during atrial fibrillation. Cardiovasc. Res. 54, 230-246.
Altomare,C., Terragni,B., Brioschi,C., Milanesi,R., Pagliuca,C., Viscomi,C., Moroni,A.,
Baruscotti,M., and DiFrancesco,D. (2003). Heteromeric HCN1-HCN4 channels: a comparison
with native pacemaker channels from the rabbit sinoatrial node. J. Physiol 549, 347-359.
Alvarez,B.V., Perez,N.G., Ennis,I.L., Camilion de Hurtado,M.C., and Cingolani,H.E. (1999).
Mechanisms underlying the increase in force and Ca(2+) transient that follow stretch of cardiac
muscle: a possible explanation of the Anrep effect. Circ. Res. 85, 716-722.
Alvarez,J.L., Aimond,F., Lorente,P., and Vassort,G. (2000). Late post-myocardial infarction
induces a tetrodotoxin-resistant Na(+)Current in rat cardiomyocytes. J. Mol. Cell Cardiol. 32,
1169-1179.
Amini,B., Bidani,A., Zwischenberger,J.B., and Clark,J.W., Jr. (2005). A model of the
pacemaking neuron of the respiratory central pattern generator. IEEE Trans. Neural Syst.
Rehabil. Eng 13, 120-124.
Amini,B., Clark,J.W., Jr., and Canavier,C.C. (1999). Calcium dynamics underlying pacemaker-
like and burst firing oscillations in midbrain dopaminergic neurons: a computational study. J.
Neurophysiol. 82, 2249-2261.
Andre,S., Boukhaddaoui,H., Campo,B., Al-Jumaily,M., Mayeux,V., Greuet,D., Valmier,J., and
Scamps,F. (2003). Axotomy-induced expression of calcium-activated chloride current in
subpopulations of mouse dorsal root ganglion neurons. J. Neurophysiol. 90, 3764-3773.
161
Angers,A., Storozhuk,M.V., Duchaine,T., Castellucci,V.F., and DesGroseillers,L. (1998).
Cloning and functional expression of an Aplysia 5-HT receptor negatively coupled to adenylate
cyclase. J. Neurosci. 18, 5586-5593.
Annunziato,L., Pignataro,G., and Di Renzo,G.F. (2004). Pharmacology of brain Na+/Ca2+
exchanger: from molecular biology to therapeutic perspectives. Pharmacol. Rev. 56, 633-654.
Arikkath,J., and Campbell,K.P. (2003). Auxiliary subunits: essential components of the voltage-
gated calcium channel complex. Curr. Opin. Neurobiol. 13, 298-307.
Artinian,J., Peret,A., Marti,G., Epsztein,J., and Crepel,V. (2011). Synaptic kainate receptors in
interplay with INaP shift the sparse firing of dentate granule cells to a sustained rhythmic mode
in temporal lobe epilepsy. J. Neurosci. 31, 10811-10818.
Atherton,J.F., and Bevan,M.D. (2005). Ionic mechanisms underlying autonomous action
potential generation in the somata and dendrites of GABAergic substantia nigra pars reticulata
neurons in vitro. J. Neurosci. 25, 8272-8281.
Ayali,A., and Harris-Warrick,R.M. (1999). Monoamine control of the pacemaker kernel and
cycle frequency in the lobster pyloric network. J. Neurosci. 19, 6712-6722.
Baartscheer,A., Schumacher,C.A., Coronel,R., and Fiolet,J.W. (2011). The Driving Force of the
Na/Ca-Exchanger during Metabolic Inhibition. Front Physiol 2, 10.
Baig,S.M., Koschak,A., Lieb,A., Gebhart,M., Dafinger,C., Nurnberg,G., Ali,A., Ahmad,I.,
Sinnegger-Brauns,M.J., Brandt,N., Engel,J., Mangoni,M.E., Farooq,M., Khan,H.U., Nurnberg,P.,
Striessnig,J., and Bolz,H.J. (2011). Loss of Ca(v)1.3 (CACNA1D) function in a human
channelopathy with bradycardia and congenital deafness. Nat. Neurosci. 14, 77-84.
Baker,T.L., and Mitchell,G.S. (2000). Episodic but not continuous hypoxia elicits long-term
facilitation of phrenic motor output in rats. J. Physiol 529 Pt 1, 215-219.
Bal,T., and McCormick,D.A. (1997). Synchronized oscillations in the inferior olive are
controlled by the hyperpolarization-activated cation current I(h). J. Neurophysiol. 77, 3145-3156.
Banasiak,K.J., Burenkova,O., and Haddad,G.G. (2004). Activation of voltage-sensitive sodium
channels during oxygen deprivation leads to apoptotic neuronal death. Neuroscience 126, 31-44.
Bangalore,R., Mehrke,G., Gingrich,K., Hofmann,F., and Kass,R.S. (1996). Influence of L-type
Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293
cells. Am. J. Physiol 270, H1521-H1528.
Banks,M.I., Pearce,R.A., and Smith,P.H. (1993). Hyperpolarization-activated cation current (Ih)
in neurons of the medial nucleus of the trapezoid body: voltage-clamp analysis and enhancement
by norepinephrine and cAMP suggest a modulatory mechanism in the auditory brain stem. J.
Neurophysiol. 70, 1420-1432.
162
Barbara,G., Alloui,A., Nargeot,J., Lory,P., Eschalier,A., Bourinet,E., and Chemin,J. (2009). T-
type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino
acids. J. Neurosci. 29, 13106-13114.
Barbuti,A., Ishii,S., Shimizu,T., Robinson,R.B., and Feinmark,S.J. (2002). Block of the
background K(+) channel TASK-1 contributes to arrhythmogenic effects of platelet-activating
factor. Am. J. Physiol Heart Circ. Physiol 282, H2024-H2030.
Barbuti,A., Terragni,B., Brioschi,C., and DiFrancesco,D. (2007). Localization of f-channels to
caveolae mediates specific beta2-adrenergic receptor modulation of rate in sinoatrial myocytes.
J. Mol. Cell Cardiol. 42, 71-78.
Barnett,D.W., Pressel,D.M., and Misler,S. (1995). Voltage-dependent Na+ and Ca2+ currents in
human pancreatic islet beta-cells: evidence for roles in the generation of action potentials and
insulin secretion. Pflugers Arch. 431, 272-282.
Baruscotti,M., Westenbroek,R., Catterall,W.A., DiFrancesco,D., and Robinson,R.B. (1997). The
newborn rabbit sino-atrial node expresses a neuronal type I-like Na+ channel. J. Physiol 498 ( Pt
3), 641-648.
Bauer,R., Walter,B., Fritz,H., and Zwiener,U. (1999). Ontogenetic aspects of traumatic brain
edema--facts and suggestions. Exp. Toxicol. Pathol. 51, 143-150.
Baukrowitz,T., and Yellen,G. (1996). Use-dependent blockers and exit rate of the last ion from
the multi-ion pore of a K+ channel. Science 271, 653-656.
Baumgarten,C.M., and Fozzard,H.A. (1981). Intracellular chloride activity in mammalian
ventricular muscle. Am. J. Physiol 241, C121-C129.
Bean,B.P. (2007). The action potential in mammalian central neurons. Nat. Rev. Neurosci. 8,
451-465.
Becker,C., Jick,S.S., and Meier,C.R. (2008). Use of antihypertensives and the risk of Parkinson
disease. Neurology 70, 1438-1444.
Bell,H.J., Inoue,T., Shum,K., Luk,C., and Syed,N.I. (2007). Peripheral oxygen-sensing cells
directly modulate the output of an identified respiratory central pattern generating neuron. Eur. J.
Neurosci. 25, 3537-3550.
Bell-Pedersen,D., Cassone,V.M., Earnest,D.J., Golden,S.S., Hardin,P.E., Thomas,T.L., and
Zoran,M.J. (2005). Circadian rhythms from multiple oscillators: lessons from diverse organisms.
Nat. Rev. Genet. 6, 544-556.
Ben-Mabrouk,F., and Tryba,A.K. (2010). Substance P modulation of TRPC3/7 channels
improves respiratory rhythm regularity and ICAN-dependent pacemaker activity. Eur. J.
Neurosci. 31, 1219-1232.
163
Bennett,B.D., Callaway,J.C., and Wilson,C.J. (2000). Intrinsic membrane properties underlying
spontaneous tonic firing in neostriatal cholinergic interneurons. J. Neurosci. 20, 8493-8503.
Bers,D.M. (2002a). Cardiac excitation-contraction coupling. Nature 415, 198-205.
Bers,D.M. (2002b). Cardiac Na/Ca exchange function in rabbit, mouse and man: what's the
difference? J. Mol. Cell Cardiol. 34, 369-373.
Bhattacharjee,A., Joiner,W.J., Wu,M., Yang,Y., Sigworth,F.J., and Kaczmarek,L.K. (2003).
Slick (Slo2.1), a rapidly-gating sodium-activated potassium channel inhibited by ATP. J.
Neurosci. 23, 11681-11691.
Biagi,B.A., and Enyeart,J.J. (1990). Gadolinium blocks low- and high-threshold calcium currents
in pituitary cells. Am. J. Physiol 259, C515-C520.
Bichet,D., Cornet,V., Geib,S., Carlier,E., Volsen,S., Hoshi,T., Mori,Y., and De,W.M. (2000).
The I-II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention
signal antagonized by the beta subunit. Neuron 25, 177-190.
Biel,M., Wahl-Schott,C., Michalakis,S., and Zong,X. (2009). Hyperpolarization-activated cation
channels: from genes to function. Physiol Rev. 89, 847-885.
Birmingham,A., Anderson,E., Sullivan,K., Reynolds,A., Boese,Q., Leake,D., Karpilow,J., and
Khvorova,A. (2007). A protocol for designing siRNAs with high functionality and specificity.
Nat. Protoc. 2, 2068-2078.
Birmingham,A., Anderson,E.M., Reynolds,A., Ilsley-Tyree,D., Leake,D., Fedorov,Y.,
Baskerville,S., Maksimova,E., Robinson,K., Karpilow,J., Marshall,W.S., and Khvorova,A.
(2006). 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat.
Methods 3, 199-204.
Black,J.L., III (2003). The voltage-gated calcium channel gamma subunits: a review of the
literature. J. Bioenerg. Biomembr. 35, 649-660.
Bleakman,D., Bowman,D., Bath,C.P., Brust,P.F., Johnson,E.C., Deal,C.R., Miller,R.J.,
Ellis,S.B., Harpold,M.M., Hans,M., and . (1995). Characteristics of a human N-type calcium
channel expressed in HEK293 cells. Neuropharmacology 34, 753-765.
Bleeker,W.K., Mackaay,A.J., Masson-Pevet,M., Bouman,L.N., and Becker,A.E. (1980).
Functional and morphological organization of the rabbit sinus node. Circ. Res. 46, 11-22.
Blitz,D.M., and Ramirez,J.M. (2002). Long-term modulation of respiratory network activity
following anoxia in vitro. J. Neurophysiol. 87, 2964-2971.
Bogdanov,K.Y., Vinogradova,T.M., and Lakatta,E.G. (2001). Sinoatrial nodal cell ryanodine
receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation. Circ. Res.
88, 1254-1258.
164
Borisova,M.P., Brutyan,R.A., and Ermishkin,L.N. (1986). Mechanism of anion-cation selectivity
of amphotericin B channels. J. Membr. Biol. 90, 13-20.
Borlak,J., and Thum,T. (2003). Hallmarks of ion channel gene expression in end-stage heart
failure. FASEB J. 17, 1592-1608.
Bosch,R.F., Zeng,X., Grammer,J.B., Popovic,K., Mewis,C., and Kuhlkamp,V. (1999). Ionic
mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc. Res. 44, 121-131.
BoSmith,R.E., Briggs,I., and Sturgess,N.C. (1993). Inhibitory actions of ZENECA ZD7288 on
whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial
node cells. Br. J. Pharmacol. 110, 343-349.
Bouhours,M., Po,M.D., Gao,S., Hung,W., Li,H., Georgiou,J., Roder,J.C., and Zhen,M. (2011). A
co-operative regulation of neuronal excitability by UNC-7 Innexin and NCA/NALCN leak
channel. Mol. Brain 4, 16.
Bowden,S.E., Fletcher,S., Loane,D.J., and Marrion,N.V. (2001). Somatic colocalization of rat
SK1 and D class (Ca(v)1.2) L-type calcium channels in rat CA1 hippocampal pyramidal
neurons. J. Neurosci. 21, RC175.
Boyett,M.R., Dobrzynski,H., Lancaster,M.K., Jones,S.A., Honjo,H., and Kodama,I. (2003).
Sophisticated architecture is required for the sinoatrial node to perform its normal pacemaker
function. J. Cardiovasc. Electrophysiol. 14, 104-106.
Boyett,M.R., Honjo,H., and Kodama,I. (2000). The sinoatrial node, a heterogeneous pacemaker
structure. Cardiovasc. Res. 47, 658-687.
Boyett,M.R., Honjo,H., Yamamoto,M., Nikmaram,M.R., Niwa,R., and Kodama,I. (1998).
Regional differences in effects of 4-aminopyridine within the sinoatrial node. Am. J. Physiol
275, H1158-H1168.
Boyett,M.R., Inada,S., Yoo,S., Li,J., Liu,J., Tellez,J., Greener,I.D., Honjo,H., Billeter,R., Lei,M.,
Zhang,H., Efimov,I.R., and Dobrzynski,H. (2006). Connexins in the sinoatrial and
atrioventricular nodes. Adv. Cardiol. 42, 175-197.
Brickley,S.G., Aller,M.I., Sandu,C., Veale,E.L., Alder,F.G., Sambi,H., Mathie,A., and
Wisden,W. (2007a). TASK-3 two-pore domain potassium channels enable sustained high-
frequency firing in cerebellar granule neurons. J. Neurosci. 27, 9329-9340.
Brickley,S.G., Aller,M.I., Sandu,C., Veale,E.L., Alder,F.G., Sambi,H., Mathie,A., and
Wisden,W. (2007b). TASK-3 two-pore domain potassium channels enable sustained high-
frequency firing in cerebellar granule neurons. J. Neurosci. 27, 9329-9340.
Bristow,D.G., and Clark,J.W. (1982). A mathematical model of primary pacemaking cell in SA
node of the heart. Am. J. Physiol 243, H207-H218.
165
Brockhaus,J., and Ballanyi,K. (1998). Synaptic inhibition in the isolated respiratory network of
neonatal rats. Eur. J. Neurosci. 10, 3823-3839.
Brodde,O.E., and Michel,M.C. (1999). Adrenergic and muscarinic receptors in the human heart.
Pharmacol. Rev. 51, 651-690.
Brown,H.F., DiFrancesco,D., and Noble,S.J. (1979). How does adrenaline accelerate the heart?
Nature 280, 235-236.
Brown,T.G. (1911). The intrinsic factors in the act of progression in the mammal. Proceedings of
the Royal Society of London. Series B 84, 308-319.
Brown,T.G. (1922). THE PHYSIOLOGY OF STEPPING: I.-THE PRODUCTION OF
RHYTHM. J. Neurol. Psychopathol. 3, 112-116.
Buckler,K.J., Williams,B.A., and Honore,E. (2000). An oxygen-, acid- and anaesthetic-sensitive
TASK-like background potassium channel in rat arterial chemoreceptor cells. J. Physiol 525 Pt
1, 135-142.
Bungay,S.D., and Campbell,S.A. (2009). Modelling a respiratory central pattern generator
neuron in Lymnaea stagnalis. Canadian Applied Mathematics Quarterly 17, 283-291.
Bureau,M.A., Zinman,R., Foulon,P., and Begin,R. (1984). Diphasic ventilatory response to
hypoxia in newborn lambs. J. Appl. Physiol 56, 84-90.
Butera,R.J., Jr., Rinzel,J., and Smith,J.C. (1999). Models of respiratory rhythm generation in the
pre-Botzinger complex. I. Bursting pacemaker neurons. J. Neurophysiol. 82, 382-397.
Byerly,L., and Moody,W.J. (1984). Intracellular calcium ions and calcium currents in perfused
neurones of the snail, Lymnaea stagnalis. J. Physiol 352, 637-652.
Caeser,M., Brown,D.A., Gahwiler,B.H., and Knopfel,T. (1993). Characterization of a calcium-
dependent current generating a slow afterdepolarization of CA3 pyramidal cells in rat
hippocampal slice cultures. Eur. J. Neurosci. 5, 560-569.
Caldwell,R.A., Clemo,H.F., and Baumgarten,C.M. (1998). Using gadolinium to identify stretch-
activated channels: technical considerations. Am. J. Physiol 275, C619-C621.
Cao,G., Xiao,X., Xu,Y., Fu,M., and Nie,L. (2003). [Study on the total activity of PKC and the
quantity of PKC (alpha, beta) in left auricle tissues of the patients with mitral disease and atrial
fibrillation]. Sichuan. Da. Xue. Xue. Bao. Yi. Xue. Ban. 34, 688-690.
Cao,K.Y., Zwillich,C.W., Berthon-Jones,M., and Sullivan,C.E. (1992). Increased normoxic
ventilation induced by repetitive hypoxia in conscious dogs. J. Appl. Physiol 73, 2083-2088.
Cassone,V.M. (1998). Melatonin's role in vertebrate circadian rhythms. Chronobiol. Int. 15, 457-
473.
166
Cassone,V.M., and Menaker,M. (1984). Is the avian circadian system a neuroendocrine loop? J.
Exp. Zool. 232, 539-549.
Catterall,W.A. (2000). From ionic currents to molecular mechanisms: the structure and function
of voltage-gated sodium channels. Neuron 26, 13-25.
Catterall,W.A. (2011). Voltage-gated calcium channels. Cold Spring Harb. Perspect. Biol. 3,
a003947.
Catterall,W.A., Goldin,A.L., and Waxman,S.G. (2005). International Union of Pharmacology.
XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels.
Pharmacol. Rev. 57, 397-409.
Caulfield,M.P. (1993). Muscarinic receptors--characterization, coupling and function.
Pharmacol. Ther. 58, 319-379.
Cha,T.J., Ehrlich,J.R., Zhang,L., and Nattel,S. (2004). Atrial ionic remodeling induced by atrial
tachycardia in the presence of congestive heart failure. Circulation 110, 1520-1526.
Chan,C.S., Guzman,J.N., Ilijic,E., Mercer,J.N., Rick,C., Tkatch,T., Meredith,G.E., and
Surmeier,D.J. (2007). 'Rejuvenation' protects neurons in mouse models of Parkinson's disease.
Nature 447, 1081-1086.
Chan,C.S., Shigemoto,R., Mercer,J.N., and Surmeier,D.J. (2004). HCN2 and HCN1 channels
govern the regularity of autonomous pacemaking and synaptic resetting in globus pallidus
neurons. J. Neurosci. 24, 9921-9932.
Chandler,N., Aslanidi,O., Buckley,D., Inada,S., Birchall,S., Atkinson,A., Kirk,D., Monfredi,O.,
Molenaar,P., Anderson,R., Sharma,V., Sigg,D., Zhang,H., Boyett,M., and Dobrzynski,H. (2011).
Computer three-dimensional anatomical reconstruction of the human sinus node and a novel
paranodal area. Anat. Rec. (Hoboken. ) 294, 970-979.
Chandler,N.J., Greener,I.D., Tellez,J.O., Inada,S., Musa,H., Molenaar,P., DiFrancesco,D.,
Baruscotti,M., Longhi,R., Anderson,R.H., Billeter,R., Sharma,V., Sigg,D.C., Boyett,M.R., and
Dobrzynski,H. (2009). Molecular architecture of the human sinus node: insights into the function
of the cardiac pacemaker. Circulation 119, 1562-1575.
Chen,J., Mitcheson,J.S., Lin,M., and Sanguinetti,M.C. (2000). Functional roles of charged
residues in the putative voltage sensor of the HCN2 pacemaker channel. J. Biol. Chem. 275,
36465-36471.
Chen,P.A., Ernstorm,G., Watanabe,S., and Jorgensen,E.M. UNC-79 and UNC-80 are required
for NCA-1 function. 2010 Neuroscience Meeting Planner. 2010.
Ref Type: Conference Proceeding
Chiappe de,C.G., Morgan,P., Mundina-Weilenmann,C., Casey,J., Fujinaga,J., Camilion de,H.M.,
and Cingolani,H. (2001). Hyperactivity and altered mRNA isoform expression of the Cl(-
)/HCO(3)(-) anion-exchanger in the hypertrophied myocardium. Cardiovasc. Res. 51, 71-79.
167
Chiba,S., Simmons,T.W., and Levy,M.N. (1976). Chronotropic responses to experimental
ischemia of the canine sino auricular node. Arch. Int. Physiol Biochim. 84, 81-88.
Chien,A.J., Zhao,X., Shirokov,R.E., Puri,T.S., Chang,C.F., Sun,D., Rios,E., and Hosey,M.M.
(1995). Roles of a membrane-localized beta subunit in the formation and targeting of functional
L-type Ca2+ channels. J. Biol. Chem. 270, 30036-30044.
Choi,K.L., Mossman,C., Aube,J., and Yellen,G. (1993). The internal quaternary ammonium
receptor site of Shaker potassium channels. Neuron 10, 533-541.
Christ,T., Boknik,P., Wohrl,S., Wettwer,E., Graf,E.M., Bosch,R.F., Knaut,M., Schmitz,W.,
Ravens,U., and Dobrev,D. (2004). L-type Ca2+ current downregulation in chronic human atrial
fibrillation is associated with increased activity of protein phosphatases. Circulation 110, 2651-
2657.
Ciani,S., Nishikawa,K., and Kidokoro,Y. (1997). Permeation of organic cations and ammonium
through the glutamate receptor channel in Drosophila larval muscle. Jpn. J. Physiol 47, 189-198.
Cifelli,C., Rose,R.A., Zhang,H., Voigtlaender-Bolz,J., Bolz,S.S., Backx,P.H., and Heximer,S.P.
(2008). RGS4 regulates parasympathetic signaling and heart rate control in the sinoatrial node.
Circ. Res. 103, 527-535.
Civelli,O., Saito,Y., Wang,Z., Nothacker,H.P., and Reinscheid,R.K. (2006). Orphan GPCRs and
their ligands. Pharmacol. Ther. 110, 525-532.
Clapham,D.E., Montell,C., Schultz,G., and Julius,D. (2003). International Union of
Pharmacology. XLIII. Compendium of voltage-gated ion channels: transient receptor potential
channels. Pharmacol. Rev. 55, 591-596.
Clark,R.B., Mangoni,M.E., Lueger,A., Couette,B., Nargeot,J., and Giles,W.R. (2004). A rapidly
activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial
node cells. Am. J. Physiol Heart Circ. Physiol 286, H1757-H1766.
Clemens,M.J., and Elia,A. (1997). The double-stranded RNA-dependent protein kinase PKR:
structure and function. J. Interferon Cytokine Res. 17, 503-524.
Cohen,A., Ben-Abu,Y., Hen,S., and Zilberberg,N. (2008). A novel mechanism for human
K2P2.1 channel gating. Facilitation of C-type gating by protonation of extracellular histidine
residues. J. Biol. Chem. 283, 19448-19455.
Cohen,A., Ben-Abu,Y., and Zilberberg,N. (2009a). Gating the pore of potassium leak channels.
Eur. Biophys. J. 39, 61-73.
Cohen,A., Sagron,R., Somech,E., Segal-Hayoun,Y., and Zilberberg,N. (2009b). Pain-associated
signals, acidosis and lysophosphatidic acid, modulate the neuronal K(2P)2.1 channel. Mol. Cell
Neurosci. 40, 382-389.
168
Cohen,A.H., and Wallen,P. (1980). The neuronal correlate of locomotion in fish. "Fictive
swimming" induced in an in vitro preparation of the lamprey spinal cord. Exp. Brain Res. 41, 11-
18.
Cohen,B.E. (1986). Concentration- and time-dependence of amphotericin-B induced
permeability changes across ergosterol-containing liposomes. Biochim. Biophys. Acta 857, 117-
122.
Cohen,C.J., Fozzard,H.A., and Sheu,S.S. (1982). Increase in intracellular sodium ion activity
during stimulation in mammalian cardiac muscle. Circ. Res. 50, 651-662.
Colmers,W.F., Lewis,D.V., Jr., and Wilson,W.A. (1982). Cs+ loading reveals Na+-dependent
persistent inward current and negative slope resistance region in Aplysia giant neurons. J.
Neurophysiol. 48, 1191-1200.
Comunanza,V., Marcantoni,A., Vandael,D.H., Mahapatra,S., Gavello,D., Carabelli,V., and
Carbone,E. (2010). CaV1.3 as pacemaker channels in adrenal chromaffin cells: specific role on
exo- and endocytosis? Channels (Austin. ) 4, 440-446.
Connelly,C.A., Ellenberger,H.H., and Feldman,J.L. (1990). Respiratory activity in retrotrapezoid
nucleus in cat. Am. J. Physiol 258, L33-L44.
Connor,J.A., Prosser,C.L., and Weems,W.A. (1974). A study of pace-maker activity in intestinal
smooth muscle. J. Physiol 240, 671-701.
Connor,J.A., and Stevens,C.F. (1971). Prediction of repetitive firing behaviour from voltage
clamp data on an isolated neurone soma. J. Physiol 213, 31-53.
Cosens,D.J., and Manning,A. (1969). Abnormal electroretinogram from a Drosophila mutant.
Nature 224, 285-287.
Cota,G. (1986). Calcium channel currents in pars intermedia cells of the rat pituitary gland.
Kinetic properties and washout during intracellular dialysis. J. Gen. Physiol 88, 83-105.
Coumoul,X., and Deng,C.X. (2006). RNAi in mice: a promising approach to decipher gene
functions in vivo. Biochimie 88, 637-643.
Cowen,D.S. (2007). Serotonin and neuronal growth factors - a convergence of signaling
pathways. J. Neurochem. 101, 1161-1171.
Crepel,V., Aniksztejn,L., Ben-Ari,Y., and Hammond,C. (1994). Glutamate metabotropic
receptors increase a Ca(2+)-activated nonspecific cationic current in CA1 hippocampal neurons.
J. Neurophysiol. 72, 1561-1569.
Crill,W.E. (1996). Persistent sodium current in mammalian central neurons. Annu. Rev. Physiol
58, 349-362.
169
Crowder,E.A., Saha,M.S., Pace,R.W., Zhang,H., Prestwich,G.D., and Del Negro,C.A. (2007).
Phosphatidylinositol 4,5-bisphosphate regulates inspiratory burst activity in the neonatal mouse
preBotzinger complex. J. Physiol 582, 1047-1058.
Cummins,T.R., Xia,Y., and Haddad,G.G. (1994). Functional properties of rat and human
neocortical voltage-sensitive sodium currents. J. Neurophysiol. 71, 1052-1064.
Currie,K.P., Wootton,J.F., and Scott,R.H. (1995). Activation of Ca(2+)-dependent Cl- currents in
cultured rat sensory neurones by flash photolysis of DM-nitrophen. J. Physiol 482 ( Pt 2), 291-
307.
D'Souza,M., Garza,M.A., Xie,M., Weinstock,J., Xiang,Q., and Robinson,P. (2007). Substance P
is associated with heart enlargement and apoptosis in murine dilated cardiomyopathy induced by
Taenia crassiceps infection. J. Parasitol. 93, 1121-1127.
Dahdal,D., Reeves,D.C., Ruben,M., Akabas,M.H., and Blau,J. (2010). Drosophila pacemaker
neurons require g protein signaling and GABAergic inputs to generate twenty-four hour
behavioral rhythms. Neuron 68, 964-977.
Dale,N. (1993). A large, sustained Na(+)- and voltage-dependent K+ current in spinal neurons of
the frog embryo. J. Physiol 462, 349-372.
Davis,L.M., Rodefeld,M.E., Green,K., Beyer,E.C., and Saffitz,J.E. (1995). Gap junction protein
phenotypes of the human heart and conduction system. J. Cardiovasc. Electrophysiol. 6, 813-
822.
Davis,R.E., and Stuart,A.E. (1988). A persistent, TTX-sensitive sodium current in an
invertebrate neuron with neurosecretory ultrastructure. J. Neurosci. 8, 3978-3991.
de Bakker,J.M., van Capelle,F.J., Janse,M.J., Wilde,A.A., Coronel,R., Becker,A.E.,
Dingemans,K.P., van Hemel,N.M., and Hauer,R.N. (1988). Reentry as a cause of ventricular
tachycardia in patients with chronic ischemic heart disease: electrophysiologic and anatomic
correlation. Circulation 77, 589-606.
De Jongh,K.S., Warner,C., and Catterall,W.A. (1990). Subunits of purified calcium channels.
Alpha 2 and delta are encoded by the same gene. J. Biol. Chem. 265, 14738-14741.
De,G.R., Sarnelli,G., Corinaldesi,R., and Stanghellini,V. (2004). Advances in our understanding
of the pathology of chronic intestinal pseudo-obstruction. Gut 53, 1549-1552.
de,K.B., and Demel,R.A. (1974). Polyene antibiotic-sterol interactions in membranes of
Acholeplasma laidlawii cells and lecithin liposomes. 3. Molecular structure of the polyene
antibiotic-cholesterol complexes. Biochim. Biophys. Acta 339, 57-70.
de,K.B., Gerritsen,W.J., Oerlemans,A., Demel,R.A., and Van Deenen,L.L. (1974). Polyene
antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin
liposomes. I. Specificity of the membrane permeability changes induced by the polyene
antibiotics. Biochim. Biophys. Acta 339, 30-43.
170
Dedman,A., Sharif-Naeini,R., Folgering,J.H., Duprat,F., Patel,A., and Honore,E. (2009). The
mechano-gated K(2P) channel TREK-1. Eur. Biophys. J. 38, 293-303.
Dekin,M.S. (1993). Inward rectification and its effects on the repetitive firing properties of
bulbospinal neurons located in the ventral part of the nucleus tractus solitarius. J. Neurophysiol.
70, 590-601.
Del Negro,C.A., Hayes,J.A., Pace,R.W., Brush,B.R., Teruyama,R., and Feldman,J.L. (2010).
Synaptically activated burst-generating conductances may underlie a group-pacemaker
mechanism for respiratory rhythm generation in mammals. Prog. Brain Res. 187, 111-136.
Del Negro,C.A., Koshiya,N., Butera,R.J., Jr., and Smith,J.C. (2002). Persistent sodium current,
membrane properties and bursting behavior of pre-botzinger complex inspiratory neurons in
vitro. J. Neurophysiol. 88, 2242-2250.
Del Negro,C.A., Morgado-Valle,C., Hayes,J.A., Mackay,D.D., Pace,R.W., Crowder,E.A., and
Feldman,J.L. (2005). Sodium and calcium current-mediated pacemaker neurons and respiratory
rhythm generation. J. Neurosci. 25, 446-453.
Demion,M., Bois,P., Launay,P., and Guinamard,R. (2007). TRPM4, a Ca2+-activated
nonselective cation channel in mouse sino-atrial node cells. Cardiovasc. Res. 73, 531-538.
Desilets,M., and Baumgarten,C.M. (1986). K+, Na+, and Cl- activities in ventricular myocytes
isolated from rabbit heart. Am. J. Physiol 251, C197-C208.
Despa,S., Islam,M.A., Weber,C.R., Pogwizd,S.M., and Bers,D.M. (2002). Intracellular Na(+)
concentration is elevated in heart failure but Na/K pump function is unchanged. Circulation 105,
2543-2548.
Dhein,S., van Koppen,C.J., and Brodde,O.E. (2001). Muscarinic receptors in the mammalian
heart. Pharmacol. Res. 44, 161-182.
Dickinson,P.S. (2006). Neuromodulation of central pattern generators in invertebrates and
vertebrates. Curr. Opin. Neurobiol. 16, 604-614.
Dickson,C.T., Magistretti,J., Shalinsky,M.H., Fransen,E., Hasselmo,M.E., and Alonso,A. (2000).
Properties and role of I(h) in the pacing of subthreshold oscillations in entorhinal cortex layer II
neurons. J. Neurophysiol. 83, 2562-2579.
DiFrancesco,D. (2005). Cardiac pacemaker I(f) current and its inhibition by heart rate-reducing
agents. Curr. Med. Res. Opin. 21, 1115-1122.
DiFrancesco,D. (2006). Funny channels in the control of cardiac rhythm and mode of action of
selective blockers. Pharmacol. Res. 53, 399-406.
DiFrancesco,D., and Camm,J.A. (2004). Heart rate lowering by specific and selective I(f) current
inhibition with ivabradine: a new therapeutic perspective in cardiovascular disease. Drugs 64,
1757-1765.
171
DiFrancesco,D., Ducouret,P., and Robinson,R.B. (1989). Muscarinic modulation of cardiac rate
at low acetylcholine concentrations. Science 243, 669-671.
DiFrancesco,D., and Tortora,P. (1991). Direct activation of cardiac pacemaker channels by
intracellular cyclic AMP. Nature 351, 145-147.
Do,M.T., and Bean,B.P. (2003). Subthreshold sodium currents and pacemaking of subthalamic
neurons: modulation by slow inactivation. Neuron 39, 109-120.
Do,M.T., and Bean,B.P. (2004). Sodium currents in subthalamic nucleus neurons from Nav1.6-
null mice. J. Neurophysiol. 92, 726-733.
Dobler,T., Springauf,A., Tovornik,S., Weber,M., Schmitt,A., Sedlmeier,R., Wischmeyer,E., and
Doring,F. (2007). TRESK two-pore-domain K+ channels constitute a significant component of
background potassium currents in murine dorsal root ganglion neurones. J. Physiol 585, 867-879.
Dobrev,D., Knuschke,D., Richter,F., Wettwer,E., Christ,T., Knaut,M., and
Ravens,U. (2002). Functional identification of m1 and m3 muscarinic acetylcholine receptors in
human atrial myocytes: influence of chronic atrial fibrillation. Circulation, 19,II–154.
Dobrzynski,H., Li,J., Tellez,J., Greener,I.D., Nikolski,V.P., Wright,S.E., Parson,S.H.,
Jones,S.A., Lancaster,M.K., Yamamoto,M., Honjo,H., Takagishi,Y., Kodama,I., Efimov,I.R.,
Billeter,R., and Boyett,M.R. (2005b). Computer three-dimensional reconstruction of the
sinoatrial node. Circulation 111, 846-854.
Dobrzynski,H., Li,J., Tellez,J., Greener,I.D., Nikolski,V.P., Wright,S.E., Parson,S.H.,
Jones,S.A., Lancaster,M.K., Yamamoto,M., Honjo,H., Takagishi,Y., Kodama,I., Efimov,I.R.,
Billeter,R., and Boyett,M.R. (2005c). Computer three-dimensional reconstruction of the
sinoatrial node. Circulation 111, 846-854.
Dobrzynski,H., Li,J., Tellez,J., Greener,I.D., Nikolski,V.P., Wright,S.E., Parson,S.H.,
Jones,S.A., Lancaster,M.K., Yamamoto,M., Honjo,H., Takagishi,Y., Kodama,I., Efimov,I.R.,
Billeter,R., and Boyett,M.R. (2005a). Computer three-dimensional reconstruction of the
sinoatrial node. Circulation 111, 846-854.
Doi,A., and Ramirez,J.M. (2008). Neuromodulation and the orchestration of the respiratory
rhythm. Respir. Physiol Neurobiol. 164, 96-104.
Drew,L.J., Wood,J.N., and Cesare,P. (2002). Distinct mechanosensitive properties of capsaicin-
sensitive and -insensitive sensory neurons. J. Neurosci. 22, RC228.
Dryer,S.E. (1994). Na(+)-activated K+ channels: a new family of large-conductance ion
channels. Trends Neurosci. 17, 155-160.
Dryer,S.E. (2003). Molecular identification of the Na+-activated K+ channel. Neuron 37, 727-
728.
172
Du,Y., Huang,X., Wang,T., Han,K., Zhang,J., Xi,Y., Wu,G., and Ma,A. (2007). Downregulation
of neuronal sodium channel subunits Nav1.1 and Nav1.6 in the sinoatrial node from volume-
overloaded heart failure rat. Pflugers Arch. 454, 451-459.
Duan,D. (2009). Phenomics of cardiac chloride channels: the systematic study of chloride
channel function in the heart. J. Physiol 587, 2163-2177.
Duan,D., Hume,J.R., and Nattel,S. (1997a). Evidence that outwardly rectifying Cl- channels
underlie volume-regulated Cl- currents in heart. Circ. Res. 80, 103-113.
Duan,D., Winter,C., Cowley,S., Hume,J.R., and Horowitz,B. (1997b). Molecular identification
of a volume-regulated chloride channel. Nature 390, 417-421.
Duan,D., Ye,L., Britton,F., Horowitz,B., and Hume,J.R. (2000). A novel anionic inward rectifier
in native cardiac myocytes. Circ. Res. 86, E63-E71.
Dunmyre,J.R., Del Negro,C.A., and Rubin,J.E. (2011). Interactions of persistent sodium and
calcium-activated nonspecific cationic currents yield dynamically distinct bursting regimes in a
model of respiratory neurons. J. Comput. Neurosci. 31, 305-328.
Dutzler,R., Campbell,E.B., Cadene,M., Chait,B.T., and MacKinnon,R. (2002). X-ray structure of
a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature 415, 287-
294.
Dzurik,M.V., Diedrich,A., Black,B., Paranjape,S.Y., Raj,S.R., Byrne,D.W., and Robertson,D.
(2007). Endogenous substance P modulates human cardiovascular regulation at rest and during
orthostatic load. J. Appl. Physiol 102, 2092-2097.
Eggermann,E., Bayer,L., Serafin,M., Saint-Mleux,B., Bernheim,L., Machard,D., Jones,B.E., and
Muhlethaler,M. (2003). The wake-promoting hypocretin-orexin neurons are in an intrinsic state
of membrane depolarization. J. Neurosci. 23, 1557-1562.
Eggermont,J. (2004). Calcium-activated chloride channels: (un)known, (un)loved? Proc. Am.
Thorac. Soc. 1, 22-27.
Eisner,D.A., Choi,H.S., Diaz,M.E., O'Neill,S.C., and Trafford,A.W. (2000). Integrative analysis
of calcium cycling in cardiac muscle. Circ. Res. 87, 1087-1094.
El-Sharkaway,T.Y., and Daniel,E.E. (1975). Ionic mechanisms of intestinal electrical control
activity. Am. J. Physiol 229, 1287-1298.
Elinder,F., and Arhem,P. (1994). Effects of gadolinium on ion channels in the myelinated axon
of Xenopus laevis: four sites of action. Biophys. J. 67, 71-83.
Ellinor,P.T., Yang,J., Sather,W.A., Zhang,J.F., and Tsien,R.W. (1995). Ca2+ channel selectivity
at a single locus for high-affinity Ca2+ interactions. Neuron 15, 1121-1132.
173
Elliott,A.A., and Elliott,J.R. (1993). Characterization of TTX-sensitive and TTX-resistant
sodium currents in small cells from adult rat dorsal root ganglia. J. Physiol 463, 39-56.
Enyedi,P., and Czirjak,G. (2010). Molecular background of leak K+ currents: two-pore domain
potassium channels. Physiol Rev. 90, 559-605.
Eshraghian,A., and Eshraghian,H. (2011). Interstitial cells of Cajal: a novel hypothesis for the
pathophysiology of irritable bowel syndrome. Can. J. Gastroenterol. 25, 277-279.
Fahlke,C., Knittle,T., Gurnett,C.A., Campbell,K.P., and George,A.L., Jr. (1997). Subunit
stoichiometry of human muscle chloride channels. J. Gen. Physiol 109, 93-104.
Farkas,R.H., Chien,P.Y., Nakajima,S., and Nakajima,Y. (1996). Properties of a slow
nonselective cation conductance modulated by neurotensin and other neurotransmitters in
midbrain dopaminergic neurons. J. Neurophysiol. 76, 1968-1981.
Fechner,H., Sipo,I., Westermann,D., Pinkert,S., Wang,X., Suckau,L., Kurreck,J., Zeichhardt,H.,
Muller,O., Vetter,R., Erdmann,V., Tschope,C., and Poller,W. (2008). Cardiac-targeted RNA
interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3
cardiomyopathy. J. Mol. Med. (Berl) 86, 987-997.
Fedorov,Y., Anderson,E.M., Birmingham,A., Reynolds,A., Karpilow,J., Robinson,K., Leake,D.,
Marshall,W.S., and Khvorova,A. (2006). Off-target effects by siRNA can induce toxic
phenotype. RNA. 12, 1188-1196.
Fei,G., Guo,C., Sun,H.S., and Feng,Z.P. (2007). Chronic hypoxia stress-induced differential
modulation of heat-shock protein 70 and presynaptic proteins. J. Neurochem. 100, 50-61.
Felder,C.C. (1995). Muscarinic acetylcholine receptors: signal transduction through multiple
effectors. FASEB J. 9, 619-625.
Feldman,J.L., and Del Negro,C.A. (2006). Looking for inspiration: new perspectives on
respiratory rhythm. Nat. Rev. Neurosci. 7, 232-242.
Felix,R., Gurnett,C.A., De,W.M., and Campbell,K.P. (1997). Dissection of functional domains
of the voltage-dependent Ca2+ channel alpha2delta subunit. J. Neurosci. 17, 6884-6891.
Feng,Z.P., Arnot,M.I., Doering,C.J., and Zamponi,G.W. (2001). Calcium channel beta subunits
differentially regulate the inhibition of N-type channels by individual Gbeta isoforms. J. Biol.
Chem. 276, 45051-45058.
Feng,Z.P., Grigoriev,N., Munno,D., Lukowiak,K., MacVicar,B.A., Goldberg,J.I., and Syed,N.I.
(2002). Development of Ca2+ hotspots between Lymnaea neurons during synaptogenesis. J.
Physiol 539, 53-65.
Feng,Z.P., Klumperman,J., Lukowiak,K., and Syed,N.I. (1997). In vitro synaptogenesis between
the somata of identified Lymnaea neurons requires protein synthesis but not extrinsic growth
factors or substrate adhesion molecules. J. Neurosci. 17, 7839-7849.
174
Fink,K.B., and Gothert,M. (2007). 5-HT receptor regulation of neurotransmitter release.
Pharmacol. Rev. 59, 360-417.
Fink,M., Duprat,F., Lesage,F., Reyes,R., Romey,G., Heurteaux,C., and Lazdunski,M. (1996).
Cloning, functional expression and brain localization of a novel unconventional outward rectifier
K+ channel. EMBO J. 15, 6854-6862.
Fire,A., Xu,S., Montgomery,M.K., Kostas,S.A., Driver,S.E., and Mello,C.C. (1998). Potent and
specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391,
806-811.
Fisahn,A., Yamada,M., Duttaroy,A., Gan,J.W., Deng,C.X., McBain,C.J., and Wess,J. (2002).
Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to
two mixed cation currents. Neuron 33, 615-624.
Forti,L., Cesana,E., Mapelli,J., and D'Angelo,E. (2006). Ionic mechanisms of autorhythmic
firing in rat cerebellar Golgi cells. J. Physiol 574, 711-729.
Frampton,J.E., Harrison,S.M., Boyett,M.R., and Orchard,C.H. (1991). Ca2+ and Na+ in rat
myocytes showing different force-frequency relationships. Am. J. Physiol 261, C739-C750.
Franceschetti,S., Lavazza,T., Curia,G., Aracri,P., Panzica,F., Sancini,G., Avanzini,G., and
Magistretti,J. (2003). Na+-activated K+ current contributes to postexcitatory hyperpolarization in
neocortical intrinsically bursting neurons. J. Neurophysiol. 89, 2101-2111.
French,C.R., Sah,P., Buckett,K.J., and Gage,P.W. (1990). A voltage-dependent persistent sodium
current in mammalian hippocampal neurons. J. Gen. Physiol 95, 1139-1157.
Friedman,J.E., and Haddad,G.G. (1994). Anoxia induces an increase in intracellular sodium in
rat central neurons in vitro. Brain Res. 663, 329-334.
Frings,S., Reuter,D., and Kleene,S.J. (2000). Neuronal Ca2+ -activated Cl- channels--homing in
on an elusive channel species. Prog. Neurobiol. 60, 247-289.
Fung,M.L., Croning,M.D., and Haddad,G.G. (1999). Sodium homeostasis in rat hippocampal
slices during oxygen and glucose deprivation: role of voltage-sensitive sodium channels.
Neurosci. Lett. 275, 41-44.
Garcia,A.J., III, Zanella,S., Koch,H., Doi,A., and Ramirez,J.M. (2011). Chapter 3--networks
within networks: the neuronal control of breathing. Prog. Brain Res. 188, 31-50.
Gardner,P.I., Ursell,P.C., Fenoglio,J.J., Jr., and Wit,A.L. (1985). Electrophysiologic and
anatomic basis for fractionated electrograms recorded from healed myocardial infarcts.
Circulation 72, 596-611.
Gasparini,S., and DiFrancesco,D. (1997). Action of the hyperpolarization-activated current (Ih)
blocker ZD 7288 in hippocampal CA1 neurons. Pflugers Arch. 435, 99-106.
175
Gaul,G.B., Gruska,M., Titscher,G., Blazek,G., Havelec,L., Marktl,W., Muellner,W., and Kaff,A.
(1996). Prediction of survival after out-of-hospital cardiac arrest: results of a community-based
study in Vienna. Resuscitation 32, 169-176.
Gauss,R., Seifert,R., and Kaupp,U.B. (1998). Molecular identification of a hyperpolarization-
activated channel in sea urchin sperm. Nature 393, 583-587.
Gerhardt,C.C., Leysen,J.E., Planta,R.J., Vreugdenhil,E., and Van,H.H. (1996). Functional
characterisation of a 5-HT2 receptor cDNA cloned from Lymnaea stagnalis. Eur. J. Pharmacol.
311, 249-258.
Ghamari-Langroudi,M., and Bourque,C.W. (2002). Flufenamic acid blocks depolarizing
afterpotentials and phasic firing in rat supraoptic neurones. J. Physiol 545, 537-542.
Gidh-Jain,M., Huang,B., Jain,P., Battula,V., and El-Sherif,N. (1995). Reemergence of the fetal
pattern of L-type calcium channel gene expression in non infarcted myocardium during left
ventricular remodeling. Biochem. Biophys. Res. Commun. 216, 892-897.
Gidh-Jain,M., Huang,B., Jain,P., Gick,G., and El-Sherif,N. (1998). Alterations in cardiac gene
expression during ventricular remodeling following experimental myocardial infarction. J. Mol.
Cell Cardiol. 30, 627-637.
Gillen,C.M., Brill,S., Payne,J.A., and Forbush,B., III (1996). Molecular cloning and functional
expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-
chloride cotransporter family. J. Biol. Chem. 271, 16237-16244.
Gilon,P., and Rorsman,P. (2009). NALCN: a regulated leak channel. EMBO Rep. 10, 963-964.
Goldman,D.E. (1943). POTENTIAL, IMPEDANCE, AND RECTIFICATION IN
MEMBRANES. J. Gen. Physiol 27, 37-60.
Goldstein,S.A., Bockenhauer,D., O'Kelly,I., and Zilberberg,N. (2001). Potassium leak channels
and the KCNK family of two-P-domain subunits. Nat. Rev. Neurosci. 2, 175-184.
Gray,P.A., Janczewski,W.A., Mellen,N., McCrimmon,D.R., and Feldman,J.L. (2001a). Normal
breathing requires preBotzinger complex neurokinin-1 receptor-expressing neurons. Nat.
Neurosci. 4, 927-930.
Gray,P.A., Rekling,J.C., Bocchiaro,C.M., and Feldman,J.L. (1999b). Modulation of respiratory
frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex. Science
286, 1566-1568.
Gray,P.A., Rekling,J.C., Bocchiaro,C.M., and Feldman,J.L. (1999a). Modulation of respiratory
frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex. Science
286, 1566-1568.
176
Gray,P.A., Rekling,J.C., Bocchiaro,C.M., and Feldman,J.L. (1999c). Modulation of respiratory
frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex. Science
286, 1566-1568.
Gray,R.P., McIntyre,H., Sheridan,D.S., and Fry,C.H. (2001b). Intracellular sodium and
contractile function in hypertrophied human and guinea-pig myocardium. Pflugers Arch. 442,
117-123.
Grima,B., Chelot,E., Xia,R., and Rouyer,F. (2004). Morning and evening peaks of activity rely
on different clock neurons of the Drosophila brain. Nature 431, 869-873.
Guinamard,R., Demion,M., and Launay,P. (2010). Physiological roles of the TRPM4 channel
extracted from background currents. Physiology. (Bethesda. ) 25, 155-164.
Guo,C.H., Senzel,A., Li,K., and Feng,Z.P. (2010). De novo protein synthesis of syntaxin-1 and
dynamin-1 in long-term memory formation requires CREB1 gene transcription in Lymnaea
stagnalis. Behav. Genet. 40, 680-693.
Guo,D., and Lu,Z. (2000). Mechanism of IRK1 channel block by intracellular polyamines. J.
Gen. Physiol 115, 799-814.
Guo,T., Ai,X., Shannon,T.R., Pogwizd,S.M., and Bers,D.M. (2007). Intra-sarcoplasmic
reticulum free [Ca2+] and buffering in arrhythmogenic failing rabbit heart. Circ. Res. 101, 802-
810.
Gutman,G.A., Chandy,K.G., Grissmer,S., Lazdunski,M., McKinnon,D., Pardo,L.A.,
Robertson,G.A., Rudy,B., Sanguinetti,M.C., Stuhmer,W., and Wang,X. (2005). International
Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated
potassium channels. Pharmacol. Rev. 57, 473-508.
Gyori,J., Platoshyn,O., Carpenter,D.O., and Salanki,J. (2000). Effect of inorganic and organic tin
compounds on ACh- and voltage-activated Na currents. Cell Mol. Neurobiol. 20, 591-604.
Haas,J.S., Dorval,A.D., and White,J.A. (2007). Contributions of Ih to feature selectivity in layer
II stellate cells of the entorhinal cortex. J. Comput. Neurosci. 22, 161-171.
Hagiwara,N., Irisawa,H., Kasanuki,H., and Hosoda,S. (1992). Background current in sino-atrial
node cells of the rabbit heart. J. Physiol 448, 53-72.
Halliwell,J.V., and Adams,P.R. (1982). Voltage-clamp analysis of muscarinic excitation in
hippocampal neurons. Brain Res. 250, 71-92.
Hallworth,N.E., Wilson,C.J., and Bevan,M.D. (2003). Apamin-sensitive small conductance
calcium-activated potassium channels, through their selective coupling to voltage-gated calcium
channels, are critical determinants of the precision, pace, and pattern of action potential
generation in rat subthalamic nucleus neurons in vitro. J. Neurosci. 23, 7525-7542.
177
Haque,Z., Lee,T.K., Inoue,T., Luk,C., Hasan,S.U., Lukowiak,K., and Syed,N.I. (2006). An
identified central pattern-generating neuron co-ordinates sensory-motor components of
respiratory behavior in Lymnaea. Eur. J. Neurosci. 23, 94-104.
Harris,D.P., Balasubramaniam,A., Badr,M.S., and Mateika,J.H. (2006). Long-term facilitation of
ventilation and genioglossus muscle activity is evident in the presence of elevated levels of
carbon dioxide in awake humans. Am. J. Physiol Regul. Integr. Comp Physiol 291, R1111-
R1119.
Harris,N.C., and Constanti,A. (1995). Mechanism of block by ZD 7288 of the hyperpolarization-
activated inward rectifying current in guinea pig substantia nigra neurons in vitro. J.
Neurophysiol. 74, 2366-2378.
Harris,N.C., Libri,V., and Constanti,A. (1994). Selective blockade of the hyperpolarization-
activated cationic current (Ih) in guinea pig substantia nigra pars compacta neurones by a novel
bradycardic agent, Zeneca ZM 227189. Neurosci. Lett. 176, 221-225.
Harris-Warrick,R.M. (2010). General principles of rhythmogenesis in central pattern generator
networks. Prog. Brain Res. 187, 213-222.
Harris-Warrick,R.M. (2002). Voltage-sensitive ion channels in rhythmic motor systems. Curr.
Opin. Neurobiol. 12, 646-651.
Hartzell,C., Putzier,I., and Arreola,J. (2005). Calcium-activated chloride channels. Annu. Rev.
Physiol 67, 719-758.
Hayar,A., Piguet,P., and Feltz,P. (1996). GABA-induced responses in electrophysiologically
characterized neurons within the rat rostro-ventrolateral medulla in vitro. Brain Res. 709, 173-
183.
Heginbotham,L., Abramson,T., and MacKinnon,R. (1992). A functional connection between the
pores of distantly related ion channels as revealed by mutant K+ channels. Science 258, 1152-
1155.
Heginbotham,L., Lu,Z., Abramson,T., and MacKinnon,R. (1994). Mutations in the K+ channel
signature sequence. Biophys. J. 66, 1061-1067.
Heginbotham,L., and MacKinnon,R. (1992). The aromatic binding site for tetraethylammonium
ion on potassium channels. Neuron 8, 483-491.
Heinemann,S.H., Terlau,H., Stuhmer,W., Imoto,K., and Numa,S. (1992). Calcium channel
characteristics conferred on the sodium channel by single mutations. Nature 356, 441-443.
Helton,T.D., Xu,W., and Lipscombe,D. (2005). Neuronal L-type calcium channels open quickly
and are inhibited slowly. J. Neurosci. 25, 10247-10251.
Henriquez,C.S. (1993). Simulating the electrical behavior of cardiac tissue using the bidomain
model. Crit Rev. Biomed. Eng 21, 1-77.
178
Herrmann,S., Stieber,J., Stockl,G., Hofmann,F., and Ludwig,A. (2007). HCN4 provides a
'depolarization reserve' and is not required for heart rate acceleration in mice. EMBO J. 26,
4423-4432.
Hill,A.A., Lu,J., Masino,M.A., Olsen,O.H., and Calabrese,R.L. (2001). A model of a segmental
oscillator in the leech heartbeat neuronal network. J. Comput. Neurosci. 10, 281-302.
Ho,W.K., Brown,H.F., and Noble,D. (1994). High selectivity of the i(f) channel to Na+ and K+
in rabbit isolated sinoatrial node cells. Pflugers Arch. 426, 68-74.
Hodgkin,A.L., and Huxley,A.F. (1947). Potassium leakage from an active nerve fibre. J. Physiol
106, 341-367.
Hodgkin,A.L., and Huxley,A.F. (1952). The dual effect of membrane potential on sodium
conductance in the giant axon of Loligo. J. Physiol 116, 497-506.
Hodgkin,A.L., and KATZ,B. (1949). The effect of sodium ions on the electrical activity of giant
axon of the squid. J. Physiol 108, 37-77.
Hofmann,M.E., and Frazier,C.J. (2010). Muscarinic receptor activation modulates the
excitability of hilar mossy cells through the induction of an afterdepolarization. Brain Res. 1318,
42-51.
Holzer,P. (2011). TRP channels in the digestive system. Curr. Pharm. Biotechnol. 12, 24-34.
Hongo,K., Pascarel,C., Cazorla,O., Gannier,F., Le Guennec,J.Y., and White,E. (1997).
Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular
myocytes. Exp. Physiol 82, 647-656.
Honore,E. (2007). The neuronal background K2P channels: focus on TREK1. Nat. Rev.
Neurosci. 8, 251-261.
Hotson,J.R., Prince,D.A., and Schwartzkroin,P.A. (1979). Anomalous inward rectification in
hippocampal neurons. J. Neurophysiol. 42, 889-895.
Huang,B., El-Sherif,T., Gidh-Jain,M., Qin,D., and El-Sherif,N. (2001). Alterations of sodium
channel kinetics and gene expression in the postinfarction remodeled myocardium. J.
Cardiovasc. Electrophysiol. 12, 218-225.
Huang,C.J., and Moczydlowski,E. (2001). Cytoplasmic polyamines as permeant blockers and
modulators of the voltage-gated sodium channel. Biophys. J. 80, 1262-1279.
Huang,Z.M., Prasad,C., Britton,F.C., Ye,L.L., Hatton,W.J., and Duan,D. (2009). Functional role
of CLC-2 chloride inward rectifier channels in cardiac sinoatrial nodal pacemaker cells. J. Mol.
Cell Cardiol. 47, 121-132.
179
Hubner,C.A., Stein,V., Hermans-Borgmeyer,I., Meyer,T., Ballanyi,K., and Jentsch,T.J. (2001).
Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic
inhibition. Neuron 30, 515-524.
Hui,K., Fei,G.H., Saab,B.J., Su,J., Roder,J.C., and Feng,Z.P. (2007). Neuronal calcium sensor-1
modulation of optimal calcium level for neurite outgrowth. Development 134, 4479-4489.
Huizinga,J.D., Zhu,Y., Ye,J., and Molleman,A. (2002). High-conductance chloride channels
generate pacemaker currents in interstitial cells of Cajal. Gastroenterology 123, 1627-1636.
Hume,J.R., Duan,D., Collier,M.L., Yamazaki,J., and Horowitz,B. (2000). Anion transport in
heart. Physiol Rev. 80, 31-81.
Humphrey,J.A., Hamming,K.S., Thacker,C.M., Scott,R.L., Sedensky,M.M., Snutch,T.P.,
Morgan,P.G., and Nash,H.A. (2007). A putative cation channel and its novel regulator: cross-
species conservation of effects on general anesthesia. Curr. Biol. 17, 624-629.
Hurley,M.J., and Dexter,D.T. (2012). Voltage-gated calcium channels and Parkinson's disease.
Pharmacol. Ther. 133, 324-333.
HUTTER,O.F., and Noble,D. (1961). Anion conductance of cardiac muscle. J. Physiol 157, 335-
350.
Im,S.H., Li,W., and Taghert,P.H. (2011). PDFR and CRY signaling converge in a subset of
clock neurons to modulate the amplitude and phase of circadian behavior in Drosophila. PLoS.
One. 6, e18974.
Inoue,T., Haque,Z., Lukowiak,K., and Syed,N.I. (2001). Hypoxia-induced respiratory patterned
activity in Lymnaea originates at the periphery. J. Neurophysiol. 86, 156-163.
Inoue,T., Takasaki,M., Lukowiak,K., and Syed,N. (1996). Inhibition of the respiratory pattern-
generating neurons by an identified whole-body withdrawal interneuron of Lymnaea stagnalis. J.
Exp. Biol. 199, 1887-1898.
Ishii,T.M., Takano,M., Xie,L.H., Noma,A., and Ohmori,H. (1999). Molecular characterization of
the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J. Biol. Chem. 274,
12835-12839.
Isom,L.L., De Jongh,K.S., and Catterall,W.A. (1994). Auxiliary subunits of voltage-gated ion
channels. Neuron 12, 1183-1194.
Isom,L.L., De Jongh,K.S., Patton,D.E., Reber,B.F., Offord,J., Charbonneau,H., Walsh,K.,
Goldin,A.L., and Catterall,W.A. (1992). Primary structure and functional expression of the beta
1 subunit of the rat brain sodium channel. Science 256, 839-842.
Itagaki,K., Koch,W.J., Bodi,I., Klockner,U., Slish,D.F., and Schwartz,A. (1992). Native-type
DHP-sensitive calcium channel currents are produced by cloned rat aortic smooth muscle and
180
cardiac alpha 1 subunits expressed in Xenopus laevis oocytes and are regulated by alpha 2- and
beta-subunits. FEBS Lett. 297, 221-225.
Jackson,A.C., and Bean,B.P. (2007). State-dependent enhancement of subthreshold A-type
potassium current by 4-aminopyridine in tuberomammillary nucleus neurons. J. Neurosci. 27,
10785-10796.
Jackson,A.C., Yao,G.L., and Bean,B.P. (2004). Mechanism of spontaneous firing in dorsomedial
suprachiasmatic nucleus neurons. J. Neurosci. 24, 7985-7998.
Jahnsen,H., and Llinas,R. (1984). Ionic basis for the electro-responsiveness and oscillatory
properties of guinea-pig thalamic neurones in vitro. J. Physiol 349, 227-247.
James,T.N. (2003). Structure and function of the sinus node, AV node and his bundle of the
human heart: part II--function. Prog. Cardiovasc. Dis. 45, 327-360.
James,T.N., and Nadeau,R.A. (1963). Sinus bradycardia during injections directly into the sinus
node artery. Am. J. Physiol 204, 9-15.
Janczewski,W.A., and Feldman,J.L. (2006b). Novel data supporting the two respiratory rhythm
oscillator hypothesis. Focus on "respiration-related rhythmic activity in the rostral medulla of
newborn rats". J. Neurophysiol. 96, 1-2.
Janczewski,W.A., and Feldman,J.L. (2006a). Distinct rhythm generators for inspiration and
expiration in the juvenile rat. J. Physiol 570, 407-420.
Janse,M.J. (2004). Electrophysiological changes in heart failure and their relationship to
arrhythmogenesis. Cardiovasc. Res. 61, 208-217.
Janse,M.J., and Wit,A.L. (1989). Electrophysiological mechanisms of ventricular arrhythmias
resulting from myocardial ischemia and infarction. Physiol Rev. 69, 1049-1169.
Jay,S.D., Sharp,A.H., Kahl,S.D., Vedvick,T.S., Harpold,M.M., and Campbell,K.P. (1991).
Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and
the associated delta peptides. J. Biol. Chem. 266, 3287-3293.
Jelicks,L.A., and Gupta,R.K. (1994). Nuclear magnetic resonance measurement of intracellular
sodium in the perfused normotensive and spontaneously hypertensive rat heart. Am. J.
Hypertens. 7, 429-435.
Jentsch,T.J., Stein,V., Weinreich,F., and Zdebik,A.A. (2002). Molecular structure and
physiological function of chloride channels. Physiol Rev. 82, 503-568.
Jin,X., Huguenard,J.R., and Prince,D.A. (2005). Impaired Cl- extrusion in layer V pyramidal
neurons of chronically injured epileptogenic neocortex. J. Neurophysiol. 93, 2117-2126.
Johnson,S.M. (2007). Glutamatergic synaptic inputs and ICAN: the basis for an emergent
property underlying respiratory rhythm generation? J. Physiol 582, 5-6.
181
JONES,J.D. (1961). Aspects of respiration in Planorbis corneus L. and Lymnaea stagnalis L.
(Gastropoda: Pulmonata). Comp Biochem. Physiol 4, 1-29.
Jones,S.W. (1989). On the resting potential of isolated frog sympathetic neurons. Neuron 3, 153-
161.
Jospin,M., Watanabe,S., Joshi,D., Young,S., Hamming,K., Thacker,C., Snutch,T.P.,
Jorgensen,E.M., and Schuske,K. (2007). UNC-80 and the NCA ion channels contribute to
endocytosis defects in synaptojanin mutants. Curr. Biol. 17, 1595-1600.
Joyner,R.W., and van Capelle,F.J. (1986). Propagation through electrically coupled cells. How a
small SA node drives a large atrium. Biophys. J. 50, 1157-1164.
Ju,Y.K., and Allen,D.G. (1998). Intracellular calcium and Na+-Ca2+ exchange current in
isolated toad pacemaker cells. J. Physiol 508 ( Pt 1), 153-166.
Ju,Y.K., Chu,Y., Chaulet,H., Lai,D., Gervasio,O.L., Graham,R.M., Cannell,M.B., and
Allen,D.G. (2007). Store-operated Ca2+ influx and expression of TRPC genes in mouse
sinoatrial node. Circ. Res. 100, 1605-1614.
Kandel,E.R. (1977). Cellular Basis of Behaviour (San Francisco: W.H.Freeman and Co Ltd).
Kannel,W.B., Ho,K., and Thom,T. (1994). Changing epidemiological features of cardiac failure.
Br. Heart J. 72, S3-S9.
Kaprielian,R., Sah,R., Nguyen,T., Wickenden,A.D., and Backx,P.H. (2002). Myocardial
infarction in rat eliminates regional heterogeneity of AP profiles, I(to) K(+) currents, and
[Ca(2+)](i) transients. Am. J. Physiol Heart Circ. Physiol 283, H1157-H1168.
Kavanaugh,M.P., Varnum,M.D., Osborne,P.B., Christie,M.J., Busch,A.E., Adelman,J.P., and
North,R.A. (1991). Interaction between tetraethylammonium and amino acid residues in the pore
of cloned voltage-dependent potassium channels. J. Biol. Chem. 266, 7583-7587.
Keith,A., and Flack,M. (1907). The Form and Nature of the Muscular Connections between the
Primary Divisions of the Vertebrate Heart. J. Anat. Physiol 41, 172-189.
Kent,R.L., Hoober,J.K., and Cooper,G. (1989). Load responsiveness of protein synthesis in adult
mammalian myocardium: role of cardiac deformation linked to sodium influx. Circ. Res. 64, 74-
85.
Kerschbaum,H.H., Kozak,J.A., and Cahalan,M.D. (2003). Polyvalent cations as permeant probes
of MIC and TRPM7 pores. Biophys. J. 84, 2293-2305.
Keynes,R.D. (1951). The ionic movements during nervous activity. J. Physiol 114, 119-150.
Khakh,B.S., Bao,X.R., Labarca,C., and Lester,H.A. (1999). Neuronal P2X transmitter-gated
cation channels change their ion selectivity in seconds. Nat. Neurosci. 2, 322-330.
182
Khaliq,Z.M., and Bean,B.P. (2010). Pacemaking in dopaminergic ventral tegmental area
neurons: depolarizing drive from background and voltage-dependent sodium conductances. J.
Neurosci. 30, 7401-7413.
Khaliq,Z.M., Gouwens,N.W., and Raman,I.M. (2003). The contribution of resurgent sodium
current to high-frequency firing in Purkinje neurons: an experimental and modeling study. J.
Neurosci. 23, 4899-4912.
Kharche,S., Yu,J., Lei,M., and Zhang,H. (2011). A mathematical model of action potentials of
mouse sinoatrial node cells with molecular bases. Am. J. Physiol Heart Circ. Physiol 301, H945-
H963.
Kiehn,O., Dougherty,K.J., Hagglund,M., Borgius,L., Talpalar,A., and Restrepo,C.E. (2010).
Probing spinal circuits controlling walking in mammals. Biochem. Biophys. Res. Commun. 396,
11-18.
Kiehn,O., and Harris-Warrick,R.M. (1992). 5-HT modulation of hyperpolarization-activated
inward current and calcium-dependent outward current in a crustacean motor neuron. J.
Neurophysiol. 68, 496-508.
Kim,B.J., Chang,I.Y., Choi,S., Jun,J.Y., Jeon,J.H., Xu,W.X., Kwon,Y.K., Ren,D., and So,I.
(2012). Involvement of Na-leak Channel in Substance P-induced Depolarization of Pacemaking
Activity in Interstitial Cells of Cajal. Cell Physiol Biochem. 29, 501-510.
Kim,B.J., Lim,H.H., Yang,D.K., Jun,J.Y., Chang,I.Y., Park,C.S., So,I., Stanfield,P.R., and
Kim,K.W. (2005). Melastatin-type transient receptor potential channel 7 is required for intestinal
pacemaking activity. Gastroenterology 129, 1504-1517.
Kim,D., Song,I., Keum,S., Lee,T., Jeong,M.J., Kim,S.S., McEnery,M.W., and Shin,H.S. (2001).
Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in
mice lacking alpha(1G) T-type Ca(2+) channels. Neuron 31, 35-45.
Kirsch,G.E., Yeh,J.Z., and Oxford,G.S. (1986). Modulation of aminopyridine block of potassium
currents in squid axon. Biophys. J. 50, 637-644.
Kiss,T., Laszlo,Z., and Szabadics,J. (2002). Mechanism of 4-aminopyridine block of the
transient outward K-current in identified Helix neuron. Brain Res. 927, 168-179.
Kits,K.S., Lodder,J.C., van der Schors,R.C., Li,K.W., Geraerts,W.P., and Fainzilber,M. (1996).
Novel omega-conotoxins block dihydropyridine-insensitive high voltage-activated calcium
channels in molluscan neurons. J. Neurochem. 67, 2155-2163.
Kiyosue,T., Spindler,A.J., Noble,S.J., and Noble,D. (1993). Background inward current in
ventricular and atrial cells of the guinea-pig. Proc. Biol. Sci. 252, 65-74.
Klugbauer,N., Lacinova,L., Marais,E., Hobom,M., and Hofmann,F. (1999). Molecular diversity
of the calcium channel alpha2delta subunit. J. Neurosci. 19, 684-691.
183
Koch,M.C., Steinmeyer,K., Lorenz,C., Ricker,K., Wolf,F., Otto,M., Zoll,B., Lehmann-Horn,F.,
Grzeschik,K.H., and Jentsch,T.J. (1992). The skeletal muscle chloride channel in dominant and
recessive human myotonia. Science 257, 797-800.
Kodama,I., and Boyett,M.R. (1985). Regional differences in the electrical activity of the rabbit
sinus node. Pflugers Arch. 404, 214-226.
Kodama,I., Boyett,M.R., Nikmaram,M.R., Yamamoto,M., Honjo,H., and Niwa,R. (1999).
Regional differences in effects of E-4031 within the sinoatrial node. Am. J. Physiol 276, H793-
H802.
Kodama,I., Nikmaram,M.R., Boyett,M.R., Suzuki,R., Honjo,H., and Owen,J.M. (1997).
Regional differences in the role of the Ca2+ and Na+ currents in pacemaker activity in the
sinoatrial node. Am. J. Physiol 272, H2793-H2806.
Koizumi,H., and Smith,J.C. (2008). Persistent Na+ and K+-dominated leak currents contribute to
respiratory rhythm generation in the pre-Botzinger complex in vitro. J. Neurosci. 28, 1773-1785.
Korneev,S.A., Kemenes,I., Straub,V., Staras,K., Korneeva,E.I., Kemenes,G., Benjamin,P.R., and
O'Shea,M. (2002). Suppression of nitric oxide (NO)-dependent behavior by double-stranded
RNA-mediated silencing of a neuronal NO synthase gene. J. Neurosci. 22, RC227.
Koschak,A., Reimer,D., Huber,I., Grabner,M., Glossmann,H., Engel,J., and Striessnig,J. (2001).
alpha 1D (Cav1.3) subunits can form l-type Ca2+ channels activating at negative voltages. J.
Biol. Chem. 276, 22100-22106.
Krejci,A., and Tucek,S. (2002). Quantitation of mRNAs for M(1) to M(5) subtypes of
muscarinic receptors in rat heart and brain cortex. Mol. Pharmacol. 61, 1267-1272.
Kreuzberg,M.M., Schrickel,J.W., Ghanem,A., Kim,J.S., Degen,J., Janssen-Bienhold,U.,
Lewalter,T., Tiemann,K., and Willecke,K. (2006a). Connexin30.2 containing gap junction
channels decelerate impulse propagation through the atrioventricular node. Proc. Natl. Acad. Sci.
U. S. A 103, 5959-5964.
Kreuzberg,M.M., Sohl,G., Kim,J.S., Verselis,V.K., Willecke,K., and Bukauskas,F.F. (2005).
Functional properties of mouse connexin30.2 expressed in the conduction system of the heart.
Circ. Res. 96, 1169-1177.
Kreuzberg,M.M., Willecke,K., and Bukauskas,F.F. (2006b). Connexin-mediated cardiac impulse
propagation: connexin 30.2 slows atrioventricular conduction in mouse heart. Trends
Cardiovasc. Med. 16, 266-272.
Krieger,J., Strobel,J., Vogl,A., Hanke,W., and Breer,H. (1999). Identification of a cyclic
nucleotide- and voltage-activated ion channel from insect antennae. Insect Biochem. Mol. Biol.
29, 255-267.
184
Krishnan,K.S., and Nash,H.A. (1990). A genetic study of the anesthetic response: mutants of
Drosophila melanogaster altered in sensitivity to halothane. Proc. Natl. Acad. Sci. U. S. A 87,
8632-8636.
Kristan,W.B., Jr., Calabrese,R.L., and Friesen,W.O. (2005). Neuronal control of leech behavior.
Prog. Neurobiol. 76, 279-327.
Kurata,Y., Hisatome,I., Imanishi,S., and Shibamoto,T. (2002). Dynamical description of
sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell. Am. J.
Physiol Heart Circ. Physiol 283, H2074-H2101.
Kurokawa,J., Motoike,H.K., Rao,J., and Kass,R.S. (2004). Regulatory actions of the A-kinase
anchoring protein Yotiao on a heart potassium channel downstream of PKA phosphorylation.
Proc. Natl. Acad. Sci. U. S. A 101, 16374-16378.
Lacampagne,A., Gannier,F., Argibay,J., Garnier,D., and Le Guennec,J.Y. (1994). The stretch-
activated ion channel blocker gadolinium also blocks L-type calcium channels in isolated
ventricular myocytes of the guinea-pig. Biochim. Biophys. Acta 1191, 205-208.
Lamas,G.A., Lee,K., Sweeney,M., Leon,A., Yee,R., Ellenbogen,K., Greer,S., Wilber,D.,
Silverman,R., Marinchak,R., Bernstein,R., Mittleman,R.S., Lieberman,E.H., Sullivan,C.,
Zorn,L., Flaker,G., Schron,E., Orav,E.J., and Goldman,L. (2000). The mode selection trial
(MOST) in sinus node dysfunction: design, rationale, and baseline characteristics of the first
1000 patients. Am. Heart J. 140, 541-551.
Large,W.A., and Wang,Q. (1996). Characteristics and physiological role of the Ca(2+)-activated
Cl- conductance in smooth muscle. Am. J. Physiol 271, C435-C454.
Larkman,P.M., Kelly,J.S., and Takahashi,T. (1995). Adenosine 3':5'-cyclic monophosphate
mediates a 5-hydroxytryptamine-induced response in neonatal rat motoneurones. Pflugers Arch.
430, 763-769.
Launay,P., Fleig,A., Perraud,A.L., Scharenberg,A.M., Penner,R., and Kinet,J.P. (2002). TRPM4
is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell
109, 397-407.
Lawonn,P., Hoffmann,E.K., Hougaard,C., and Wehner,F. (2003). A cell shrinkage-induced non-
selective cation conductance with a novel pharmacology in Ehrlich-Lettre-ascites tumour cells.
FEBS Lett. 539, 115-119.
Le Bon-Jego,M., and Yuste,R. (2007). Persistently active, pacemaker-like neurons in neocortex.
Front Neurosci. 1, 123-129.
Lear,B.C., Lin,J.M., Keath,J.R., McGill,J.J., Raman,I.M., and Allada,R. (2005). The ion channel
narrow abdomen is critical for neural output of the Drosophila circadian pacemaker. Neuron 48,
965-976.
185
Lee,C.R., and Tepper,J.M. (2007). A calcium-activated nonselective cation conductance
underlies the plateau potential in rat substantia nigra GABAergic neurons. J. Neurosci. 27, 6531-
6541.
Lee,J.H., Cribbs,L.L., and Perez-Reyes,E. (1999). Cloning of a novel four repeat protein related
to voltage-gated sodium and calcium channels. FEBS Lett. 445, 231-236.
Legendre,P., Guzman,A., Dupouy,B., and Vincent,J.D. (1989). Excitatory effect of serotonin on
pacemaker neurons in spinal cord cell culture. Neuroscience 28, 201-209.
Lei,M., Jones,S.A., Liu,J., Lancaster,M.K., Fung,S.S., Dobrzynski,H., Camelliti,P., Maier,S.K.,
Noble,D., and Boyett,M.R. (2004). Requirement of neuronal- and cardiac-type sodium channels
for murine sinoatrial node pacemaking. J. Physiol 559, 835-848.
Leibovitch,B.A., Campbell,D.B., Krishnan,K.S., and Nash,H.A. (1995). Mutations that affect ion
channels change the sensitivity of Drosophila melanogaster to volatile anesthetics. J. Neurogenet.
10, 1-13.
Lesage,F. (2003). Pharmacology of neuronal background potassium channels.
Neuropharmacology 44, 1-7.
Lesage,F., Guillemare,E., Fink,M., Duprat,F., Lazdunski,M., Romey,G., and Barhanin,J. (1996).
TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure.
EMBO J. 15, 1004-1011.
Lesage,F., Terrenoire,C., Romey,G., and Lazdunski,M. (2000). Human TREK2, a 2P domain
mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids,
lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors. J. Biol. Chem. 275, 28398-
28405.
Lesauter,J., Silver,R., Cloues,R., and Witkovsky,P. (2011). Light exposure induces short- and
long-term changes in the excitability of retinorecipient neurons in suprachiasmatic nucleus. J.
Neurophysiol. 106, 576-588.
Lewis,T. (1910). Galvanometric curves yielded by cardiac beats generated in various areas of the
auricular musculature: The pacemaker of the heart. Heart 2, 23-46.
Li,G.R., Lau,C.P., Ducharme,A., Tardif,J.C., and Nattel,S. (2002). Transmural action potential
and ionic current remodeling in ventricles of failing canine hearts. Am. J. Physiol Heart Circ.
Physiol 283, H1031-H1041.
Li,G.R., Lau,C.P., Leung,T.K., and Nattel,S. (2004). Ionic current abnormalities associated with
prolonged action potentials in cardiomyocytes from diseased human right ventricles. Heart
Rhythm. 1, 460-468.
Li,Q., Luo,X., and Muallem,S. (2005a). Regulation of the P2X7 receptor permeability to large
molecules by extracellular Cl- and Na+. J. Biol. Chem. 280, 26922-26927.
186
Li,T., ter,V.F., Nurnberger,H.R., and Wehner,F. (2005b). A novel hypertonicity-induced cation
channel in primary cultures of human hepatocytes. FEBS Lett. 579, 2087-2091.
Liss,B., Franz,O., Sewing,S., Bruns,R., Neuhoff,H., and Roeper,J. (2001). Tuning pacemaker
frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription. EMBO J.
20, 5715-5724.
Liu,R.J., van den Pol,A.N., and Aghajanian,G.K. (2002). Hypocretins (orexins) regulate
serotonin neurons in the dorsal raphe nucleus by excitatory direct and inhibitory indirect actions.
J. Neurosci. 22, 9453-9464.
Llinas,R., and Yarom,Y. (1981). Electrophysiology of mammalian inferior olivary neurones in
vitro. Different types of voltage-dependent ionic conductances. J. Physiol 315, 549-567.
Llinas,R.R., Sugimori,M., and Cherksey,B. (1989). Voltage-dependent calcium conductances in
mammalian neurons. The P channel. Ann. N. Y. Acad. Sci. 560, 103-111.
Loirand,G., Mironneau,C., Mironneau,J., and Pacaud,P. (1989). Two types of calcium currents in
single smooth muscle cells from rat portal vein. J. Physiol 412, 333-349.
Lopes,C.M., Remon,J.I., Matavel,A., Sui,J.L., Keselman,I., Medei,E., Shen,Y., Rosenhouse-
Dantsker,A., Rohacs,T., and Logothetis,D.E. (2007). Protein kinase A modulates PLC-dependent
regulation and PIP2-sensitivity of K+ channels. Channels (Austin. ) 1, 124-134.
Lopes,C.M., Rohacs,T., Czirjak,G., Balla,T., Enyedi,P., and Logothetis,D.E. (2005). PIP2
hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain
K+ channels. J. Physiol 564, 117-129.
Lopes,C.M., Zilberberg,N., and Goldstein,S.A. (2001). Block of Kcnk3 by protons. Evidence
that 2-P-domain potassium channel subunits function as homodimers. J. Biol. Chem. 276, 24449-
24452.
Lu,B., Su,Y., Das,S., Liu,J., Xia,J., and Ren,D. (2007). The neuronal channel NALCN
contributes resting sodium permeability and is required for normal respiratory rhythm. Cell 129,
371-383.
Lu,B., Su,Y., Das,S., Wang,H., Wang,Y., Liu,J., and Ren,D. (2009). Peptide neurotransmitters
activate a cation channel complex of NALCN and UNC-80. Nature 457, 741-744.
Lu,B., Zhang,Q., Wang,H., Wang,Y., Nakayama,M., and Ren,D. (2010). Extracellular calcium
controls background current and neuronal excitability via an UNC79-UNC80-NALCN cation
channel complex. Neuron 68, 488-499.
Lu,T.Z., and Feng,Z.P. (2011). A sodium leak current regulates pacemaker activity of adult
central pattern generator neurons in Lymnaea stagnalis. PLoS. One. 6, e18745.
Lu,T.Z., and Feng,Z.P. (2012). NALCN: A Regulator of Pacemaker Activity. Mol. Neurobiol.
187
Ludwig,A., Budde,T., Stieber,J., Moosmang,S., Wahl,C., Holthoff,K., Langebartels,A.,
Wotjak,C., Munsch,T., Zong,X., Feil,S., Feil,R., Lancel,M., Chien,K.R., Konnerth,A.,
Pape,H.C., Biel,M., and Hofmann,F. (2003). Absence epilepsy and sinus dysrhythmia in mice
lacking the pacemaker channel HCN2. EMBO J. 22, 216-224.
Ludwig,A., Zong,X., Hofmann,F., and Biel,M. (1999a). Structure and function of cardiac
pacemaker channels. Cell Physiol Biochem. 9, 179-186.
Ludwig,A., Zong,X., Jeglitsch,M., Hofmann,F., and Biel,M. (1998). A family of
hyperpolarization-activated mammalian cation channels. Nature 393, 587-591.
Ludwig,A., Zong,X., Stieber,J., Hullin,R., Hofmann,F., and Biel,M. (1999b). Two pacemaker
channels from human heart with profoundly different activation kinetics. EMBO J. 18, 2323-
2329.
Lund,J.P., and Kolta,A. (2006). Generation of the central masticatory pattern and its
modification by sensory feedback. Dysphagia 21, 167-174.
Luzhkov,V.B., and Aqvist,J. (2001). Mechanisms of tetraethylammonium ion block in the KcsA
potassium channel. FEBS Lett. 495, 191-196.
Maccaferri,G., Mangoni,M., Lazzari,A., and DiFrancesco,D. (1993). Properties of the
hyperpolarization-activated current in rat hippocampal CA1 pyramidal cells. J. Neurophysiol. 69,
2129-2136.
Maccaferri,G., and McBain,C.J. (1996). The hyperpolarization-activated current (Ih) and its
contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones.
J. Physiol 497 ( Pt 1), 119-130.
MacLean,J.N., Zhang,Y., Goeritz,M.L., Casey,R., Oliva,R., Guckenheimer,J., and Harris-
Warrick,R.M. (2005). Activity-independent coregulation of IA and Ih in rhythmically active
neurons. J. Neurophysiol. 94, 3601-3617.
Macri,V., and Accili,E.A. (2004). Structural elements of instantaneous and slow gating in
hyperpolarization-activated cyclic nucleotide-gated channels. J. Biol. Chem. 279, 16832-16846.
Macri,V., Proenza,C., Agranovich,E., Angoli,D., and Accili,E.A. (2002). Separable gating
mechanisms in a Mammalian pacemaker channel. J. Biol. Chem. 277, 35939-35946.
Maguy,A., Hebert,T.E., and Nattel,S. (2006). Involvement of lipid rafts and caveolae in cardiac
ion channel function. Cardiovasc. Res. 69, 798-807.
Mahapatra,S., Marcantoni,A., Vandael,D.H., Striessnig,J., and Carbone,E. (2011). Are Ca(v)1.3
pacemaker channels in chromaffin cells? Possible bias from resting cell conditions and DHP
blockers usage. Channels (Austin. ) 5, 219-224.
Maingret,F., Fosset,M., Lesage,F., Lazdunski,M., and Honore,E. (1999a). TRAAK is a
mammalian neuronal mechano-gated K+ channel. J. Biol. Chem. 274, 1381-1387.
188
Maingret,F., Lauritzen,I., Patel,A.J., Heurteaux,C., Reyes,R., Lesage,F., Lazdunski,M., and
Honore,E. (2000a). TREK-1 is a heat-activated background K(+) channel. EMBO J. 19, 2483-
2491.
Maingret,F., Patel,A.J., Lesage,F., Lazdunski,M., and Honore,E. (2000b). Lysophospholipids
open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK. J. Biol. Chem.
275, 10128-10133.
Maingret,F., Patel,A.J., Lesage,F., Lazdunski,M., and Honore,E. (1999b). Mechano- or acid
stimulation, two interactive modes of activation of the TREK-1 potassium channel. J. Biol.
Chem. 274, 26691-26696.
Malasics,A., Boda,D., Valisko,M., Henderson,D., and Gillespie,D. (2010). Simulations of
calcium channel block by trivalent cations: Gd(3+) competes with permeant ions for the
selectivity filter. Biochim. Biophys. Acta 1798, 2013-2021.
Maltsev,V.A., Vinogradova,T.M., Bogdanov,K.Y., Lakatta,E.G., and Stern,M.D. (2004).
Diastolic calcium release controls the beating rate of rabbit sinoatrial node cells: numerical
modeling of the coupling process. Biophys. J. 86, 2596-2605.
Mangoni,M.E., Couette,B., Bourinet,E., Platzer,J., Reimer,D., Striessnig,J., and Nargeot,J.
(2003). Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc.
Natl. Acad. Sci. U. S. A 100, 5543-5548.
Mangoni,M.E., Couette,B., Marger,L., Bourinet,E., Striessnig,J., and Nargeot,J. (2006a).
Voltage-dependent calcium channels and cardiac pacemaker activity: from ionic currents to
genes. Prog. Biophys. Mol. Biol. 90, 38-63.
Mangoni,M.E., and Nargeot,J. (2001). Properties of the hyperpolarization-activated current (I(f))
in isolated mouse sino-atrial cells. Cardiovasc. Res. 52, 51-64.
Mangoni,M.E., and Nargeot,J. (2008). Genesis and regulation of the heart automaticity. Physiol
Rev. 88, 919-982.
Mangoni,M.E., Traboulsie,A., Leoni,A.L., Couette,B., Marger,L., Le,Q.K., Kupfer,E., Cohen-
Solal,A., Vilar,J., Shin,H.S., Escande,D., Charpentier,F., Nargeot,J., and Lory,P. (2006b).
Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-
type calcium channels. Circ. Res. 98, 1422-1430.
Mannikko,R., Elinder,F., and Larsson,H.P. (2002). Voltage-sensing mechanism is conserved
among ion channels gated by opposite voltages. Nature 419, 837-841.
Marcantoni,A., Vandael,D.H., Mahapatra,S., Carabelli,V., Sinnegger-Brauns,M.J., Striessnig,J.,
and Carbone,E. (2010). Loss of Cav1.3 channels reveals the critical role of L-type and BK
channel coupling in pacemaking mouse adrenal chromaffin cells. J. Neurosci. 30, 491-504.
Marger,L., Mesirca,P., Alig,J., Torrente,A., Dubel,S., Engeland,B., Kanani,S., Fontanaud,P.,
Striessnig,J., Shin,H.S., Isbrandt,D., Ehmke,H., Nargeot,J., and Mangoni,M.E. (2011).
189
Functional roles of Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of mouse
atrioventricular cells: insights into the atrioventricular pacemaker mechanism. Channels (Austin.
) 5, 251-261.
Marionneau,C., Couette,B., Liu,J., Li,H., Mangoni,M.E., Nargeot,J., Lei,M., Escande,D., and
Demolombe,S. (2005). Specific pattern of ionic channel gene expression associated with
pacemaker activity in the mouse heart. J. Physiol 562, 223-234.
Marks,A.R., Marx,S.O., and Reiken,S. (2002). Regulation of ryanodine receptors via
macromolecular complexes: a novel role for leucine/isoleucine zippers. Trends Cardiovasc. Med.
12, 166-170.
Marrion,N.V., and Tavalin,S.J. (1998). Selective activation of Ca2+-activated K+ channels by
co-localized Ca2+ channels in hippocampal neurons. Nature 395, 900-905.
Marx,S.O., Reiken,S., Hisamatsu,Y., Jayaraman,T., Burkhoff,D., Rosemblit,N., and Marks,A.R.
(2000). PKA phosphorylation dissociates FKBP12.6 from the calcium release channel
(ryanodine receptor): defective regulation in failing hearts. Cell 101, 365-376.
Marx,T., Gisselmann,G., Stortkuhl,K.F., Hovemann,B.T., and Hatt,H. (1999). Molecular cloning
of a putative voltage- and cyclic nucleotide-gated ion channel present in the antennae and eyes of
Drosophila melanogaster. Invert. Neurosci. 4, 55-63.
Mathie,A. (2007). Neuronal two-pore-domain potassium channels and their regulation by G
protein-coupled receptors. J. Physiol 578, 377-385.
Matsuoka,S., Sarai,N., Kuratomi,S., Ono,K., and Noma,A. (2003). Role of individual ionic
current systems in ventricular cells hypothesized by a model study. Jpn. J. Physiol 53, 105-123.
Matsuura,H., Ehara,T., Ding,W.G., Omatsu-Kanbe,M., and Isono,T. (2002). Rapidly and slowly
activating components of delayed rectifier K(+) current in guinea-pig sino-atrial node pacemaker
cells. J. Physiol 540, 815-830.
Maurice,N., Tkatch,T., Meisler,M., Sprunger,L.K., and Surmeier,D.J. (2001). D1/D5 dopamine
receptor activation differentially modulates rapidly inactivating and persistent sodium currents in
prefrontal cortex pyramidal neurons. J. Neurosci. 21, 2268-2277.
Mayer,M.L. (1985). A calcium-activated chloride current generates the after-depolarization of rat
sensory neurones in culture. J. Physiol 364, 217-239.
McComb,C., Meems,R., Syed,N., and Lukowiak,K. (2003). Electrophysiological differences in
the CpG aerial respiratory behavior between juvenile and adult Lymnaea. J. Neurophysiol. 90,
983-992.
McCormick,D.A., and Bal,T. (1997). Sleep and arousal: thalamocortical mechanisms. Annu.
Rev. Neurosci. 20, 185-215.
190
McCormick,D.A., and Huguenard,J.R. (1992). A model of the electrophysiological properties of
thalamocortical relay neurons. J. Neurophysiol. 68, 1384-1400.
McCormick,D.A., and Pape,H.C. (1990). Properties of a hyperpolarization-activated cation
current and its role in rhythmic oscillation in thalamic relay neurones. J. Physiol 431, 291-318.
Mercer,J.N., Chan,C.S., Tkatch,T., Held,J., and Surmeier,D.J. (2007). Nav1.6 sodium channels
are critical to pacemaking and fast spiking in globus pallidus neurons. J. Neurosci. 27, 13552-
13566.
Mereu,G., Lilliu,V., Casula,A., Vargiu,P.F., Diana,M., Musa,A., and Gessa,G.L. (1997).
Spontaneous bursting activity of dopaminergic neurons in midbrain slices from immature rats:
role of N-methyl-D-aspartate receptors. Neuroscience 77, 1029-1036.
Milanesi,R., Baruscotti,M., Gnecchi-Ruscone,T., and DiFrancesco,D. (2006). Familial sinus
bradycardia associated with a mutation in the cardiac pacemaker channel. N. Engl. J. Med. 354,
151-157.
Miller,C. (1982). Open-state substructure of single chloride channels from Torpedo electroplax.
Philos. Trans. R. Soc. Lond B Biol. Sci. 299, 401-411.
Miller,C., and White,M.M. (1984). Dimeric structure of single chloride channels from Torpedo
electroplax. Proc. Natl. Acad. Sci. U. S. A 81, 2772-2775.
Mironov,S.L. (2008). Metabotropic glutamate receptors activate dendritic calcium waves and
TRPM channels which drive rhythmic respiratory patterns in mice. J. Physiol 586, 2277-2291.
Mlinar,B., and Enyeart,J.J. (1993). Block of current through T-type calcium channels by trivalent
metal cations and nickel in neural rat and human cells. J. Physiol 469, 639-652.
Moccia,F., Di,C.C., Winlow,W., and Di,C.A. (2009). GABA(A)- and AMPA-like receptors
modulate the activity of an identified neuron within the central pattern generator of the pond
snail Lymnaea stagnalis. Invert. Neurosci. 9, 29-41.
Moffat,M.P. (1987). Concentration-dependent effects of prostacyclin on the response of the
isolated guinea pig heart to ischemia and reperfusion: possible involvement of the slow inward
current. J. Pharmacol. Exp. Ther. 242, 292-299.
Mommersteeg,M.T., Hoogaars,W.M., Prall,O.W., de Gier-de,V.C., Wiese,C., Clout,D.E.,
Papaioannou,V.E., Brown,N.A., Harvey,R.P., Moorman,A.F., and Christoffels,V.M. (2007).
Molecular pathway for the localized formation of the sinoatrial node. Circ. Res. 100, 354-362.
Montell,C. (2005). The TRP superfamily of cation channels. Sci. STKE. 2005, re3.
Morgado-Valle,C., and Feldman,J.L. (2004). Depletion of substance P and glutamate by
capsaicin blocks respiratory rhythm in neonatal rat in vitro. J. Physiol 555, 783-792.
191
Morris,C., and Lecar,H. (1981). Voltage oscillations in the barnacle giant muscle fiber. Biophys.
J. 35, 193-213.
Mrejeru,A., Wei,A., and Ramirez,J.M. (2011). Calcium-activated non-selective cation currents
are involved in generation of tonic and bursting activity in dopamine neurons of the substantia
nigra pars compacta. J. Physiol 589, 2497-2514.
Munch,G., Bolck,B., Sugaru,A., Brixius,K., Bloch,W., and Schwinger,R.H. (2001). Increased
expression of isoform 1 of the sarcoplasmic reticulum Ca(2+)-release channel in failing human
heart. Circulation 103, 2739-2744.
Murbartian,J., Lei,Q., Sando,J.J., and Bayliss,D.A. (2005). Sequential phosphorylation mediates
receptor- and kinase-induced inhibition of TREK-1 background potassium channels. J. Biol.
Chem. 280, 30175-30184.
Nakayama,S., Kajioka,S., Goto,K., Takaki,M., and Liu,H.N. (2007). Calcium-associated
mechanisms in gut pacemaker activity. J. Cell Mol. Med. 11, 958-968.
Narahashi,T., Moore,J.W., and SCOTT,W.R. (1964). TETRODOTOXIN BLOCKAGE OF
SODIUM CONDUCTANCE INCREASE IN LOBSTER GIANT AXONS. J. Gen. Physiol 47,
965-974.
Nash,H.A., Scott,R.L., Lear,B.C., and Allada,R. (2002). An unusual cation channel mediates
photic control of locomotion in Drosophila. Curr. Biol. 12, 2152-2158.
Nattel,S., Maguy,A., Le,B.S., and Yeh,Y.H. (2007). Arrhythmogenic ion-channel remodeling in
the heart: heart failure, myocardial infarction, and atrial fibrillation. Physiol Rev. 87, 425-456.
Nejatbakhsh,N., Guo,C.H., Lu,T.Z., Pei,L., Smit,A.B., Sun,H.S., van Kesteren,R.E., and
Feng,Z.P. (2011). Caltubin, a novel molluscan tubulin-interacting protein, promotes axonal
growth and attenuates axonal degeneration of rodent neurons. J. Neurosci. 31, 15231-15244.
Neubauer,J.A., Melton,J.E., and Edelman,N.H. (1990). Modulation of respiration during brain
hypoxia. J. Appl. Physiol 68, 441-451.
Newland,C.F., Adelman,J.P., Tempel,B.L., and Almers,W. (1992). Repulsion between
tetraethylammonium ions in cloned voltage-gated potassium channels. Neuron 8, 975-982.
Nikitin,E.S., Kiss,T., Staras,K., O'Shea,M., Benjamin,P.R., and Kemenes,G. (2006). Persistent
sodium current is a target for cAMP-induced neuronal plasticity in a state-setting modulatory
interneuron. J. Neurophysiol. 95, 453-463.
Nikmaram,M.R., Boyett,M.R., Kodama,I., Suzuki,R., and Honjo,H. (1997). Variation in effects
of Cs+, UL-FS-49, and ZD-7288 within sinoatrial node. Am. J. Physiol 272, H2782-H2792.
Nilius,B., and Owsianik,G. (2010). Transient receptor potential channelopathies. Pflugers Arch.
460, 437-450.
192
Nilius,B., Prenen,J., Droogmans,G., Voets,T., Vennekens,R., Freichel,M., Wissenbach,U., and
Flockerzi,V. (2003). Voltage dependence of the Ca2+-activated cation channel TRPM4. J. Biol.
Chem. 278, 30813-30820.
Nilius,B., Prenen,J., Janssens,A., Owsianik,G., Wang,C., Zhu,M.X., and Voets,T. (2005a). The
selectivity filter of the cation channel TRPM4. J. Biol. Chem. 280, 22899-22906.
Nilius,B., Prenen,J., Voets,T., and Droogmans,G. (2004). Intracellular nucleotides and
polyamines inhibit the Ca2+-activated cation channel TRPM4b. Pflugers Arch. 448, 70-75.
Nilius,B., Talavera,K., Owsianik,G., Prenen,J., Droogmans,G., and Voets,T. (2005b). Gating of
TRP channels: a voltage connection? J. Physiol 567, 35-44.
Nilius,B., Vennekens,R., Prenen,J., Hoenderop,J.G., Bindels,R.J., and Droogmans,G. (2000).
Whole-cell and single channel monovalent cation currents through the novel rabbit epithelial
Ca2+ channel ECaC. J. Physiol 527 Pt 2, 239-248.
Nitabach,M.N., Blau,J., and Holmes,T.C. (2002). Electrical silencing of Drosophila pacemaker
neurons stops the free-running circadian clock. Cell 109, 485-495.
Noble,D. (1960). Cardiac action and pacemaker potentials based on the Hodgkin-Huxley
equations. Nature 188, 495-497.
Noble,D. (1962). A modification of the Hodgkin--Huxley equations applicable to Purkinje fibre
action and pace-maker potentials. J. Physiol 160, 317-352.
Noble,D., Denyer,J.C., Brown,H.F., and DiFrancesco,D. (1992). Reciprocal role of the inward
currents ib, Na and i(f) in controlling and stabilizing pacemaker frequency of rabbit sino-atrial
node cells. Proc. Biol. Sci. 250, 199-207.
Noble,D., DiFrancesco,D., and Denyer,J.C. (1989). Ionic mechanisms in normal and abnormal
cardiac pacemaker activity. In Neuronal and Cellular Oscillators, J.W. Jacklet, ed. Marcel
Dekker Inc), pp. 59-85.
Noble,D., and Noble,S.J. (1984). A model of sino-atrial node electrical activity based on a
modification of the DiFrancesco-Noble (1984) equations. Proc. R. Soc. Lond B Biol. Sci. 222,
295-304.
Nof,E., Luria,D., Brass,D., Marek,D., Lahat,H., Reznik-Wolf,H., Pras,E., Dascal,N., Eldar,M.,
and Glikson,M. (2007). Point mutation in the HCN4 cardiac ion channel pore affecting synthesis,
trafficking, and functional expression is associated with familial asymptomatic sinus
bradycardia. Circulation 116, 463-470.
Nolan,M.F., Malleret,G., Dudman,J.T., Buhl,D.L., Santoro,B., Gibbs,E., Vronskaya,S.,
Buzsaki,G., Siegelbaum,S.A., Kandel,E.R., and Morozov,A. (2004). A behavioral role for
dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal
dendrites of CA1 pyramidal neurons. Cell 119, 719-732.
193
Noma,A., and Irisawa,H. (1976). Membrane currents in the rabbit sinoatrial node cell as studied
by the double microelectrode method. Pflugers Arch. 364, 45-52.
Nowycky,M.C., Fox,A.P., and Tsien,R.W. (1985). Three types of neuronal calcium channel with
different calcium agonist sensitivity. Nature 316, 440-443.
Nuss,H.B., Kaab,S., Kass,D.A., Tomaselli,G.F., and Marban,E. (1999). Cellular basis of
ventricular arrhythmias and abnormal automaticity in heart failure. Am. J. Physiol 277, H80-
H91.
Ogata,N., and Tatebayashi,H. (1993). Kinetic analysis of two types of Na+ channels in rat dorsal
root ganglia. J. Physiol 466, 9-37.
Onimaru,H., Arata,A., and Homma,I. (1987). Localization of respiratory rhythm-generating
neurons in the medulla of brainstem-spinal cord preparations from newborn rats. Neurosci. Lett.
78, 151-155.
Onimaru,H., Arata,A., and Homma,I. (1988). Primary respiratory rhythm generator in the
medulla of brainstem-spinal cord preparation from newborn rat. Brain Res. 445, 314-324.
Onimaru,H., Arata,A., and Homma,I. (1997). Neuronal mechanisms of respiratory rhythm
generation: an approach using in vitro preparation. Jpn. J. Physiol 47, 385-403.
Onimaru,H., Arata,A., and Homma,I. (1990). Inhibitory synaptic inputs to the respiratory rhythm
generator in the medulla isolated from newborn rats. Pflugers Arch. 417, 425-432.
Onimaru,H., and Homma,I. (2003). A novel functional neuron group for respiratory rhythm
generation in the ventral medulla. J. Neurosci. 23, 1478-1486.
Onizuka,S., Kasaba,T., Hamakawa,T., and Takasaki,M. (2005). Lidocaine excites both pre- and
postsynaptic neurons of reconstructed respiratory pattern generator in Lymnaea stagnalis.
Anesth. Analg. 100, 175-182.
Ono,K., and Ito,H. (1995). Role of rapidly activating delayed rectifier K+ current in sinoatrial
node pacemaker activity. Am. J. Physiol 269, H453-H462.
Opthof,T., Coronel,R., Rademaker,H.M., Vermeulen,J.T., Wilms-Schopman,F.J., and Janse,M.J.
(2000). Changes in sinus node function in a rabbit model of heart failure with ventricular
arrhythmias and sudden death. Circulation 101, 2975-2980.
Opthof,T., de,J.B., Jongsma,H.J., and Bouman,L.N. (1987). Functional morphology of the
mammalian sinuatrial node. Eur. Heart J. 8, 1249-1259.
Ornato,J.P., and Peberdy,M.A. (1996). The mystery of bradyasystole during cardiac arrest. Ann.
Emerg. Med. 27, 576-587.
194
Paarmann,I., Frermann,D., Keller,B.U., and Hollmann,M. (2000). Expression of 15 glutamate
receptor subunits and various splice variants in tissue slices and single neurons of brainstem
nuclei and potential functional implications. J. Neurochem. 74, 1335-1345.
Paarmann,I., Frermann,D., Keller,B.U., Villmann,C., Breitinger,H.G., and Hollmann,M. (2005).
Kinetics and subunit composition of NMDA receptors in respiratory-related neurons. J.
Neurochem. 93, 812-824.
Pace,R.W., Mackay,D.D., Feldman,J.L., and Del Negro,C.A. (2007). Role of persistent sodium
current in mouse preBotzinger Complex neurons and respiratory rhythm generation. J. Physiol
580, 485-496.
Pang,D.S., Robledo,C.J., Carr,D.R., Gent,T.C., Vyssotski,A.L., Caley,A., Zecharia,A.Y.,
Wisden,W., Brickley,S.G., and Franks,N.P. (2009). An unexpected role for TASK-3 potassium
channels in network oscillations with implications for sleep mechanisms and anesthetic action.
Proc. Natl. Acad. Sci. U. S. A 106, 17546-17551.
Papp,Z., Sipido,K.R., Callewaert,G., and Carmeliet,E. (1995). Two components of [Ca2+]i-
activated Cl- current during large [Ca2+]i transients in single rabbit heart Purkinje cells. J.
Physiol 483 ( Pt 2), 319-330.
Partridge,L.D., and Valenzuela,C.F. (2000). Block of hippocampal CAN channels by
flufenamate. Brain Res. 867, 143-148.
Patel,A.J., Honore,E., Lesage,F., Fink,M., Romey,G., and Lazdunski,M. (1999). Inhalational
anesthetics activate two-pore-domain background K+ channels. Nat. Neurosci. 2, 422-426.
Paton,J.F., Abdala,A.P., Koizumi,H., Smith,J.C., and St-John,W.M. (2006). Respiratory rhythm
generation during gasping depends on persistent sodium current. Nat. Neurosci. 9, 311-313.
Payne,J.A., Xu,J.C., Haas,M., Lytle,C.Y., Ward,D., and Forbush,B., III (1995). Primary
structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-
K-Cl cotransporter in human colon. J. Biol. Chem. 270, 17977-17985.
Pena,F., and Aguileta,M.A. (2007). Effects of riluzole and flufenamic acid on eupnea and
gasping of neonatal mice in vivo. Neurosci. Lett. 415, 288-293.
Pena,F., Parkis,M.A., Tryba,A.K., and Ramirez,J.M. (2004). Differential contribution of
pacemaker properties to the generation of respiratory rhythms during normoxia and hypoxia.
Neuron 43, 105-117.
Pena,F., and Ramirez,J.M. (2004). Substance P-mediated modulation of pacemaker properties in
the mammalian respiratory network. J. Neurosci. 24, 7549-7556.
Pena,F., and Ramirez,J.M. (2002). Endogenous activation of serotonin-2A receptors is required
for respiratory rhythm generation in vitro. J. Neurosci. 22, 11055-11064.
195
Peng,Y., Stoleru,D., Levine,J.D., Hall,J.C., and Rosbash,M. (2003). Drosophila free-running
rhythms require intercellular communication. PLoS. Biol. 1, E13.
Peralta,E.G., Ashkenazi,A., Winslow,J.W., Smith,D.H., Ramachandran,J., and Capon,D.J.
(1987). Distinct primary structures, ligand-binding properties and tissue-specific expression of
four human muscarinic acetylcholine receptors. EMBO J. 6, 3923-3929.
Perez,N.G., Alvarez,B.V., Camilion de Hurtado,M.C., and Cingolani,H.E. (1995). pHi regulation
in myocardium of the spontaneously hypertensive rat. Compensated enhanced activity of the
Na(+)-H+ exchanger. Circ. Res. 77, 1192-1200.
Perez-Reyes,E. (2003). Molecular physiology of low-voltage-activated t-type calcium channels.
Physiol Rev. 83, 117-161.
Perkel,D.H., and Mulloney,B. (1974). Motor pattern production in reciprocally inhibitory
neurons exhibiting postinhibitory rebound. Science 185, 181-183.
Pfaffl,M.W. (2001). A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Res. 29, e45.
Pieske,B., Maier,L.S., Piacentino,V., III, Weisser,J., Hasenfuss,G., and Houser,S. (2002). Rate
dependence of [Na+]i and contractility in nonfailing and failing human myocardium. Circulation
106, 447-453.
Pignatelli,A., Ackman,J.B., Vigetti,D., Beltrami,A.P., Zucchini,S., and Belluzzi,O. (2009). A
potential reservoir of immature dopaminergic replacement neurons in the adult mammalian
olfactory bulb. Pflugers Arch. 457, 899-915.
Platzer,J., Engel,J., Schrott-Fischer,A., Stephan,K., Bova,S., Chen,H., Zheng,H., and
Striessnig,J. (2000). Congenital deafness and sinoatrial node dysfunction in mice lacking class D
L-type Ca2+ channels. Cell 102, 89-97.
Pogwizd,S.M., Schlotthauer,K., Li,L., Yuan,W., and Bers,D.M. (2001). Arrhythmogenesis and
contractile dysfunction in heart failure: Roles of sodium-calcium exchange, inward rectifier
potassium current, and residual beta-adrenergic responsiveness. Circ. Res. 88, 1159-1167.
Potse,M., Dube,B., Richer,J., Vinet,A., and Gulrajani,R.M. (2006). A comparison of
monodomain and bidomain reaction-diffusion models for action potential propagation in the
human heart. IEEE Trans. Biomed. Eng 53, 2425-2435.
Pragnell,M., De,W.M., Mori,Y., Tanabe,T., Snutch,T.P., and Campbell,K.P. (1994). Calcium
channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-
subunit. Nature 368, 67-70.
Prinz,A.A., Bucher,D., and Marder,E. (2004). Similar network activity from disparate circuit
parameters. Nat. Neurosci. 7, 1345-1352.
196
Proenza,C., Angoli,D., Agranovich,E., Macri,V., and Accili,E.A. (2002). Pacemaker channels
produce an instantaneous current. J. Biol. Chem. 277, 5101-5109.
Ptak,K., Yamanishi,T., Aungst,J., Milescu,L.S., Zhang,R., Richerson,G.B., and Smith,J.C.
(2009). Raphe neurons stimulate respiratory circuit activity by multiple mechanisms via
endogenously released serotonin and substance P. J. Neurosci. 29, 3720-3737.
Puopolo,M., Raviola,E., and Bean,B.P. (2007). Roles of subthreshold calcium current and
sodium current in spontaneous firing of mouse midbrain dopamine neurons. J. Neurosci. 27, 645-
656.
Quik,M., and Wonnacott,S. (2011). alpha6beta2* and alpha4beta2* nicotinic acetylcholine
receptors as drug targets for Parkinson's disease. Pharmacol. Rev. 63, 938-966.
Rae,J., Cooper,K., Gates,P., and Watsky,M. (1991). Low access resistance perforated patch
recordings using amphotericin B. J. Neurosci. Methods 37, 15-26.
Raman,I.M., Gustafson,A.E., and Padgett,D. (2000). Ionic currents and spontaneous firing in
neurons isolated from the cerebellar nuclei. J. Neurosci. 20, 9004-9016.
Raman,I.M., Sprunger,L.K., Meisler,M.H., and Bean,B.P. (1997). Altered subthreshold sodium
currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice. Neuron 19, 881-
891.
Ramsey,I.S., Delling,M., and Clapham,D.E. (2006). An introduction to TRP channels. Annu.
Rev. Physiol 68, 619-647.
Randall,A., and Tsien,R.W. (1995). Pharmacological dissection of multiple types of Ca2+
channel currents in rat cerebellar granule neurons. J. Neurosci. 15, 2995-3012.
Rekling,J.C., Funk,G.D., Bayliss,D.A., Dong,X.W., and Feldman,J.L. (2000). Synaptic control
of motoneuronal excitability. Physiol Rev. 80, 767-852.
Ren,D. (2011). Sodium leak channels in neuronal excitability and rhythmic behaviors. Neuron
72, 899-911.
Reynolds,A., Leake,D., Boese,Q., Scaringe,S., Marshall,W.S., and Khvorova,A. (2004). Rational
siRNA design for RNA interference. Nat. Biotechnol. 22, 326-330.
Richter,D.W., Bischoff,A., Anders,K., Bellingham,M., and Windhorst,U. (1991). Response of
the medullary respiratory network of the cat to hypoxia. J. Physiol 443, 231-256.
Rinke,I., Artmann,J., and Stein,V. (2010). ClC-2 voltage-gated channels constitute part of the
background conductance and assist chloride extrusion. J. Neurosci. 30, 4776-4786.
Rivera,C., Voipio,J., Payne,J.A., Ruusuvuori,E., Lahtinen,H., Lamsa,K., Pirvola,U., Saarma,M.,
and Kaila,K. (1999). The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during
neuronal maturation. Nature 397, 251-255.
197
Robbins,M., Judge,A., and Maclachlan,I. (2009). siRNA and innate immunity. Oligonucleotides.
19, 89-102.
Robinson,R.B., and Siegelbaum,S.A. (2003). Hyperpolarization-activated cation currents: from
molecules to physiological function. Annu. Rev. Physiol 65, 453-480.
Roffman,R.C., Norris,B.J., and Calabrese,R.L. (2012). Animal-to-animal variability of
connection strength in the leech heartbeat central pattern generator. J. Neurophysiol. 107, 1681-
1693.
Rohl,C.A., Boeckman,F.A., Baker,C., Scheuer,T., Catterall,W.A., and Klevit,R.E. (1999).
Solution structure of the sodium channel inactivation gate. Biochemistry 38, 855-861.
Rosati,B., Yan,Q., Lee,M.S., Liou,S.R., Ingalls,B., Foell,J., Kamp,T.J., and McKinnon,D.
(2011). Robust L-type calcium current expression following heterozygous knockout of the
Cav1.2 gene in adult mouse heart. J. Physiol 589, 3275-3288.
Rose,R.A., Kabir,M.G., and Backx,P.H. (2007). Altered heart rate and sinoatrial node function in
mice lacking the cAMP regulator phosphoinositide 3-kinase-gamma. Circ. Res. 101, 1274-1282.
Rose,R.M., and Hindmarsh,J.L. (1985). A model of a thalamic neuron. Proc. R. Soc. Lond B
Biol. Sci. 225, 161-193.
Ruan,Y., Liu,N., and Priori,S.G. (2009). Sodium channel mutations and arrhythmias. Nat. Rev.
Cardiol. 6, 337-348.
Rubin,J.E., Hayes,J.A., Mendenhall,J.L., and Del Negro,C.A. (2009). Calcium-activated
nonspecific cation current and synaptic depression promote network-dependent burst
oscillations. Proc. Natl. Acad. Sci. U. S. A 106, 2939-2944.
Russell,D.F. (1979). CNS control of pattern generators in the lobster stomatogastric ganglion.
Brain Res. 177, 598-602.
Russo,M.J., Mugnaini,E., and Martina,M. (2007). Intrinsic properties and mechanisms of
spontaneous firing in mouse cerebellar unipolar brush cells. J. Physiol 581, 709-724.
Rybak,I.A., Abdala,A.P., Markin,S.N., Paton,J.F., and Smith,J.C. (2007). Spatial organization
and state-dependent mechanisms for respiratory rhythm and pattern generation. Prog. Brain Res.
165, 201-220.
Rybak,I.A., Shevtsova,N.A., Ptak,K., and McCrimmon,D.R. (2004). Intrinsic bursting activity in
the pre-Botzinger complex: role of persistent sodium and potassium currents. Biol. Cybern. 90,
59-74.
Sabourin,J., Robin,E., and Raddatz,E. (2011). A key role of TRPC channels in the regulation of
electromechanical activity of the developing heart. Cardiovasc. Res. 92, 226-236.
198
Sacchi,O., Rossi,M.L., Canella,R., and Fesce,R. (1999). Participation of a chloride conductance
in the subthreshold behavior of the rat sympathetic neuron. J. Neurophysiol. 82, 1662-1675.
Safronov,B.V., and Vogel,W. (1996). Properties and functions of Na(+)-activated K+ channels
in the soma of rat motoneurones. J. Physiol 497 ( Pt 3), 727-734.
Sah,R., Ramirez,R.J., Oudit,G.Y., Gidrewicz,D., Trivieri,M.G., Zobel,C., and Backx,P.H.
(2003). Regulation of cardiac excitation-contraction coupling by action potential repolarization:
role of the transient outward potassium current (I(to)). J. Physiol 546, 5-18.
Sakakibara,M., Okuda,F., Nomura,K., Watanabe,K., Meng,H., Horikoshi,T., and Lukowiak,K.
(2005). Potassium currents in isolated statocyst neurons and RPeD1 in the pond snail, Lymnaea
stagnalis. J. Neurophysiol. 94, 3884-3892.
Salkoff,L., Butler,A., Ferreira,G., Santi,C., and Wei,A. (2006). High-conductance potassium
channels of the SLO family. Nat. Rev. Neurosci. 7, 921-931.
Sanders,K.M., Koh,S.D., and Ward,S.M. (2006a). Interstitial cells of cajal as pacemakers in the
gastrointestinal tract. Annu. Rev. Physiol 68, 307-343.
Sanders,L., Rakovic,S., Lowe,M., Mattick,P.A., and Terrar,D.A. (2006b). Fundamental
importance of Na+-Ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial
node. J. Physiol 571, 639-649.
Sandmann,S., Yu,M., Kaschina,E., Blume,A., Bouzinova,E., Aalkjaer,C., and Unger,T. (2001).
Differential effects of angiotensin AT1 and AT2 receptors on the expression, translation and
function of the Na+-H+ exchanger and Na+-. J. Am. Coll. Cardiol. 37, 2154-2165.
Santoro,B., Liu,D.T., Yao,H., Bartsch,D., Kandel,E.R., Siegelbaum,S.A., and Tibbs,G.R. (1998).
Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell
93, 717-729.
Sarai,N., Matsuoka,S., Kuratomi,S., Ono,K., and Noma,A. (2003). Role of individual ionic
current systems in the SA node hypothesized by a model study. Jpn. J. Physiol 53, 125-134.
Sarna,S.K. (2008). Are interstitial cells of Cajal plurifunction cells in the gut? Am. J. Physiol
Gastrointest. Liver Physiol 294, G372-G390.
Satin,L.S., Tavalin,S.J., and Smolen,P.D. (1994). Inactivation of HIT cell Ca2+ current by a
simulated burst of Ca2+ action potentials. Biophys. J. 66, 141-148.
Satoh,T.O., and Yamada,M. (2000). A bradycardiac agent ZD7288 blocks the hyperpolarization-
activated current (I(h)) in retinal rod photoreceptors. Neuropharmacology 39, 1284-1291.
Schulze-Bahr,E., Neu,A., Friederich,P., Kaupp,U.B., Breithardt,G., Pongs,O., and Isbrandt,D.
(2003). Pacemaker channel dysfunction in a patient with sinus node disease. J. Clin. Invest 111,
1537-1545.
199
Schwartz,G., and Fehlings,M.G. (2002). Secondary injury mechanisms of spinal cord trauma: a
novel therapeutic approach for the management of secondary pathophysiology with the sodium
channel blocker riluzole. Prog. Brain Res. 137, 177-190.
Scott,R.H., Sutton,K.G., Griffin,A., Stapleton,S.R., and Currie,K.P. (1995). Aspects of calcium-
activated chloride currents: a neuronal perspective. Pharmacol. Ther. 66, 535-565.
Seifert,R., Scholten,A., Gauss,R., Mincheva,A., Lichter,P., and Kaupp,U.B. (1999). Molecular
characterization of a slowly gating human hyperpolarization-activated channel predominantly
expressed in thalamus, heart, and testis. Proc. Natl. Acad. Sci. U. S. A 96, 9391-9396.
Selverston,A.I. (2010). Invertebrate central pattern generator circuits. Philos. Trans. R. Soc.
Lond B Biol. Sci. 365, 2329-2345.
Seyama,I. (1977). The effect of Na, K and Cl ions on the resting membrane potential of sino-
atrial node cell of the rabbit. Jpn. J. Physiol 27, 577-588.
Seyama,I. (1979). Characteristics of the anion channel in the sino-atrial node cell of the rabbit. J.
Physiol 294, 447-460.
Shah,M., and Haylett,D.G. (2000). Ca(2+) channels involved in the generation of the slow
afterhyperpolarization in cultured rat hippocampal pyramidal neurons. J. Neurophysiol. 83,
2554-2561.
Shampine,L.F., and Reichelt,M.W. (1997). The MATLAB ODE Suite. SIAM J. on Scientific
Computing 18, 1-22.
Shao,X.M., and Feldman,J.L. (2005). Cholinergic neurotransmission in the preBotzinger
Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm.
Neuroscience 130, 1069-1081.
Shao,X.M., and Feldman,J.L. (2009b). Central cholinergic regulation of respiration: nicotinic
receptors. Acta Pharmacol. Sin. 30, 761-770.
Shao,X.M., and Feldman,J.L. (1997). Respiratory rhythm generation and synaptic inhibition of
expiratory neurons in pre-Botzinger complex: differential roles of glycinergic and GABAergic
neural transmission. J. Neurophysiol. 77, 1853-1860.
Shao,X.M., and Feldman,J.L. (2009a). Central cholinergic regulation of respiration: nicotinic
receptors. Acta Pharmacol. Sin. 30, 761-770.
Shao,X.M., Tan,W., Xiu,J., Puskar,N., Fonck,C., Lester,H.A., and Feldman,J.L. (2008). Alpha4*
nicotinic receptors in preBotzinger complex mediate cholinergic/nicotinic modulation of
respiratory rhythm. J. Neurosci. 28, 519-528.
Shen,K.Z., and North,R.A. (1992b). Substance P opens cation channels and closes potassium
channels in rat locus coeruleus neurons. Neuroscience 50, 345-353.
200
Shen,K.Z., and North,R.A. (1992a). Muscarine increases cation conductance and decreases
potassium conductance in rat locus coeruleus neurones. J. Physiol 455, 471-485.
SHERRINGTON,C.S. (1910). Flexion-reflex of the limb, crossed extension-reflex, and reflex
stepping and standing. J. Physiol 40, 28-121.
Shi,H., Wang,H., and Wang,Z. (1999a). Identification and characterization of multiple subtypes
of muscarinic acetylcholine receptors and their physiological functions in canine hearts. Mol.
Pharmacol. 55, 497-507.
Shi,W., Wymore,R., Yu,H., Wu,J., Wymore,R.T., Pan,Z., Robinson,R.B., Dixon,J.E.,
McKinnon,D., and Cohen,I.S. (1999b). Distribution and prevalence of hyperpolarization-
activated cation channel (HCN) mRNA expression in cardiac tissues. Circ. Res. 85, e1-e6.
Shiga,N., and Wangemann,P. (1995). Ion selectivity of volume regulatory mechanisms present
during a hypoosmotic challenge in vestibular dark cells. Biochim. Biophys. Acta 1240, 48-54.
Shin,K.S., Rothberg,B.S., and Yellen,G. (2001). Blocker state dependence and trapping in
hyperpolarization-activated cation channels: evidence for an intracellular activation gate. J. Gen.
Physiol 117, 91-101.
Shirokov,R., Ferreira,G., Yi,J., and Rios,E. (1998). Inactivation of gating currents of L-type
calcium channels. Specific role of the alpha 2 delta subunit. J. Gen. Physiol 111, 807-823.
Shistik,E., Ivanina,T., Puri,T., Hosey,M., and Dascal,N. (1995). Ca2+ current enhancement by
alpha 2/delta and beta subunits in Xenopus oocytes: contribution of changes in channel gating
and alpha 1 protein level. J. Physiol 489 ( Pt 1), 55-62.
Silverman,M.E., Grove,D., and Upshaw,C.B., Jr. (2006). Why does the heart beat? The
discovery of the electrical system of the heart. Circulation 113, 2775-2781.
Singer,D., Biel,M., Lotan,I., Flockerzi,V., Hofmann,F., and Dascal,N. (1991). The roles of the
subunits in the function of the calcium channel. Science 253, 1553-1557.
Sinke,A.P., Caputo,C., Tsaih,S.W., Yuan,R., Ren,D., Deen,P.M., and Korstanje,R. (2011).
Genetic analysis of mouse strains with variable serum sodium concentrations identifies the Nalcn
sodium channel as a novel player in osmoregulation. Physiol Genomics 43, 265-270.
Sipido,K.R., Callewaert,G., and Carmeliet,E. (1993). [Ca2+]i transients and [Ca2+]i-dependent
chloride current in single Purkinje cells from rabbit heart. J. Physiol 468, 641-667.
Sipila,S.T., Huttu,K., Voipio,J., and Kaila,K. (2006). Intrinsic bursting of immature CA3
pyramidal neurons and consequent giant depolarizing potentials are driven by a persistent Na+
current and terminated by a slow Ca2+ -activated K+ current. Eur. J. Neurosci. 23, 2330-2338.
Skinner,F.K., Kopell,N., and Marder,E. (1994). Mechanisms for oscillation and frequency
control in reciprocally inhibitory model neural networks. J. Comput. Neurosci. 1, 69-87.
201
Smith,D.C., Priola,D.V., and Blomquist,T.M. (1992). Substance P modulates autonomic nerve
activity in canine hearts. Am. J. Physiol 262, H1663-H1668.
Smith,J.C., Abdala,A.P., Koizumi,H., Rybak,I.A., and Paton,J.F. (2007). Spatial and functional
architecture of the mammalian brain stem respiratory network: a hierarchy of three oscillatory
mechanisms. J. Neurophysiol. 98, 3370-3387.
Smith,J.C., Ellenberger,H.H., Ballanyi,K., Richter,D.W., and Feldman,J.L. (1991). Pre-Botzinger
complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254,
726-729.
Song,J.H., Huang,C.S., Nagata,K., Yeh,J.Z., and Narahashi,T. (1997). Differential action of
riluzole on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. J. Pharmacol. Exp.
Ther. 282, 707-714.
Spafford,J.D., Munno,D.W., van,N.P., Feng,Z.P., Jarvis,S.E., Gallin,W.J., Smit,A.B.,
Zamponi,G.W., and Syed,N.I. (2003). Calcium channel structural determinants of synaptic
transmission between identified invertebrate neurons. J. Biol. Chem. 278, 4258-4267.
Spear,J.F., Michelson,E.L., and Moore,E.N. (1983a). Cellular electrophysiologic characteristics
of chronically infarcted myocardium in dogs susceptible to sustained ventricular
tachyarrhythmias. J. Am. Coll. Cardiol. 1, 1099-1110.
Spear,J.F., Michelson,E.L., and Moore,E.N. (1983b). Reduced space constant in slowly
conducting regions of chronically infarcted canine myocardium. Circ. Res. 53, 176-185.
Speca,D.J., Chihara,D., Ashique,A.M., Bowers,M.S., Pierce-Shimomura,J.T., Lee,J., Rabbee,N.,
Speed,T.P., Gularte,R.J., Chitwood,J., Medrano,J.F., Liao,M., Sonner,J.M., Eger,E.I.,
Peterson,A.S., and McIntire,S.L. (2010). Conserved role of unc-79 in ethanol responses in
lightweight mutant mice. PLoS. Genet. 6.
Spencer,G.E., Syed,N.I., and Lukowiak,K. (1999). Neural changes after operant conditioning of
the aerial respiratory behavior in Lymnaea stagnalis. J. Neurosci. 19, 1836-1843.
St-John,W.M., and Paton,J.F. (2004). Role of pontile mechanisms in the neurogenesis of eupnea.
Respir. Physiol Neurobiol. 143, 321-332.
Stafstrom,C.E., Schwindt,P.C., and Crill,W.E. (1982). Negative slope conductance due to a
persistent subthreshold sodium current in cat neocortical neurons in vitro. Brain Res. 236, 221-
226.
Staras,K., Gyori,J., and Kemenes,G. (2002). Voltage-gated ionic currents in an identified
modulatory cell type controlling molluscan feeding. Eur. J. Neurosci. 15, 109-119.
Stark,G.R., Kerr,I.M., Williams,B.R., Silverman,R.H., and Schreiber,R.D. (1998). How cells
respond to interferons. Annu. Rev. Biochem. 67, 227-264.
202
Stea,A., Dubel,S.J., Pragnell,M., Leonard,J.P., Campbell,K.P., and Snutch,T.P. (1993). A beta-
subunit normalizes the electrophysiological properties of a cloned N-type Ca2+ channel alpha 1-
subunit. Neuropharmacology 32, 1103-1116.
Steinmeyer,K., Klocke,R., Ortland,C., Gronemeier,M., Jockusch,H., Grunder,S., and Jentsch,T.J.
(1991). Inactivation of muscle chloride channel by transposon insertion in myotonic mice.
Nature 354, 304-308.
Steriade,M., and Timofeev,I. (2003). Neuronal plasticity in thalamocortical networks during
sleep and waking oscillations. Neuron 37, 563-576.
Stieber,J., Herrmann,S., Feil,S., Loster,J., Feil,R., Biel,M., Hofmann,F., and Ludwig,A. (2003).
The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker
action potentials in the embryonic heart. Proc. Natl. Acad. Sci. U. S. A 100, 15235-15240.
Stoleru,D., Peng,Y., Nawathean,P., and Rosbash,M. (2005). A resetting signal between
Drosophila pacemakers synchronizes morning and evening activity. Nature 438, 238-242.
Strupp,M., Kalla,R., Glasauer,S., Wagner,J., Hufner,K., Jahn,K., and Brandt,T. (2008).
Aminopyridines for the treatment of cerebellar and ocular motor disorders. Prog. Brain Res. 171,
535-541.
Stuhmer,W., Conti,F., Suzuki,H., Wang,X.D., Noda,M., Yahagi,N., Kubo,H., and Numa,S.
(1989). Structural parts involved in activation and inactivation of the sodium channel. Nature
339, 597-603.
Suckau,L., Fechner,H., Chemaly,E., Krohn,S., Hadri,L., Kockskamper,J., Westermann,D.,
Bisping,E., Ly,H., Wang,X., Kawase,Y., Chen,J., Liang,L., Sipo,I., Vetter,R., Weger,S.,
Kurreck,J., Erdmann,V., Tschope,C., Pieske,B., Lebeche,D., Schultheiss,H.P., Hajjar,R.J., and
Poller,W.C. (2009). Long-term cardiac-targeted RNA interference for the treatment of heart
failure restores cardiac function and reduces pathological hypertrophy. Circulation 119, 1241-
1252.
Swandulla,D., and Lux,H.D. (1985). Activation of a nonspecific cation conductance by
intracellular Ca2+ elevation in bursting pacemaker neurons of Helix pomatia. J. Neurophysiol.
54, 1430-1443.
Swayne,L.A., Mezghrani,A., Lory,P., Nargeot,J., and Monteil,A. (2010). The NALCN ion
channel is a new actor in pancreatic beta-cell physiology. Islets. 2, 54-56.
Swayne,L.A., Mezghrani,A., Varrault,A., Chemin,J., Bertrand,G., Dalle,S., Bourinet,E., Lory,P.,
Miller,R.J., Nargeot,J., and Monteil,A. (2009). The NALCN ion channel is activated by M3
muscarinic receptors in a pancreatic beta-cell line. EMBO Rep. 10, 873-880.
Syed,N.I., Bulloch,A.G., and Lukowiak,K. (1992). The respiratory central pattern generator
(CPG) of Lymnaea reconstructed in vitro. Acta Biol. Hung. 43, 409-419.
203
Syed,N.I., Bulloch,A.G., and Lukowiak,K. (1990). In vitro reconstruction of the respiratory
central pattern generator of the mollusk Lymnaea. Science 250, 282-285.
Syed,N.I., and Winlow,W. (1991). Coordination of locomotor and cardiorespiratory networks of
Lymnaea stagnalis by a pair of identified interneurones. J. Exp. Biol. 158, 37-62.
Takeuchi,A., Tatsumi,S., Sarai,N., Terashima,K., Matsuoka,S., and Noma,A. (2007). Role of
Ca2+ transporters and channels in the cardiac cell volume regulation. Ann. N. Y. Acad. Sci.
1099, 377-382.
Tan,W., Janczewski,W.A., Yang,P., Shao,X.M., Callaway,E.M., and Feldman,J.L. (2008).
Silencing preBotzinger complex somatostatin-expressing neurons induces persistent apnea in
awake rat. Nat. Neurosci. 11, 538-540.
Tanoue,S., Krishnan,P., Chatterjee,A., and Hardin,P.E. (2008). G protein-coupled receptor
kinase 2 is required for rhythmic olfactory responses in Drosophila. Curr. Biol. 18, 787-794.
Taylor,B.E., and Lukowiak,K. (2000). The respiratory central pattern generator of Lymnaea: a
model, measured and malleable. Respir. Physiol 122, 197-207.
Tazerart,S., Vinay,L., and Brocard,F. (2008). The persistent sodium current generates pacemaker
activities in the central pattern generator for locomotion and regulates the locomotor rhythm. J.
Neurosci. 28, 8577-8589.
Tepper,J.M., Martin,L.P., and Anderson,D.R. (1995). GABAA receptor-mediated inhibition of
rat substantia nigra dopaminergic neurons by pars reticulata projection neurons. J. Neurosci. 15,
3092-3103.
Tessier,S., Karczewski,P., Krause,E.G., Pansard,Y., Acar,C., Lang-Lazdunski,M., Mercadier,J.J.,
and Hatem,S.N. (1999). Regulation of the transient outward K(+) current by Ca(2+)/calmodulin-
dependent protein kinases II in human atrial myocytes. Circ. Res. 85, 810-819.
Thoby-Brisson,M., and Ramirez,J.M. (2000). Role of inspiratory pacemaker neurons in
mediating the hypoxic response of the respiratory network in vitro. J. Neurosci. 20, 5858-5866.
Thoby-Brisson,M., Telgkamp,P., and Ramirez,J.M. (2000). The role of the hyperpolarization-
activated current in modulating rhythmic activity in the isolated respiratory network of mice. J.
Neurosci. 20, 2994-3005.
Thomas,M.J., Sjaastad,I., Andersen,K., Helm,P.J., Wasserstrom,J.A., Sejersted,O.M., and
Ottersen,O.P. (2003). Localization and function of the Na+/Ca2+-exchanger in normal and
detubulated rat cardiomyocytes. J. Mol. Cell Cardiol. 35, 1325-1337.
Thompson,S. (1982). Aminopyridine block of transient potassium current. J. Gen. Physiol 80, 1-
18.
204
Thomsen,L., Robinson,T.L., Lee,J.C., Farraway,L.A., Hughes,M.J., Andrews,D.W., and
Huizinga,J.D. (1998). Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat. Med.
4, 848-851.
Tokimasa,T., and Akasu,T. (1990). Cyclic AMP regulates an inward rectifying sodium-
potassium current in dissociated bull-frog sympathetic neurones. J. Physiol 420, 409-429.
Tokimasa,T., and North,R.A. (1996). Effects of barium, lanthanum and gadolinium on
endogenous chloride and potassium currents in Xenopus oocytes. J. Physiol 496 ( Pt 3), 677-686.
Toporikova,N., and Butera,R.J. (2011). Two types of independent bursting mechanisms in
inspiratory neurons: an integrative model. J. Comput. Neurosci. 30, 515-528.
Torres,A.M., Bansal,P.S., Sunde,M., Clarke,C.E., Bursill,J.A., Smith,D.J., Bauskin,A.,
Breit,S.N., Campbell,T.J., Alewood,P.F., Kuchel,P.W., and Vandenberg,J.I. (2003). Structure of
the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type
inactivation. J. Biol. Chem. 278, 42136-42148.
Tryba,A.K., Pena,F., and Ramirez,J.M. (2006). Gasping activity in vitro: a rhythm dependent on
5-HT2A receptors. J. Neurosci. 26, 2623-2634.
Tryba,A.K., and Ramirez,J.M. (2004). Background sodium current stabilizes bursting in
respiratory pacemaker neurons. J. Neurobiol. 60, 481-489.
Tsien,R.W., Hess,P., McCleskey,E.W., and Rosenberg,R.L. (1987). Calcium channels:
mechanisms of selectivity, permeation, and block. Annu. Rev. Biophys. Biophys. Chem. 16,
265-290.
Tsien,R.W., Lipscombe,D., Madison,D.V., Bley,K.R., and Fox,A.P. (1988). Multiple types of
neuronal calcium channels and their selective modulation. Trends Neurosci. 11, 431-438.
Turner,D., and Stuenkel,E.L. (1998). Effects of depolarization evoked Na+ influx on intracellular
Na+ concentration at neurosecretory nerve endings. Neuroscience 86, 547-556.
Turner,D.L., and Mitchell,G.S. (1997). Long-term facilitation of ventilation following repeated
hypoxic episodes in awake goats. J. Physiol 499 ( Pt 2), 543-550.
Turner,D.L., and Sumners,D.P. (2002). Associative conditioning of the exercise ventilatory
response in humans. Respir. Physiol Neurobiol. 132, 159-168.
Ueda,K., Nakamura,K., Hayashi,T., Inagaki,N., Takahashi,M., Arimura,T., Morita,H.,
Higashiuesato,Y., Hirano,Y., Yasunami,M., Takishita,S., Yamashina,A., Ohe,T., Sunamori,M.,
Hiraoka,M., and Kimura,A. (2004). Functional characterization of a trafficking-defective HCN4
mutation, D553N, associated with cardiac arrhythmia. J. Biol. Chem. 279, 27194-27198.
Ulens,C., and Tytgat,J. (2001). Functional heteromerization of HCN1 and HCN2 pacemaker
channels. J. Biol. Chem. 276, 6069-6072.
205
Ullrich,N.D., Voets,T., Prenen,J., Vennekens,R., Talavera,K., Droogmans,G., and Nilius,B.
(2005). Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and
TRPM5 from mice. Cell Calcium 37, 267-278.
Undrovinas,A.I., Maltsev,V.A., and Sabbah,H.N. (1999). Repolarization abnormalities in
cardiomyocytes of dogs with chronic heart failure: role of sustained inward current. Cell Mol.
Life Sci. 55, 494-505.
Urbani,A., and Belluzzi,O. (2000). Riluzole inhibits the persistent sodium current in mammalian
CNS neurons. Eur. J. Neurosci. 12, 3567-3574.
Urthaler,F., Katholi,C.R., Macy,J., Jr., and James,T.N. (1974). Electrophysiological and
mathematical characteristics of the escape rhythm during complete AV block. Cardiovasc. Res.
8, 173-186.
Vaca,L., Stieber,J., Zong,X., Ludwig,A., Hofmann,F., and Biel,M. (2000). Mutations in the S4
domain of a pacemaker channel alter its voltage dependence. FEBS Lett. 479, 35-40.
van den,T.M., Lee,K., Whyment,A.D., Blanks,A.M., and Spanswick,D. (2004). Orexigen-
sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat. Neurosci. 7,
493-494.
van Diepen,M.T., Spencer,G.E., van,M.J., Gouwenberg,Y., Bouwman,J., Smit,A.B., and van
Kesteren,R.E. (2005). The molluscan RING-finger protein L-TRIM is essential for neuronal
outgrowth. Mol. Cell Neurosci. 29, 74-81.
Van,W.A., and Matthews,G. (2006). Impaired firing and cell-specific compensation in neurons
lacking nav1.6 sodium channels. J. Neurosci. 26, 7172-7180.
Vandael,D.H., Marcantoni,A., Mahapatra,S., Caro,A., Ruth,P., Zuccotti,A., Knipper,M., and
Carbone,E. (2010). Ca(v)1.3 and BK channels for timing and regulating cell firing. Mol.
Neurobiol. 42, 185-198.
Vassilev,P., Scheuer,T., and Catterall,W.A. (1989). Inhibition of inactivation of single sodium
channels by a site-directed antibody. Proc. Natl. Acad. Sci. U. S. A 86, 8147-8151.
Vega-Saenz de Miera EC, Rudy,B., Sugimori,M., and Llinas,R. (1997). Molecular
characterization of the sodium channel subunits expressed in mammalian cerebellar Purkinje
cells. Proc. Natl. Acad. Sci. U. S. A 94, 7059-7064.
Verdonck,F., Volders,P.G., Vos,M.A., and Sipido,K.R. (2003). Increased Na+ concentration and
altered Na/K pump activity in hypertrophied canine ventricular cells. Cardiovasc. Res. 57, 1035-
1043.
Vergara,C., Latorre,R., Marrion,N.V., and Adelman,J.P. (1998). Calcium-activated potassium
channels. Curr. Opin. Neurobiol. 8, 321-329.
206
Vermeulen,J.T., McGuire,M.A., Opthof,T., Coronel,R., de Bakker,J.M., Klopping,C., and
Janse,M.J. (1994). Triggered activity and automaticity in ventricular trabeculae of failing human
and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.
Vest,J.A., Wehrens,X.H., Reiken,S.R., Lehnart,S.E., Dobrev,D., Chandra,P., Danilo,P.,
Ravens,U., Rosen,M.R., and Marks,A.R. (2005). Defective cardiac ryanodine receptor regulation
during atrial fibrillation. Circulation 111, 2025-2032.
Viemari,J.C., and Ramirez,J.M. (2006). Norepinephrine differentially modulates different types
of respiratory pacemaker and nonpacemaker neurons. J. Neurophysiol. 95, 2070-2082.
Villarroel,A., Burnashev,N., and Sakmann,B. (1995). Dimensions of the narrow portion of a
recombinant NMDA receptor channel. Biophys. J. 68, 866-875.
Virginio,C., MacKenzie,A., Rassendren,F.A., North,R.A., and Surprenant,A. (1999). Pore
dilation of neuronal P2X receptor channels. Nat. Neurosci. 2, 315-321.
Wagoner,P.K., and Oxford,G.S. (1990). Aminopyridines block an inactivating potassium current
having slow recovery kinetics. Biophys. J. 58, 1481-1489.
Wahl-Schott,C., and Biel,M. (2009). HCN channels: structure, cellular regulation and
physiological function. Cell Mol. Life Sci. 66, 470-494.
Walker,R.L., Koh,S.D., Sergeant,G.P., Sanders,K.M., and Horowitz,B. (2002). TRPC4 currents
have properties similar to the pacemaker current in interstitial cells of Cajal. Am. J. Physiol Cell
Physiol 283, C1637-C1645.
Wallace,R.H., Wang,D.W., Singh,R., Scheffer,I.E., George,A.L., Jr., Phillips,H.A., Saar,K.,
Reis,A., Johnson,E.W., Sutherland,G.R., Berkovic,S.F., and Mulley,J.C. (1998). Febrile seizures
and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene
SCN1B. Nat. Genet. 19, 366-370.
Wang,D.Y., Chae,S.W., Gong,Q.Y., and Lee,C.O. (1988). Role of aiNa in positive force-
frequency staircase in guinea pig papillary muscle. Am. J. Physiol 255, C798-C807.
Wang,H., and Ren,D. (2009). UNC80 functions as a scaffold for Src kinases in NALCN channel
function. Channels (Austin. ) 3, 161-163.
Wang,H., Lu,Y., and Wang,Z. (2007). Function of cardiac M3 receptors. Auton Autacoid
Pharmacol. 27(1), 1-11.
Wang,H., Shi,H., Lu,Y., Yang,B., and Wang,Z. (1999). Pilocarpine modulates the cellular
electrical properties of mammalian hearts by activating a cardiac M3 receptor and a K+ current.
Br. J. Pharmacol. 126, 1725-1734.
Wang,K.S., Liu,X.F., and Aragam,N. (2010). A genome-wide meta-analysis identifies novel loci
associated with schizophrenia and bipolar disorder. Schizophr. Res. 124, 192-199.
207
Wang,Z., Wong,N.C., Cheng,Y., Kehl,S.J., and Fedida,D. (2009). Control of voltage-gated K+
channel permeability to NMDG+ by a residue at the outer pore. J. Gen. Physiol 133, 361-374.
Watanabe,E.I., Honjo,H., Anno,T., Boyett,M.R., Kodama,I., and Toyama,J. (1995). Modulation
of pacemaker activity of sinoatrial node cells by electrical load imposed by an atrial cell model.
Am. J. Physiol 269, H1735-H1742.
Wei,X.Y., Perez-Reyes,E., Lacerda,A.E., Schuster,G., Brown,A.M., and Birnbaumer,L. (1991).
Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and
gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels. J. Biol. Chem.
266, 21943-21947.
Whalley,K. (2011). Circadian rhythms: Calcium sets the tempo. Nat. Rev. Neurosci. 12, 434-
435.
Whitaker,G.M., Angoli,D., Nazzari,H., Shigemoto,R., and Accili,E.A. (2007). HCN2 and HCN4
isoforms self-assemble and co-assemble with equal preference to form functional pacemaker
channels. J. Biol. Chem. 282, 22900-22909.
Wicher,D., Agricola,H.J., Schonherr,R., Heinemann,S.H., and Derst,C. (2006). TRPgamma
channels are inhibited by cAMP and contribute to pacemaking in neurosecretory insect neurons.
J. Biol. Chem. 281, 3227-3236.
Wiese,C., Grieskamp,T., Airik,R., Mommersteeg,M.T., Gardiwal,A., de Gier-de,V.C., Schuster-
Gossler,K., Moorman,A.F., Kispert,A., and Christoffels,V.M. (2009). Formation of the sinus
node head and differentiation of sinus node myocardium are independently regulated by Tbx18
and Tbx3. Circ. Res. 104, 388-397.
Wilders,R. (2007). Computer modelling of the sinoatrial node. Med. Biol. Eng Comput. 45, 189-
207.
Williams,M.E., Feldman,D.H., McCue,A.F., Brenner,R., Velicelebi,G., Ellis,S.B., and
Harpold,M.M. (1992). Structure and functional expression of alpha 1, alpha 2, and beta subunits
of a novel human neuronal calcium channel subtype. Neuron 8, 71-84.
Wilson,C.J., and Callaway,J.C. (2000). Coupled oscillator model of the dopaminergic neuron of
the substantia nigra. J. Neurophysiol. 83, 3084-3100.
Winlow,W., and Syed,N.I. (1992). The respiratory central pattern generator of Lymnaea. Acta
Biol. Hung. 43, 399-408.
Wolfart,J., Neuhoff,H., Franz,O., and Roeper,J. (2001). Differential expression of the small-
conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in
dopaminergic midbrain neurons. J. Neurosci. 21, 3443-3456.
Wolfart,J., and Roeper,J. (2002). Selective coupling of T-type calcium channels to SK potassium
channels prevents intrinsic bursting in dopaminergic midbrain neurons. J. Neurosci. 22, 3404-
3413.
208
Woodin,M.A., Munno,D.W., and Syed,N.I. (2002). Trophic factor-induced excitatory
synaptogenesis involves postsynaptic modulation of nicotinic acetylcholine receptors. J.
Neurosci. 22, 505-514.
Wu,J., DeChon,J., Xue,F., Li,G., Ellsworth,K., Gao,M., Liu,Q., Yang,K., Zheng,C., He,P., Tu,J.,
Kim,d.Y., Rho,J.M., Rekate,H., Kerrigan,J.F., and Chang,Y. (2008). GABA(A) receptor-
mediated excitation in dissociated neurons from human hypothalamic hamartomas. Exp. Neurol.
213, 397-404.
Xie,J., Dernovici,S., and Ribeiro,P. (2005). Mutagenesis analysis of the serotonin 5-HT2C
receptor and a Caenorhabditis elegans 5-HT2 homologue: conserved residues of helix 4 and
helix 7 contribute to agonist-dependent activation of 5-HT2 receptors. J. Neurochem. 92, 375-
387.
Xu,W., and Lipscombe,D. (2001). Neuronal Ca(V)1.3alpha(1) L-type channels activate at
relatively hyperpolarized membrane potentials and are incompletely inhibited by
dihydropyridines. J. Neurosci. 21, 5944-5951.
Yanagihara,K., and Irisawa,H. (1980). Inward current activated during hyperpolarization in the
rabbit sinoatrial node cell. Pflugers Arch. 385, 11-19.
Yanagihara,K., Noma,A., and Irisawa,H. (1980). Reconstruction of sino-atrial node pacemaker
potential based on the voltage clamp experiments. Jpn. J. Physiol 30, 841-857.
Yang,J., Ellinor,P.T., Sather,W.A., Zhang,J.F., and Tsien,R.W. (1993). Molecular determinants
of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366, 158-161.
Yang,X.C., and Sachs,F. (1989). Block of stretch-activated ion channels in Xenopus oocytes by
gadolinium and calcium ions. Science 243, 1068-1071.
Yang,Y., Chen,X., Margulies,K., Jeevanandam,V., Pollack,P., Bailey,B.A., and Houser,S.R.
(2000). L-type Ca2+ channel alpha 1c subunit isoform switching in failing human ventricular
myocardium. J. Mol. Cell Cardiol. 32, 973-984.
Yeh,E., Ng,S., Zhang,M., Bouhours,M., Wang,Y., Wang,M., Hung,W., Aoyagi,K., Melnik-
Martinez,K., Li,M., Liu,F., Schafer,W.R., and Zhen,M. (2008). A putative cation channel, NCA-
1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans. PLoS. Biol. 6, e55.
Youm,J.B., Han,J., Kim,N., Zhang,Y.H., Kim,E., Leem,C.H., Kim,S.J., and Earm,Y.E. (2005).
Role of Stretch-activated Channels in the Heart: Action Potential and Ca2+ Transients.
Yu,F.H., and Catterall,W.A. (2004). The VGL-chanome: a protein superfamily specialized for
electrical signaling and ionic homeostasis. Sci. STKE. 2004, re15.
Yu,F.H., and Catterall,W.A. (2003). Overview of the voltage-gated sodium channel family.
Genome Biol. 4, 207.
209
Zagotta,W.N., Olivier,N.B., Black,K.D., Young,E.C., Olson,R., and Gouaux,E. (2003).
Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425,
200-205.
Zhang,H., Holden,A.V., Kodama,I., Honjo,H., Lei,M., Varghese,T., and Boyett,M.R. (2000a).
Mathematical models of action potentials in the periphery and center of the rabbit sinoatrial
node. Am. J. Physiol Heart Circ. Physiol 279, H397-H421.
Zhang,H., Zhao,Y., Lei,M., Dobrzynski,H., Liu,J.H., Holden,A.V., and Boyett,M.R. (2007).
Computational evaluation of the roles of Na+ current, iNa, and cell death in cardiac pacemaking
and driving. Am. J. Physiol Heart Circ. Physiol 292, H165-H174.
Zhang,Y.H., Youm,J.B., Sung,H.K., Lee,S.H., Ryu,S.Y., Ho,W.K., and Earm,Y.E. (2000b).
Stretch-activated and background non-selective cation channels in rat atrial myocytes. J. Physiol
523 Pt 3, 607-619.
Zhang,Z., and Bourque,C.W. (2006). Calcium permeability and flux through osmosensory
transduction channels of isolated rat supraoptic nucleus neurons. Eur. J. Neurosci. 23, 1491-
1500.
Zhang,Z., Xu,Y., Song,H., Rodriguez,J., Tuteja,D., Namkung,Y., Shin,H.S., and
Chiamvimonvat,N. (2002). Functional Roles of Ca(v)1.3 (alpha(1D)) calcium channel in
sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ. Res. 90, 981-987.
Zhao,S., Golowasch,J., and Nadim,F. (2010). Pacemaker neuron and network oscillations depend
on a neuromodulator-regulated linear current. Front Behav. Neurosci. 4, 21.
Zheng,J., Lee,S., and Zhou,Z.J. (2006). A transient network of intrinsically bursting starburst
cells underlies the generation of retinal waves. Nat. Neurosci. 9, 363-371.
Zhong,N., Beaumont,V., and Zucker,R.S. (2001). Roles for mitochondrial and reverse mode
Na+/Ca2+ exchange and the plasmalemma Ca2+ ATPase in post-tetanic potentiation at crayfish
neuromuscular junctions. J. Neurosci. 21, 9598-9607.
Zhu,Y., Mucci,A., and Huizinga,J.D. (2005). Inwardly rectifying chloride channel activity in
intestinal pacemaker cells. Am. J. Physiol Gastrointest. Liver Physiol 288, G809-G821.
Zicha,S., Fernandez-Velasco,M., Lonardo,G., L'Heureux,N., and Nattel,S. (2005). Sinus node
dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart
failure model. Cardiovasc. Res. 66, 472-481.
Zilberberg,N., Ilan,N., Gonzalez-Colaso,R., and Goldstein,S.A. (2000). Opening and closing of
KCNKO potassium leak channels is tightly regulated. J. Gen. Physiol 116, 721-734.
Zygmunt,A.C., and Gibbons,W.R. (1991). Calcium-activated chloride current in rabbit
ventricular myocytes. Circ. Res. 68, 424-437.
210
Zygmunt,A.C., and Gibbons,W.R. (1992). Properties of the calcium-activated chloride current in
heart. J. Gen. Physiol 99, 391-414.
211
Appendices A: Additional recordings.
Appendix A1. TTX regulates voltage-gated Na+ channel of L. stagnalis RPeD1 neuron in a dose-dependent manner.
(A) Representative voltage-gated Na+ channel currents activated from holding voltage of -50mV to +10 mV in
presence of various TTX concentrations. Residual currents that are not blocked by 300 µM of TTX are considered
TTX-insensitive component. (B) Dose response curve of the TTX sensitive voltage-gated Na+ current. Data are fit
with Hill equation. Half-maximal inhibitory concentration (IC50) is 23.7 ± 2.1 µM.
212
Appendix A2. Na+ free subsitution reveals a Na
+-independent transient inward current. (A) Representative traces of
RPeD1 whole-cell currents under physiological saline and Na+ free saline. Voltage-step stimulation recorded from
holding potential of -50 mV and step from -40 mV to +50 mV. (B) Current-voltage relation of transient inward
current of both control and Na+ free saline.
213
Appendix A3. Partial knockdown of U-type channel reduces inward hyperpolarizing Na+ current in RPeD1 neurons.
(A) Representative Na+ current generated by subtracting the current obtained in Na
+ free condition from that in
saline from RPeD1 neurons in naive control, control dsRNA, and U-type dsRNA treatments (from the data
presented in Figure 4.2). (B) Average INa density-voltage (I-V) relations of naive control (n = 8), control dsRNA (n
= 6), and U-type dsRNA (n = 8) treatment. U-type knockdown significantly reduces inward INa densities at all
hyperpolarizing voltages (P < 0.05).
214
Appendices B: Permission to Reproduce Previously Published Works
215
216
217
