Functional Consequences of Cantu Syndrome Associated

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Functional Consequences of Cantu SyndromeAssociated Mutations in the ATP SensitivePotassium ChannelPaige CooperWashington University in St. Louis

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WASHINGTON UNIVERSITY IN ST. LOUIS

Division of Biology and Biomedical Sciences

Molecular Cell Biology

Dissertation Examination Committee:

Colin G. Nichols, Chair

Sarah K. England

Chris J. Lingle

Jeanne M. Nerbonne

Maria S. Remedi

Functional Consequences of Cantu Syndrome Associated Mutations in the ATP Sensitive

Potassium Channel

by

Paige E. Cooper

A dissertation presented to the

Graduate School of Arts & Sciences

of Washington University in

partial fulfillment of the

requirements for the degree

of Doctor of Philosophy

December 2015

Saint Louis, Missouri

ii

Table of Contents

LIST OF FIGURES ....................................................................................................................... iii

LIST OF TABLES .......................................................................................................................... v

ACKNOWLEDGEMENTS ........................................................................................................... vi

ABSTRACT OF THE DISSERTATION ..................................................................................... vii

CHAPTER 1: INTRODUCTION ................................................................................................... 1

1.1 KATP Channel Protein Topology ........................................................................................... 3

1.2 KATP Channel Stoichiometry/Trafficking/Subunit Pairing ................................................... 5

1.3 KATP Channel Activity Regulation ........................................................................................ 6

1.4 KATP Channels in Tissue ..................................................................................................... 14

1.5 KATP Channels in Pathophysiology ..................................................................................... 23

CHAPTER 2: METHODS ............................................................................................................ 31

CHAPTER 3: CANTU SYNDROME RESULTING FROM ACTIVATING MUTATION IN

THE KCNJ8 GENE ...................................................................................................................... 34

3.1 Introduction ......................................................................................................................... 34

3.2 Methods ............................................................................................................................... 34

3.3 Results ................................................................................................................................. 34

3.3 Discussion ........................................................................................................................... 41

CHAPTER 4: DISEASE CAUSING SLIDE HELIX RESIDUE MUTATION IN KIR6.1 AND

KIR6.2 ........................................................................................................................................... 47

4.1 Introduction ......................................................................................................................... 47

4.2 Results ................................................................................................................................. 47

4.3 Discussion ........................................................................................................................... 56

CHAPTER 5: DIFFERENTIAL MECHANISMS OF CANTU SYNDROME-ASSOCIATED

GAIN-OF-FUNCTION MUTATIONS IN THE ABCC9 (SUR2) SUBUNIT OF THE KATP

CHANNEL ................................................................................................................................... 58

5.1 Introduction ......................................................................................................................... 58

5.2 Methods ............................................................................................................................... 58

5.3 Results ................................................................................................................................. 59

5.3 Discussion ........................................................................................................................... 72

CHAPTER 6: DISCUSSION ........................................................................................................ 78

REFERENCES ............................................................................................................................. 86

iii

LIST OF FIGURES

Figure 1.1- The genes encoding KATP channel proteins.................................................................. 2

Figure 1.2-Topology and stoichiometry of the KATP channel complex. ......................................... 4

Figure 1.3-Nucleotide Binding and Regulation of the KATP channel .............................................. 8

Figure 1.4- PKA and PKC signaling pathway regulation of the KATP channel ............................ 12

Figure 1.5-The role of KATP channel activity in hypothalamic glucose-sensing neuron function 17

Figure 1.6-Hypothesized role of KATP channels in SNr neuron function. .................................... 19

Figure 1.7-The role of KATP channels in beta cell function .......................................................... 20

Figure 1.8-The role of KATP channels in cardiomyocyte function ................................................ 21

Figure 1.9-The role of KATP channels in smooth muscle cell function ......................................... 24

Figure 1.10-The role of KATP channels with decreased activity in the function of beta cells in

PHHI patients .............................................................................................................. 26

Figure 1.11-The role of overactive KATP channels in the beta cell function of NDM patients..... 28

Figure 3.1- Gain-of-function KCNJ8 mutation Kir6.1[Cys176Ser] underlies Cantu syndrome .. 36

Figure 3.2-Increased channel activity in basal and stimulated conditions in intact cells expressing

Kir6.1[C176S]-containing KATP channels .................................................................. 39

Figure 3.3- Increased relative efflux in basal and stimulated conditions in intact cells expressing

Kir6.1[C176S]-containing KATP channels .................................................................. 40

Figure 3.4- Increased channel activity in basal and stimulated conditions in reconstituted

heteromeric KATP channels containing Cys176Ser subunits. ...................................... 42

Figure 3.5- Reduced ATP-sensitivity of reconstituted heteromeric KATP channels containing

Kir6.1[Cys176Ser] subunits ........................................................................................ 43

Figure 4.1-Homomeric Kir6.1-V65M–but not Kir6.1-V65L–expressed with SUR1 increases

KATP channel activity under basal conditions and in the presence of diazoxide in intact

cells. ............................................................................................................................ 49

Figure 4.2- Homomeric Kir6.1-V65M (but not Kir6.1-V65L) expressed with SUR2A increases

KATP channel activity under Metabolic Inhibition (MI) and in the presence of

pinacidil in intact cells. ............................................................................................... 50

Figure 4.3- Homomeric Kir6.2-V64M–but not Kir6.2-V64L– expressed with SUR1 increases

KATP channel activity in basal conditions and in the presence of Diazoxide in intact

cells ............................................................................................................................. 51

iv

Figure 4.4- Homomeric Kir6.2-V64M–but not Kir6.2-V64L–with SUR2A increases KATP

channel activity in basal conditions and in the presence of pinacidil in intact cells. .. 52

Figure 4.5- Gain of function in Kir6.2-V64M due to decreased ATP-sensitivity. ....................... 54

Figure 4.6- Increased open state stability in Kir6.2-V64M-based channels underlies decreased

ATP sensitivity............................................................................................................ 55

Figure 5.1-Cantu Syndrome mutations in SUR2. ......................................................................... 60

Figure 5.2- Increased channel activity in intact cells expressing homomeric P429L, A475V or

C1039Y-containing KATP channels ............................................................................. 61

Figure 5.3-KATP conductance is increased in basal and stimulated conditions in intact cells

expressing homomeric P429L and A475V-based KATP channels ............................... 62

Figure 5.4-Relative KATP conductance is markedly increased in basal and stimulated conditions

in intact cells expressing homomeric C1039Y KATP channels ................................... 64

Figure 5.5-ATP-sensitivity is decreased in C1039Y channels ..................................................... 66

Figure 5.6- C1039Y channels decreased ATP-sensitivity is due to increased Po. ........................ 67

Figure 5.7-P429L and A475V show enhanced MgADP activation .............................................. 68

Figure 5.8- Channel activity in cells expressing heteromeric P429L, A475V or C1039Y-based

KATP channels. ............................................................................................................. 70

Figure 5.9- KATP conductance normalized in the basal condition and in stimulated conditions for

heteromeric P429L, A475V or C1039Y-based KATP channels ................................... 71

Figure 5.10- Decreased sensitivity to glibenclamide inhibition in C1039Y channels. ................. 73

v

LIST OF TABLES

Table 1.1- Native KATP Channel Composition in Various Tissues ................................................. 7

Table 6.1-Cantu Syndrome Mutations and Clinical Symptoms. .................................................. 82

vi

ACKNOWLEDGEMENTS

First, I would like to acknowledge my committee members for their help and, more

importantly, patience in this process. I am grateful, and definitely couldn’t have been successful

without your guidance. Equally essential are the sources from which I have been funded during

my PhD, which include both the DBBS-Cell and Molecular Biology Training Grant and

Cardiovascular Training Grant.

I must acknowledge the Nichols lab, both past and present, for the many questions

answered, techniques taught, and all the fun times. Specifically, I want to thank Yu Wen, who

pulled the short straw, but still patiently taught me everything I know about inside-out patch

clamping and rubidium effluxes, both of which were critical techniques in this project. Monica,

who collaborated with me and helped translate my first manuscript into actual scientific writing.

Sun Joo and Jeff, my positive peer-pressure, from whom in attempting to replicate the energy

effort they put in, I learned my dedication to getting experiments done. William Borschel, for

teaching me all I know about PowerPoint animations, letting me use your cells when my

planning was poor, and giving a heroic effort to help with editing my thesis introduction.

I’d also like to acknowledge the best administrative ladies in all of Wash U: Paula

Reynolds and Stacy Kiel for keeping me in line and just putting up with all of my random

questions and requests. You both made sure I was functioning as a person so I could do the

science, and for that I am thankful.

Finally I’d like to acknowledge and thank Colin. From your guidance and rigorous

standard of doing, presenting and writing science I have benefitted greatly. Thank you for taking

me on as a student and providing a strong foundation on which I can continue to develop as a

scientist. I am eternally grateful.

vii

ABSTRACT OF THE DISSERTATION

Functional Consequences of Cantu Syndrome Associated Mutations in the ATP Sensitive

Potassium Channel

by

Paige E. Cooper

Doctor of Philosophy in Biology and Biomedical Sciences

Molecular Cell Biology

Washington University in St. Louis, 2015

Professor Colin G. Nichols, Chair

ATP-sensitive potassium (KATP) channels are composed of inward-rectifying potassium channel

pore-forming subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and

regulatory sulfonylurea receptor subunits (SUR1 and SUR2, encoded by ABCC8 and ABCC9,

respectively). These channels couple metabolism to excitability in multiple tissues. Mutations in

ABCC9 have been linked to Cantú syndrome (CS), a multi-organ disease characterized by

congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects.

Additionally, two ABCC9 mutation-negative patients, exhibiting clinical hallmarks of CS, have

been identified as having KCNJ8 mutations. This body of work is focused on determining the

functional consequences and molecular mechanisms of documented CS-associated ABCC9 and

identified KCNJ8 mutations. These mutations were engineered into recombinant cDNA clones

and expressed as functional channels. Macroscopic rubidium (86Rb+) efflux assay experiments

demonstrate that KATP channels formed with SUR2 and Kir6.1 mutant subunits both result in

gain of function in KATP activity. Inside-out patch-clamp electrophysiological experiments show

that there are at least 3 mechanisms by which these mutations alter KATP channel activity,

viii

including: 1) an SUR2 mutation that indirectly decreases ATP sensitivity by allosteric changes,

2) a Kir6.1 mutation that increases channel-open probability, and thereby indirectly decreases

ATP sensitivity, and 3) mutations in SUR2 that increase channel activation in response to

MgADP. Taken together, CS-associated ABCC9 or KCNJ8 mutations result in enhanced KATP

activity that underlies the cardinal features of Cantu Syndrome.

1

CHAPTER L: INTRODUCTION

Inwardly-rectifying potassium (Kir) channels are a class of channels generated by 7 gene sub-

families. All Kir channels are made of tetramers of Kir subunits and all require

phosphatidylinositol 4,5-bisphosphate (PIP2) for channel activity (Huang et al., 1998; Zhang et

al., 1999). These channels are expressed widely throughout the human body and play important

roles in regulating membrane potential. Disruption in Kir channel activity is linked to a variety of

diseases (Pattnaik et al., 2012). The Kir6.X sub-family members generate the pores of adenosine

triphosphate (ATP) sensitive potassium channels, which is the focus of this project.

The ATP-sensitive potassium current (IK,ATP) was first described, in cardiomyocytes, as

an outward current that is activated during metabolic poisoning and inhibited by ATP (Noma,

1983). Over the next several years, additional tissues were found to have IK,ATP (Cook and Hales,

1984; Spruce et al., 1985). Capitalizing on the sensitivity of the current to sulfonylurea drugs,

Aguilar-Bryan et al. identified and then purified the heteromeric complex of pore and auxillary

subunits that comprise the ATP-sensitive potassium (KATP) channel (Aguilar-Bryan and Bryan,

1999). The genes that encode the pore and auxiliary subunits are found in pairs on two different

human chromosomes. On human chromosome 11p at position 15.1, KCNJ11, which encodes the

pore-forming Kir6.2 subunit, is directly downstream of ABCC8, which encodes the auxiliary

subunit SUR1 (Aguilar-Bryan et al., 1995). Conversely, on human chromosome 12p at position

11.23, KCNJ8, which encodes the pore-forming subunit Kir6.1, is located downstream of

ABCC9, which encodes the auxiliary subunit SUR2 (Figure 1.1) (Chutkow et al., 1996; Inagaki

et al., 1995b).

2

Figure 1.1- The genes encoding KATP channel proteins. Cartoon

representation of human chromosomes 11 and 12, where the genes

encoding KATP channels are found. Each box represents the gene denoted in

italics and the name of the encoded protein is indicated above.

3

1.1 KATP Channel Protein Topology

The pore-forming subunit proteins Kir6.2 and Kir6.1, which are encoded by KCNJ11 and

KCNJ8, respectively, are members of the inward- rectifying potassium (Kir) channel family.

Crystallization of the bacterial KcsA potassium channel provided a structural model for the Kir6

family (Figure 1.2A). The N and C termini orient within the cytoplasm with two transmembrane

helices connected by a short loop (P-loop), which forms the pore and ion selectivity filter (Doyle

et al., 1998). The two Kir6.X family member proteins share ~70% sequence identity (Inagaki et

al., 1995a).

KATP channels formed with either Kir6.2 or Kir6.1 exhibit distinct gating and

conductance. Kir6.2 channels spontaneously activate in the absence of ATP; however, Kir6.1

channels require the application of activating nucleotides (Kondo et al., 1998). Second, the

single-channel conductance for Kir6.2, is double that of Kir6.1 conductance (Inagaki et al.,

1995a; Isomoto et al., 1996; Sakura et al., 1995; Yamada et al., 1997). In chimera studies,

replacing the Kir6.2 extracellular linker and the p-loop with that of Kir6.1 had no effect on

gating but lowered the unitary conductance, implying that this region determines the unitary

conductance (Kondo et al., 1998; Kono et al., 2000; Repunte et al., 1999). Conversely, additional

chimera studies demonstrate that the unique N-terminus and C-terminus of Kir6.2 are necessary

for spontaneous channel activity.

The sulfonylurea receptor (SUR) 1 and 2, the KATP auxiliary subunit protein isoforms

encoded by ABCC8 and ABCC9, respectively, are members of the ATP-binding Cassette (ABC)

family (Figure 1.1). SUR1 and SUR2 share ~68% sequence identity homology (Inagaki et al.,

1996) and are oriented in the membrane with an extracellular N terminus and a cytoplasmic C

terminus (Figure 1.2A). As first predicted by hydrophobicity mapping and later confirmed using

4

IN

OUT

NBD

P-loop TMD TMD TMD

A

B

Figure 1.2- Topology and stoichiometry of the KATP channel complex. A cartoon

of a KATP channel Kir6.X (blue) and a SURX (yellow) subunit orientation within the

membrane. The P-loop and the nucleotide binding domains (NBDs) are boxed in red

and green, respectively (A). The 4:4 stoichiometry of Kir6.X to SURX proteins

which form the KATP channel complex is depicted in (B).

5

biotinylation assays and immunofluorescence, the SUR protein contains 17 transmembrane

helices grouped into 3 domains (TMD0-2) connected and followed by cytoplasmic loops that

form two nucleotide-binding domains (NBD) (Figure 1.2A) (Conti et al., 2001; Tusnady et al.,

1997). SUR2 has two major splice variants, SUR2A and SUR2B (Inagaki et al., 1996; Isomoto et

al., 1996). Both SUR1 and SUR2 have additional splice sites, but the resulting variants are

poorly understood (Shi et al., 2005).

1.2 KATP Channel Stoichiometry/Trafficking/Subunit Pairing

The size of the purified KATP channel complex suggested that each KATP channel is formed by

four Kir6.X and four SURX subunits. 1:1 Kir6.2-SUR1 fusion proteins forming functional

channels provided supporting evidence (Clement et al., 1997) for this hypothesis. The current-

voltage profiles of channels formed with various heteromeric molar ratios of wild-type Kir6.2

were compared with a strongly rectifying Kir6.2 mutant. This work provided direct evidence

that four Kir6.X subunits form the pore (Shyng and Nichols, 1997). The electrophysiological

properties of KATP channels produced from an engineered fusion construct made of one Kir6.2

and one SUR1 were identical to those produced by co-expression of Kir6.x and SURX subunits,

thus confirming a 4:4 stoichiometry of Kir6.x and SURX subunits forming KATP channels

(Figure 1.2B) (Shyng and Nichols, 1997).

The short Arg-Lys-Arg endoplasmic reticulum (ER) retention signals in Kir6.X at the C-

terminus and in SURX between TMD1 and NBF1 are mutually masked in the full complex,,

allowing for translocation of these subunits to the plasma membrane (Zerangue et al., 1999).

The retention signal on each Kir6.X and SURX subunit also obligates correct subunit pairing and

stoichiometry before trafficking to the plasma membrane (Zerangue et al., 1999). However,

removing the last 30 amino acids from the C-terminus of Kir6.2, which includes the ER-retention

6

signal, allows Kir6.2 to traffic and function alone on the plasma membrane (Tucker et al., 1997).

As both pore and auxiliary subunits come in multiple isoforms, the resulting KATP channels can

be formed with multiple subunit combinations. Studies in heterologous expression systems have

shown that all combinations are able to traffic and function at the cell membrane (Shi et al.,

2005). Table 1.1 lists subunit combinations that have been identified in native tissues.

1.3 KATP Channel Activity Regulation

KATP channels couple cell metabolism and excitability in multiple cell types. Importantly, there

are a variety of physiological and pharmacological modulators of KATP channels, some of which

are discussed in detail below.

1.3.1 Physiological Modulators

1.3.1.i Nucleotides

In addition to forming the pore of the channel, the Kir6.X subunits bind inhibitory ATP and

adenosine diphosphate (ADP) (Mg-free) (Figure 1.3A) (Tucker et al., 1997). Truncation and

mutagenesis experiments demonstrate that both the N- and C-termini of Kir6.X contribute to the

ATP-sensitivity (Drain et al., 1998; Koster et al., 1999; Tucker et al., 1998).

Adenylylimidodiphosphate (AMP-PNP), a non-metabolizable form of ATP, produces an

inhibitory response identical to that of ATP, indicating that binding, not hydrolysis, of ATP is

sufficient for KATP inhibition (Inagaki et al., 1995a). The specificity of ATP binding and

inhibition was validated in photoaffinity labeling experiments. Additionally, numerous

triphosphates, including: guanine, inosine, cytidine or uridine, fail to inhibit IK,ATP currents in

both recombinant systems and in native tissues, further supporting the specificity of the KATP

7

Table 1.1- Native KATP Channel Composition in Various Tissues

TISSUE KATP Channel

COMPOSITION

REFERENCES

BRAIN Hippocampus

Kir6.2/SUR1

Kir6.2/SUR2

Kir6.1/SUR1

Kir6.1/SUR2

Zawar et al. J Physiol 514: 327-341, 1999

Hyllienmark, L. and Brismar, T. Journal of Physiol, 496,

155± 164, 1996

Fujimura, N. et al. Journal of Neurophysiology, 77, 378±

385, 1997

Hypothalamus Kir6.1/SUR1

Kir6.2/SUR1

Spanswick, D et al. Nature, 390, 521-525, 1997

Liss, B. et al. Journal of Physiology 527, 114P-115P, 2000

Lee, K. et al. Journal of Physiology 515, 439-452, 1999

Miki T et al. Nat Neurosci. May;4(5):507-12, 2001

Ashford, M. L et al. Pflugers Archives, 415, 479± 483,

1990

Brain Stem Kir6.2/SUR1 Karschin, A. et al. Journal of Physiology 509, 339± 346,

1998

Trapp, S. and Ballanyi, K., Journal of Physiology, 487,

37± 50, 1995

Basal Ganglia

-Striatum

-Substantia nigra

Kir6.1/SUR1

Kir6.2/SUR2B

Kir6.2/SUR1

Lee, K. et al. Journal of Physiology, 510, 441± 453, 1998

Stanford, I. M. and Lacey, M. G. Neuroscience, 74, 499±

509, 1996

Liss, B. et al. Embo Journal, 18,833± 846, 1999

Schwanstecher, C. and Panten, U. Naunyn Schmiedebergs

Archives of Pharmacology, 348, 113± 117, 1993

Roeper, J et al. Journal of Physiology, 430, P130. 1990.

HEART

Ventricle

Kir6.2/SUR2A Babenko AP et al. Circ Res 83: 1132–1143, 1998

Atrium Kir6.2/SUR1 Flagg TP et al.Circ Res 103: 1458–1465, 2008

Glukhov AV et al. J Mol Cell Cardiol 48: 152–160, 2010

Conduction

System

Kir6.1/SUR2A

Kir6.2/SUR2A

Han X et al.J Physiol 490: 337–350, 1996

Fukuzaki K et al. J Physiol 586: 2767–2778, 2008

Kakei M and Noma A. J Physiol 352: 265–284, 1984

Light PE et al. Cardiovasc Res 44: 356–369, 1999

PANCREATIC

BETA CELL

Kir6.2/SUR1 Inagaki N et al. Science Nov17;270(5239):1166-70, 1995

SKELETAL

MUSCLE

Kir6.2/SUR2A

Kir6.2/SUR1

Tricarico D et al. Proc Natl Acad Sci USA 103: 1118–

1123, 2006

VASCULAR

SMOOTH

MUSCLE

Kir6.1/SUR2B

Kir6.2/SUR2B

Isomoto S et al. J Biol Chem 271: 24321–24324, 1996

Zhang HL and Bolton TB. Br J Pharmacol 118: 105–114.

8

A

B

Figure 1.3- Nucleotide Binding and Regulation of the KATP channel.

The Kir6.X is represented in blue and the SURX subunit is represented

in yellow. ATP and ADP (No Mg2+), which both bind to the Kir6.X

subunits to inhibit the channel, are represented by red and green circles,

respectively. MgADP binds to the Nucleotide Binding Domains (NBDs)

within the SURX subunit (A). The NBD region is enlarged in the square

region and the Walker A and Walker B motifs, which are within the

NBDs, are represented by purple and gray regions, respectively. In these

Walker motifs ATP and MgADP have been shown to bind. Mg2+ is

represented by light blue hexagons attached to ADP (B).

9

channel to regulation by ATP (Koster et al., 1999; Lederer and Nichols, 1989; Tanabe et al.,

2000; Tucker et al., 1998).

Channels composed of Kir6.2 alone, which traffic after truncation of the ER retention

signal, have decreased sensitivity to ATP inhibition compared to WT channels composed of

Kir6.2 and SUR1 (Tucker et al., 1997). This finding suggests that SUR subunits also play a role

in regulating channel activity. Photoaffinity labeling using 8-azido-[α-32P]ATP or 8-azido-[γ-

32P]ATP in cells overexpressing SUR1 demonstrated that ATP binds the SUR subunit directly

(Ueda et al., 1997). MgADP works at the level of the NBDs in SUR to activate the channel

(Nichols et al., 1996). Nucleotide binding folds (NBFs), which are conserved in all ABC proteins

including SURX subunits, contain Walker A and Walker B nucleotide-binding motifs (Figure

1.3B) (Higgins, 1992). Nucleotide binding sites in SURX were identified by mutagenesis of the

conserved Walker A and Walker B regions in both NBF1 and NBF2. Free ATP binds to NBF1

and MgADP interacts with NBF2, but can also alter ATP binding to NBF1 (Ueda et al., 1997).

Disruption of the NBF2 Walker B motif or linker region within SUR1 completely

abolishes the activating effect of MgADP, without altering ATP-sensitivity. Conversely,

mutations at the equivalent positions in the NBF1 Walker B motif only alter activation kinetics

(Shyng et al., 1997). Expression of SUR1 mutagenized at lysine residue(s) within Walker A

motifs in NBF1, NBF2 or both, with Kir6.2-WT, produced KATP channels with increased ATP-

sensitivity (no Mg2+) and with MgADP inhibition instead of activation (Gribble et al., 1997).

Taken together, the Walker A motif in NBF1 and both Walker A and Walker B motifs in NBF2

are required for MgADP activation of KATP channels (Figure 1.3B). MgADP stimulation differs

among the SUR isoforms, where SUR1-based channels are more stimulated than SUR2A (Masia

et al., 2005). The differences in nucleotide-binding affinities among the SUR isoforms may

10

account for some of the diversity of behavior among the different channel compositions (Matsuo

et al., 2000).

1.3.1.ii Phosphorylation

PKC

Direct modulation via either the protein kinase C (PKC) pathway or KATP channel activity

modulates vascular tone. In addition, activation of PKC can decrease KATP channel activity

indirectly to influence tone. The response of KATP channels to PKC activation could occur

because: 1) KATP channels are directly regulated by PKC signaling or 2) PKC activation inhibits

KATP channel indirectly by depleting PIP2 from the membrane. Stable Kir6.1+SUR2B and

Kir6.2+SUR2B cell lines were used to monitor KATP channel activity response to

pharmacological activation of PKC in combination with inhibitors of the PKC signaling cascade,

providing some of the first evidence that Kir6.1 based channels are directly regulated by PKC

(Quinn et al., 2003).

Additional engineered chimera studies demonstrated that N- and C-termini of Kir6.1 are

necessary for PKC regulation. Use of electrophysiology and phosphorylation assays revealed that

individual intracellular serine residues, which are unique to the C-terminus of Kir6.1, mutated to

alanine do not prevent PKC inhibition. However, mutating these serine residues simultaneously

abolishes PKC inhibition of KATP channels. Taken together, several serine residues in the C-

terminus of Kir6.1 are required for direct PKC phosphorylation and inhibition of KATP channel

activity (Figure 1.4A) (Shi et al., 2008).

The ability of PKC to inhibit Kir6.1-based KATP channels might suggest that KATP

channel activation results from dephosphorylation by phosphatases. Electrophysiological data

11

from isolated rat aortic smooth muscle show that different structural classes of Ca2+-dependent

protein phosphatase type 2B (PP2B) inhibitors can inhibit IK,ATP. These results suggest that in

native tissue, PP2B phosphatase activity can regulate KATP channel activity (Figure 1.4A)

(Wilson et al., 2000).

PKA

At least two studies have shown that KATP channels composed of Kir6.1+SUR2B are activated

by PKA in a cAMP-dependent manner (Quinn et al., 2004; Shi et al., 2007). There is conflicting

evidence however about the actual residues phosphorylated by PKA. Quinn et al. concluded that

a serine residue in Kir6.1 and/or a serine and a threonine residue in SUR2B are phosphorylation

sites for PKA(Quinn et al., 2004). On the other hand, Shi et al. made alanine substitutions at

these residues and found no differences in PKA sensitivity of KATP channel currents, leading

them to conclude that these sites are not relevant. They went on to identify a single serine residue

in SUR2B as the only PKA phosphorylation site, which they confirmed by in vitro

phosphorylation assays (Figure 1.4B) (Shi et al., 2007).

Despite the debate over which specific Kir6.1 and/or SUR2B residue(s) PKA

phosphorylates (Quinn et al., 2004; Shi et al., 2007), it is agreed that PKA phosphorylates KATP

channels directly to regulate vascular tone. There has therefore been an interest in identifying

potential phosphatase(s) to counter activation of KATP channel after PKA phosphorylation (Orie

et al., 2009). Dephosphorylation of KATP would inhibit KATP channel activity and, noting that

Kir6.1+2B activity decreases in high intracellular Ca2+, the Ca2+-dependent phosphatase

calcineurin was tested as a potential KATP channel phosphatase. Multiple chemically unrelated

calcineurin inhibitors increase both in vitro phosphorylation of KATP channel subunits and KATP

12

A

B

Figure 1.4- PKA and PKC signaling pathway regulation of the

KATP channel. Cartoon of KATP channel regulation by PKC (A) and

PKA (B) pathways in which Kir6.1 is depicted in blue surrounded by

SUR2B in yellow. Red dots depict phosphorylation sites on Kir6.1 c-

terminus or SUR2B as described in the text.

13

channel activity (Figure 1.4B) (Orie et al., 2009). In the presence of a PKA catalytic domain that

is constitutively active, calcineurin has no effect on KATP channel activity. This observation

indicates that calcineurin directly dephosphorylates KATP channels to regulate channel activity

(Orie et al., 2009).

1.3.1.iii pH

Increases in intracellular pH result in increases in KATP channel current, in the presence or

absence of ATP, in isolated rat beta cells (Misler et al., 1989). Further evidence for KATP channel

pH sensitivity has been obtained from studies on inside-out patches from Xenopus oocytes

expressing Kir6.2+SUR1 and from isolated mouse cardiomyocytes (Baukrowitz et al., 1999).

Histidine is the most common amino acid protonated or deprotonated at physiological pH. By

mutating each of the 9 intracellular histidine residues in Kir6.2 to amino acids with non-

titratable side chains, a single residue was identified as the pH-sensing residue in KATP channels

(Baukrowitz et al., 1999).

1.3.2 Pharmacological Modulation

In addition to physiological regulation, KATP channel activity is modulated by distinct

pharmacological agents. The molecular basis and action of these modulators are discussed

below.

1.3.2.i K channel openers (KCOs)

Diverse classes of drugs called K+ channel openers (KCOs) activate KATP channels by binding to

the SUR subunits (Inagaki et al., 1996). The ability of individual KCOs to activate channels is

dependent on the SUR isoform. Whereas SUR1-containing channels are activated specifically by

diazoxide (Inagaki et al., 1995a), SUR2A -containing channels are effectively activated by all

14

KCOs except diazoxide (Inagaki et al., 1996). SUR2B-containing channels respond to all KCOs

(Isomoto et al., 1996).

Synthesis and study of SUR chimera proteins has demonstrated that KCOs such as

levcromakalim, cromakalim, P1075 and pinacidil appear to bind SUR2 at the intracellular loop

between TM13 and TM14, with two additional critical residues in TM17, SUR2-L1249 and

SUR2-T1253. These interactions have been confirmed by the observation that disruption to these

areas and/or residues alters the activating properties of the KCOs (Moreau et al., 2000; Uhde et

al., 1999). Diazoxide diverges structurally from that of the majority of KCOs, and its preferential

binding is thought to be at the NBFs (Shyng et al., 1997).

1.3.2.ii Sulfonylureas

A class of drugs called sulphonylureas also specifically binds the SUR subunit of KATP channels

but inhibit channel activity (Inagaki et al., 1996). Tolbutamide is an example of a first-generation

sulfonylurea that binds at TMD2 (Ashfield et al., 1999). Second and third generation, more

potent, sulfonylureas have been developed; an example is glibenclamide. The binding site of

glibenclamide also includes TMD2 with additional involvement of a site on the cytoplasmic loop

between TMD0 and TMD1 (Mikhailov et al., 2001). In general, sulfonylureas are most effective

at inhibiting SUR1-based channels, less so for SUR2B-, and least effective for SUR2A-based

channels.

1.4 KATP Channels in Tissue

The variation in nucleotide sensitivity, pharmacology and physiological pairings (Aguilar-Bryan

et al., 1995; Inagaki et al., 1996) of the Kir6.X and SURX subunits results in diversity of the

15

properties and functional roles of KATP channels in the tissues in which they are expressed

(Akrouh et al., 2009; Nichols, 2006).

1.4.1 Tissue Distribution

RT-PCR data from a large variety of tissues indicates the pore-forming subunit Kir6.1 is very

widely expressed (Inagaki et al., 1995c). In contrast, Kir6.2 expression is prominent in pancreatic

beta cells, brain, heart and skeletal muscle(Inagaki et al., 1995a). In native tissues, SUR1

subunits only physiologically interact with Kir6.2. Consistent with this finding, SUR1 expression

is also in pancreatic beta cells, brain, and heart (Inagaki et al., 1995a). Conversely, the SUR2

subunits pair in native tissue with either Kir6.1 or Kir6.2. The splice variant SUR2A is found in

the heart and skeletal muscle where, based on electrophysiology studies, it pairs with Kir6.2.

Like Kir6.1, SUR2B subunits are expressed widely, and electrophysiological recordings from

KATP channels in vascular smooth muscle cells appear to reflect the co-assembly of SUR2B with

Kir6.1(Table 1.1) (Chutkow et al., 1996; Inagaki et al., 1995c).

1.4.2 Role in Tissue Function

1.4.2.i Brain

KATP channel expression and activity has been reported throughout the nervous system including

the pituitary gland, basal ganglia, cerebral cortex, hippocampus, basal forebrain, striatum, and

brain stem (Liss and Roeper, 2001; Seino and Miki, 2003; Yamada and Inagaki, 2005).

Electrophysiological characterization of KATP channel currents, combined with RT-PCR analysis

of KATP channel subunit expression, suggests composition of native channels in various regions

of the brain is quite heterogeneous. Based on radiolabeled glibenclamide studies, KATP channels

are most abundant in the hypothalamus and substantia nigra pars rectculata (SNr)neurons (Liss

16

and Roeper, 2001; Seino and Miki, 2003; Yamada and Inagaki, 2005). The roles for KATP

channels in the brain will be discussed below in reference to these regions specifically.

Hypothalamus

The hypothalamus, which plays a critical role in glucose homeostasis by regulating counter-

regulatory hormones such as glucagon and catecholamines, relies on glucose-sensing neurons.

Within these neurons, the electrical firing patterns that lead to the autonomic input to release

glucagon and catecholamines are regulated by KATP channel activity (Miki et al., 2001).

Electrophysiological and RT-PCR analysis indicates that Kir6.2 and SUR1 compose the KATP

channels in these glucose-responsive neurons (Ashford et al., 1990; Miki et al., 2001; Spanswick

et al., 1997). As brain glucose levels drop, glucose-sensing neurons experience a decrease in the

intracellular ATP/ADP ratio that activates KATP channels, thereby hyperpolarizing the plasma

membrane and stimulating autonomic input for glucagon release (Figure 1.5). The release of

glucagon, from pancreatic alpha cells, stimulates gluconeogenesis and lipolysis to increase blood

glucose levels (Seino and Miki, 2003).

SNr

The basal ganglion contains SNr neurons, which are implicated in the propagation of seizures.

KATP channels are not active in SNr neurons at rest, allowing for active firing (Yamada and

Inagaki, 2005). In metabolically stressful conditions, such as hypoxia or hypoglycemia, the

activity of KATP channels is thought to be critical for protection from seizures. In metabolically

stressful conditions SNr neurons decrease ATP production, reducing the ATP/ADP ratio,

activating KATP channels and thereby hyperpolarizing the membrane. Membrane

hyperpolarization decreases the firing rate of SNr neurons inhibiting GABA release, which is

17

Figure 1.5- The role of KATP channel activity in hypothalamic glucose-

sensing neuron function. Illustrated above is the role KATP channels play in

hypothalamic glucose-sensing neurons. The KATP channel is depicted in green

and the voltage gated Ca2+ channel is in orange.

18

hypothesized to slow or prevent propagation (Figure 1.6)(Amoroso et al., 1990; Yamada et al.,

2001).

1.4.2.ii Pancreas

Within the pancreas there are clusters of endocrine cells called islets of Langerhans, of which

beta cells are the most abundant. Beta cells secrete insulin, an endocrine hormone which helps

decrease blood glucose levels. KATP channels are expressed abundantly on the plasma membrane

of beta cells, and are critical for regulating insulin release (Ashcroft and Gribble, 1999). During

or after a meal, as blood glucose levels elevate, beta cell metabolism increases, which elevates

the intracellular ATP/ADP ratio, inhibiting KATP channels. Inhibition of KATP channels leads to

membrane depolarization and activation of L-type Ca2+ channels, which increases Ca2+ entry.

The increased flux of calcium promotes fusion and release of insulin (Figure 1.7). Once in the

bloodstream insulin helps decrease blood glucose levels by facilitating glucose uptake into

muscle (Ashcroft and Gribble, 1999).

1.4.2.iii Heart

In the resting state, cardiomyocytes have elevated intracellular ATP/ADP ratios which leaves

KATP channels generally closed (Koster et al., 2001). While the role of KATP channels in

regulating heart rhythm or contraction is not fully understood, complete activation of KATP

channels in the heart can lead to profound shortening of the action potential (Figure 1.8)(Nichols

et al., 1991). During ischemia, in which a lack of oxygen or glucose disrupts the metabolic

function of cardiomyocytes, the ATP/ADP ratio decreases, activating KATP channels. Activation

of KATP channels under these conditions is thought to be cardio-protective since

hyperpolarization of the membrane will prevent excessive Ca2+ entry, thus preserving

cardiomyocyte function and promoting cell survival (Bers, 2008).

19

Figure 1.6- Hypothesized role of KATP channels in SNr neuron function. Cartoon

of the potential role KATP channels play in SNr neurons. The KATP channel is depicted

in green, the voltage-gated Ca2+ channel is in orange, and yellow circles represent

GABA.

20

Figure 1.7- The role of KATP channels in beta cell function. Cartoon of the

role KATP channels play in the pancreatic beta cell, where the KATP channel is

depicted in green, the voltage-gated Ca2+ channel is depicted in orange and

insulin is represented by purple circles.

21

Figure 1.8- The role of KATP channels in cardiomyocyte function. Cartoon of the role KATP channels play in cardiomyocytes, where the KATP

channel is depicted in green and the voltage gated Ca2+ channel is depicted

in orange.

22

1.4.2.iv Skeletal Muscle

KATP channels are not thought to play a significant role in regulating the electrical activity of

skeletal muscle in the resting state, but may be crucial during muscle fatigue (Cifelli et al., 2008;

Gong et al., 2000). KATP channel activity can preserve a polarized membrane potential,

preventing voltage-dependent Ca2+ entry and decreasing contractility (Gong et al., 2000; Matar et

al., 2000). After treadmill or swim training, Kir6.2−/− mice, but WT, experience extensive fiber

damage in muscle. This observation is also consistent with KATP channel activation being

protective during fatigue (Kane et al., 2004; Thabet et al., 2005).

There is also evidence that KATP channels play a role in glucose uptake in skeletal

muscle: 1) sulfonylureas enhance glucose uptake into isolated skeletal muscle (Miki et al., 1998);

2) Kir6.2-/- mice have increased effectiveness of insulin in lowering blood glucose in the muscle

versus WT mice (Miki et al., 2002); and 3) muscle from SUR2-/- mice have enhanced insulin-

stimulated glucose transport in vitro compared to WT (Chutkow et al., 2001).

1.4.2.v Smooth Muscle

In smooth muscle cells, KATP channel activity is thought to be important for regulating vascular

tone (Chutkow et al., 2002; Miki et al., 2002). Excised patch clamp experiments on vascular

smooth muscle cells revealed KATP channels which did not spontaneously open in the absence of

ATP, a characteristic of nucleoside-dependent Kir6.1 channels (Beech et al., 1993; Kajioka et al.,

1991; Zhang and Bolton, 1996). It is hypothesized that these KATP channels help regulate tone by

activating in response to a declining ATP/ADP ratio or input from PKA pathways (Figure 1.4B).

Outward K+ currents through these channels hyperpolarize the membrane, thereby preventing

Ca2+-entry and leading to vasodilation of the vessel and decreases in contractility (Figure 1.9). In

23

support of this proposed mechanism, treatment with the L-type Ca2+-channel inhibitor,

nifedipine, protects against the development of vasospasms in SUR2-/- animals (Chutkow et al.,

2002).

1.5 KATP Channels in Pathophysiology

KATP channels are widely expressed and provide a finely tuned link between metabolism and

electrical activity in tissues. Dysregulation of KATP channel activity (gain of function or loss of

function) results in pathophysiology. Several examples, in which KATP channel dysregulation

underlies a disease state, are described below.

1.5.1 Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI)

The first reported clinical characterization of a child with hypoglycemia that failed to improve

without treatment intervention occurred in 1956 (Cochrane et al., 1956). This disease was later

termed Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI), and is found in 1 of every

50,000 live births or up to 1 in every 3,000 live births in familial cases (Stanley and De León,

2012). Clinically, patients commonly present with increased plasma levels of insulin in

hypoglycemic conditions along with decreased levels of ketone bodies and free fatty acids

(Aguilar-Bryan and Bryan, 1999).

Mutations in KCNJ11 (Kir6.2) or ABCC8 (SUR1) account for nearly 70% of known

PHHI cases, with the majority of mutations found in ABCC8. Every PHHI-associated KCNJ11

(Kir6.2) or ABCC8 (SUR1) mutation leads to a decrease in KATP channel activity. This keeps the

beta cell membrane constantly depolarized and leads to elevated Ca2+ levels and secretion of

insulin (Figure 1.10)(Remedi and Koster, 2010).

24

Figure 1.9- The role of KATP channels in smooth muscle cell

function. Cartoon of the role KATP channels play in smooth muscle

cell function, where the KATP channel is depicted in green and the

voltage gated Ca2+ channel is depicted in orange.

25

There is no single mechanism by which all PHHI-associated KATP channel mutations reduce

channel activity. The mechanisms identified to date include trafficking defects, increased ATP

sensitivity, decreased MgADP activation and Kir6.2 inactivation(Denton and Jacobson, 2012).

Patients may be treated with the KATP channel opener diazoxide, or high doses of glucose to

maintain euglycemia (Pinney et al., 2008). However, in extreme cases in which these treatments

are ineffective, partial or complete removal of the pancreas is necessary(De Leon and Stanley,

2007).

1.5.2 Neonatal Diabetes Mellitus

Phenotypically the opposite of PHHI is a disease called Neonatal Diabetes Mellitus (NDM). Also

a condition commonly diagnosed in infants less than six months old, NDM patients present with

hyperglycemia and hypoinsulinemia. NDM incidence is estimated to be ~1 in every 100,000-

300,000 births (Naylor et al., 2011). It results from disruption in genes encoding critical proteins

for beta cell function including missense mutations in genes encoding proteins in the metabolic

pathway or involved in insulin production or secretion (Remedi and Nichols, 2009). The most

common monogenic cause of NDM is gain of function in KATP channel activity due to mutations

in either KCNJ11 (Kir6.2) or ABCC8 (SUR1) (Naylor et al., 2011). Despite elevated ATP/ADP

ratios, which results from increased glucose metabolism, the gain of function in KATP channel

activity prevents channel inhibition. Because KATP channels remain open, the membrane remains

hyperopolarized, leading to cessation of beta cell secretion of insulin (Figure 1.11) (Koster et al.,

2000).

NDM-associated KATP channel mutations are scattered throughout the Kir6.2 and SUR1

proteins (Denton and Jacobson, 2012), with multiple underlying molecular mechanisms. Kir6.2

26

A

B

Figure 1.10- The role of KATP channels with decreased activity in the

function of beta cells in PHHI patients. Cartoon of the role KATP channels

play in normal beta cell function (A) or with decreased KATP channel activity

as found in PHHI patients (B). The KATP channel is depicted in green and

the voltage-gated Ca2+ channel is depicted in orange.

27

mutations have most commonly been shown to give rise to increased channel activity by

increasing the intrinsic open probability of the channel or by decreasing the affinity or binding of

inhibitory ATP (Koster et al., 2008b). Similarly, mechanisms for SUR1 mutations also result in

overactive KATP channels, but enhanced affinity for MgADP and altered nucleotide hydrolysis at

the NBDs can additionally be responsible (Denton and Jacobson, 2012).

Originally NDM patients were treated with insulin injections as a means of managing

their blood glucose levels, but understanding the molecular basis of KATP channel-dependent

NDM cases has allowed for better/simpler treatment (Babenko et al., 2006; Sagen et al., 2004;

Zung et al., 2004). Now, instead of insulin injections, patients are typically administrated

sulphonylureas tablets orally, as an easier and more targeted treatment that directly inhibits the

overactive KATP channels. Additionally, sulfonylureas typically result in more regulated

glycemic control, relative to insulin therapy (Koster et al., 2008a). However, it should be noted

that in patients with mutations that result in extremely overactive KATP channels sulfonylureas

are not always effective (Remedi and Koster, 2010).

1.5.3 Cantu Syndrome

A disease more recently proposed to be associated with defective KATP channels is Cantu

Syndrome (CS), a rare and complex disorder. In the first description of this disorder, reported by

Dr. Cantu in 1982, four patients presented with hypertrichosis and dental abnormalities,

comparable to that seen in patients with hypertrichosis universalis and/or congenital

hypertrichosis lanuginose. Additional skeletal abnormalities in these patients establish CS as a

unique disorder (Cantu et al., 1982). The heritability of CS in both males and females from

28

A

B

Figure 1.11- The role of overactive KATP channels in the beta cell function

of NDM patients. Cartoon of the role KATP channels play in normal beta

function (A) or the role of overactive KATP channels play in the beta cell

function of patients with NDM (B). The KATP channel is depicted in green and

the voltage-gated Ca2+ channel is depicted in orange.

29

asymptomatic parents originally suggested that it can be an autosomal recessive disease (Cantu

et al., 1982). However, a familial case in which a father with CS passed it to his son, made

autosomal dominance more likely (Lazalde et al., 2000).

The number of CS patients described in the literature to date totals ~50 and the clinical

hallmarks have expanded to include: hypertrichosis, macrosomia, macrocephaly, coarse facial

appearance, cardiomegaly, and skeletal abnormalities (Nichols et al., 2013). Additional

symptoms present in subsets of patients includes patent ductus arteriosus, lymphedema, heart

vavular anomalies, and congenital hypertrophic cardiomyopathy (Grange et al., 2014). The fact

that hypertensive patients treated with (KCOs) such as Minoxidil (Mehta et al., 1975), diazoxide

(Goldberg, 1988), or pinacidil (Koblenzer and Baker, 1968) can develop similar features,

including hypertrichosis and structural defects in the heart, led to the hypothesis that CS could be

a result of dysregulated potassium channel activity (Grange et al., 2006).

It was not until 2012, however, when the genetic analysis of two separate CS patient

cohorts determined that the majority have mutations in ABCC9 (SUR2), that the K+ channel

dysregulation hypothesis was investigated. Harakalova et al. expressed Kir6.2-WT with three of

the identified SUR2 mutations and found that KATP channel activity was increased (Harakalova

et al., 2012). The data presented in the following chapters represent the first reports of KCNJ8

mutations which result in CS, and the mechanisms underlying the functional effects of these

mutations. Additionally included is the first molecular account and detailed mechanistic study of

how the gain of function in KATP channel activity arises from CS-associated SUR2 mutations.

KATP channels are highly regulated and are expressed throughout the body, and these

channels primarily function to link cell metabolism and cell excitability. This body of work is

30

focused on determining the mechanism(s) by which identified mutations in Kir6.1 and SUR2,

found in CS-patients, alter KATP channel activity. CS patients with Kir6.1 or SUR2 mutations

have overlapping symptoms, and the results presented imply that the gain of function in KATP

channel activity is causal. This work underscores the intricate role KATP channels play in tissues

and how minor changes in channel activity can have profound effects on disease presentation.

Understanding the impact these mutations have on channel activity should provide insight into

how a gain of function in KATP channels activity results in the various pathophysiological

consequences of CS and, more importantly, guide future efforts focused on inhibiting these

overactive channels as a treatment option for patients.

31

CHAPTER W: METHODS

Mutagenesis and Heterologous Expression of KATP Channels

The Quick Change II Site-Directed Mutagenesis kit (Agilent Technologies) was used to engineer

SUR2 mutations into ratSUR2A-pCMV6, Kir6.1 mutations into ratKir6.1-pcDNA3.1, and Kir6.2

mutations into mouseKir6.2-pcDNA3.1. Mutations were confirmed by direct sequencing of the

SUR2 or Kir6.X coding regions.

For channel expression, COSm6 cells were cultured in Dulbecco’s Modified Eagle’s

Medium (DMEM) supplemented with 10% fetal bovine serum, 105 units/L penicillin, and 100

mg/L streptomycin. At 60-70% confluence, cells were transfected with the relevant plasmids

using FuGENE6 transfection reagent (Promega). For experiments with homomeric CS mutant

channels, cells were co-transfected with pCDNA3.1‐mouseKir6.2-WT (0.6 µg) and wild-type

(WT) or mutant pCMV6-ratSUR2A (SUR2) (1 µg). For experiments on heteromeric channels,

cells were co-transfected with mouseKir6.2-WT, ratSUR2-WT, and mutant SUR2 at ratios of

0.6:0.5;0.5 (w:w:w). Cells transfected with GFP-pcDNA3.1 served as controls. A small amount

of GFP cDNA was included to allow for identification of GFP-expressing transfected cells

during electrophysiology experiments.

Macroscopic 86Rb+ Efflux Assays

Cells were incubated overnight at 37°C in DMEM containing 1 µCi/mL 86RbCl (PerkinElmer

Life Sciences), and then incubated in Ringers solution (mM: NaCl 118, HEPES 10, NaHCO3 25,

KCl 4.7, KH2PO4 1.2, CaCl2 2.5, MgSO4 1.2, adjusted to pH 7.4 with NaOH) in (1) the absence

(basal) or (2) the presence of 2.5 mg/mL oligomycin and 1 mM 2‐deoxy‐d‐glucose (metabolic

32

inhibition), (3) the presence of SUR1 or SUR2-specific K+ channel openers diazoxide or

pinacidil or (4) a combination of metabolic inhibition and pinacidil, to achieve maximal channel

activation. Subsequently, at selected time points (2.5, 5, 7.5, 15, 25, and 40 minutes), the solution

was collected and replaced with fresh solution. Upon completion of the assay, cells were lysed

with 2% SDS and collected, and the radioactive 86Rb+ in these samples was measured. 86Rb+

efflux is expressed as a fraction of total content. An inactivating, KATP-independent,

conductance was assumed to be present in all 86Rb+ efflux experiments, and apparent rate

constants for the KATP-independent 86Rb+ efflux were obtained from GFP transfected cells using

the equation:

[1] Rb efflux = {1 − ��∗��∗ ��.� }

where k1 and k-1 are the apparent activation and inactivation rate constants. KATP-dependent 86Rb+

efflux was also assumed to activate and inactivate with time and was obtained using the

equation:

[2] Rb efflux = {1 − ��∗��∗ �� ∗�� ∗��!.� }

where k1 and k-1 are the rate constants for KATP independent pathways (obtained from GFP-

transfected cells by Equation 1), k2 and k-2 are the activation (k2) and inactivation (k-2) rate

constants for KATP-specific K+ conductance. k2 is then assumed to be proportional to the number

of active KATP channels.

86Rb+ efflux experiments were also carried out in the presence of MI with the addition of

10 µM glibenclamide. In these experiments, the k-2 parameter was assumed to be the same for

each construct and all KATP-dependent fluxes were fitted simultaneously on any given day and

33

under any given condition. To account for day-to-day variability, k1 was determined in mock

transfected cells for each condition (Basal, MI, PIN, MI+PIN, and MI+GLIB), and incorporated

into the equation to estimate the KATP-dependent k2 for each construct.

Excised Patch Clamp

After 24-48h, transfected cells that fluoresced green under UV light were selected for analysis by

excised patch clamp experiments at room temperature. For experiments, cells were placed in a

perfusion chamber that allowed for the rapid switching of solutions. Kint solution (mM: KCl 140,

HEPES 10, and EGTA 1, to pH 7.4) was used as the standard pipette (extracellular) and bath

(cytoplasmic) solution in these experiments. ATP or ADP was added as indicated. The

appropriate amounts of MgCl2 to be added in each Mg2+-nucleotide containing solution to attain

0.5 mM free Mg2+ were calculated by means of the CaBuf program (KU Leuven). Membrane

patches were voltage-clamped using an Axopatch 1D amplifier (Molecular Devices), and

currents were recorded at −50 mV (pipette voltage, +50 mV) in the on-cell and inside-out

excised patch configurations. Data were typically filtered at 1 kHz and digitized at 5 kHz with a

Digidata 1322A (Molecular Devices) A-D converter. pClamp and Axoscope software (Molecular

Devices) were used for data acquisition. For ATP inhibition, [ATP]-response relationships (Fig.

4) were fitted by equation 3:

[3] "#�$ = {1 + &'()*+,-

./

}�0

where Irel (relative current) is the current in the presence of a given concentration of ATP relative

to current in zero ATP, Ki is the apparent ATP inhibition constant, and H is the Hill coefficient.

For PIP2 activation experiments, an ammonium salt of L-α-phosphatidylinositol-4,5-

bisphosphate from Porcine Brain (Brain PI(4,5)P2) (Avanti Polar Lipids) was dissolved in KINT

to make a 5 µg/µL working solution. For each patch, we estimated the relative Po by dividing the

maximum steady-state current in zero ATP by the maximum steady-state current in PIP2.

34

CHAPTER Z: CANTU SYNDROME RESULTING FROM ACTIVATING MUTATION IN

THE KCNJ8 GENE

Cooper PE et al. 2014

3.1 Introduction

In collaboration with Heiko Reutter we screened KCNJ8 in an ABCC9 mutation-negative patient

who also exhibited clinical hallmarks of CS (hypertrichosis, macrosomia, macrocephaly, coarse

facial appearance, cardiomegaly, and skeletal abnormalities). A de novo missense mutation

encoding Kir6.1[Cys176Ser] was identified in the patient. Kir6.1[p.Cys176Ser] channels

exhibited markedly higher activity than wild-type channels, as a result of reduced ATP

sensitivity, whether co-expressed with SUR1 or SUR2A subunits. Our results identify a novel

causal gene in CS, and also demonstrate that the cardinal features of the disease result from gain

of KATP channel function, not from a Kir6-independent SUR2 function.

3.2 Methods

All genetic analysis and patient assessments were carried out in collaboration with

Heiko Reutter’s group.

3.3 Results

Case study

The clinical phenotype of the proband at the age of 3 months was originally described by Engels

et al. (Engels et al., 2002). Following the realization that this CS patient harbored a distinct

molecular basis, we have now carried out a detailed reevaluation of the patient at the age of 13

years. The patient shows key clinical hallmarks of CS, including congenital hypertrichosis,

macrosomia at birth, macrocephaly, coarse facial appearance, cardiomegaly, skeletal

35

abnormalities, and developmental delay. At the time of reevaluation, he also had excessive

gingival hyperplasia (Figure 3.1C).

Echocardiography at 13 years of age showed normal biventricular function with signs of

noncompaction of the left ventricular apical myocardium, sonography of parenchymal abdominal

organs was within the normal range, as were ECG and 24 hr blood pressure measurements. The

patient's weight was 41 kg (25.–50. centile), height was 144 cm (10.–25. centile) and BMI was

19.5 (+0.3 SDS). He appeared disproportionate with a sitting height of 76 cm, arm span of

151.5 cm (10–25th centile) and arm span: height ratio >97th centile. His chest X-ray showed the

same broadening of ribs as initially reported (Engels et al., 2002). X-ray of his left hand showed

his bone age to be 10½ years at 13 years of age, corresponding to a delay of 2½ years.

Laboratory studies showed essentially undetectable serum IGF-1 levels (<25 ng/ml, Ref. value

131–690) and a relatively low IGFBP3 value of 1.4 μg/ml (Ref. value 2.6–8.9). Arginine

tolerance and clonidine test revealed absolute growth hormone (GH) deficiency (basal and all

stimulated GH concentrations were below 1.0 ng/ml). GHRH testing resulted in subnormal GH

secretion (max. GH 2.8 ng/ml).

Comparison of the present case with recently published CS patients carrying ABCC9

mutations (Harakalova et al., 2012; van Bon et al., 2012) shows that the present patient exhibits

all clinical hallmarks of CS, as well as 10/12 CS associated facial/cranial features; 2/5 cardiac

features; 7/17 skeletal abnormalities, visible on radiographic studies; and 3/15 additional

previously reported CS-associated features.

36

Figure 3.1- Gain-of-function KCNJ8 mutation Kir6.1[Cys176Ser]underlies Cantu

syndrome. A: Sequence chromatogram of patient and unaffected parents’ genomic

DNA confirms de novo g.21,919,406A>T transition mutation in the patient, which is

absent in the mother and father. B: Ribbon diagram of two of the four Kir6.1 subunits

that form the K+-selective pore in KATP, based on the crystal structures of KirBac1.1

(PDB ID: 1BL8) and cytoplasmic domain of Kir3.1 (PDB ID: 1N9P). Shown are

Kir6.1 residues mutated in CS (Cys176Ser), and reported in association with the J-

wave syndrome (p.Ser422Leu).C: Clinical phenotype of the patient. Photographs of

the patient at 13 years reveal extensive hypertrichosis, macrocephaly, coarse facial

appearance, long arm- and torso-to-height ratio, and gingival hyperplasia with

thickened lips.

37

Genetic analysis

To date, mutations in ABCC9 are the only known genetic cause of CS (Czeschik et al., 2013;

Harakalova et al., 2012; van Bon et al., 2012). In our initial study, we found that nine out of 12

patients diagnosed with CS carry missense mutations in ABCC9 (van Bon et al., 2012).We

performed Sanger sequencing of the three coding exons of KCNJ8 in the three unexplained

patients, including the present case. KCNJ8 was chosen as the most promising candidate gene

because of the functional considerations stated above. The study was approved by the local

ethics committees, and all participating patients gave written informed consent. Parental consent

was given on behalf of patients younger than 18 years of age; the study was explained to children

capable of giving assent, and they provided oral assent.

In the proband, we identified a mutation Chr12(GRCh37):g.21919406A>T

(NM_004982.2:c.526T>A), in the KCNJ8 gene, encoding a missense mutation (Cys176Ser) in

the Kir6.1 protein (Figure 3.1).We did not observe the mutation in any of 2,096 in-house

exomes, nor is it reported in any of 6,503 individuals from the exome variant server (Exome

Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA

(URL: http://evs.gs.washington.edu/EVS/) [March 2014]). The variant has been deposited in the

LOVD 3.0 (http://databases.lovd.nl/). The mutation is predicted to be “deleterious” by SIFT

(score 0.03), and probably damaging by PolyPhen2 (score 1.0). The affected amino acid is fully

conserved in both mammalian Kir6.1 and Kir6.2 proteins, suggesting a key role in channel

function. Paternity was proven and sample mix-up excluded by STR marker analysis. The

mutation was not present in maternal or paternal DNA and hence was presumed to have arisen de

novo. Primer sequences and PCR conditions are available upon request.

38

Functional analysis of KCNJ8 mutations

To assess the effect of the Cys176Ser mutation on channel activity, mutant and wild type KCNJ8

(Kir6.1) cDNAs were co-transfected with ABCC8 (SUR1) or ABCC9 (SUR2A) cDNAs. Channel

activity was assessed under normal metabolic conditions, in metabolically inhibited conditions

(mimicking tissue ischemia), and in the presence of pharmacological potassium channel openers

(KCOs), using 86Rb+ efflux assays (see Methods). We also examined the channel activity

resulting from Kir6.1[Ser422Leu], a mutation reported in several studies to be associated with J-

wave abnormalities in the electrocardiogram (Barajas-Martinez et al., 2012; Medeiros-Domingo

et al., 2010). As shown in Figure 3.2 and 3.3, very similar fluxes were measured for WT Kir6.1

and Ser422Leu channels. Measurable basal conductance was present for WT Kir6.1 with SUR1,

but only MI- or KCO-stimulated fluxes were present for WT plus SUR2A. In contrast,

significant fluxes were present for Kir6.1[Cys176Ser] with SUR1, and both MI- and KCO-

stimulated fluxes were higher for each Kir6.1[Cys176Ser]/SUR combination. These experiments

establish that the Cys176Ser mutation indeed causes a gain- of-function in the Kir6.1 channel,

leading to markedly enhanced channel activity even under basal metabolic conditions, in co-

expression with either SUR1 or SUR2A subunits.

To gain further insight to the mechanism by which the mutation enhances channel

activity, we turned to patch-clamp experiments. The majority of KATP channels in vivo are

probably formed as homomeric Kir6.1 tetramers or Kir6.2 tetramers, although there is evidence

that heteromeric Kir6.1/Kir6.2 combinations may also be present in cardiac (Bao et al., 2011)

and smooth (Flagg et al., 2010; Insuk et al., 2003) muscle, for instance. Since the original

cloning and expression of KATP subunit genes, it has been clear that Kir6.1 channels are

experimentally considerably more labile in excised patches than are Kir6.2 channels, and channel

39

Figure 3.2- Increased channel activity in basal and stimulated conditions in intact cells

expressing Kir6.1[C176S]-containing KATP channels. Relative efflux of 86Rb+ as a

function of time in basal conditions (left), in the presence of potassium channel openers

(center), or in metabolic inhibition (right) for reconstituted wild-type and mutant KATP

channels (with SUR1 (above) or SUR2A (below)), and untransfected controls (mean +

s.e.m., from 4-6 experiments). Data were fit with a double-exponential equation to obtain

rate constants for KATP-dependent efflux, k2 (see Methods). Lines show mean fitted equations

40

Figure 3.3- Increased relative efflux in basal and stimulated conditions in

intact cells expressing Kir6.1[C176S]-containing KATP channels. Relative

efflux rate constants for KATP-dependent 86Rb+efflux (k2)of 86Rb+ as a function of

time in basal conditions (black), in the presence of potassium channel openers

(white), or in metabolic inhibition (gray) for reconstituted wild-type and mutant

KATP channels (with SUR1 (A) or SUR2A (B)), and untransfected controls in

basal conditions relative to metabolic inhibition (MI) for WT and mutant

KATP channels (mean ± s.e.m., from 4 to 6 experiments). *P < 0.01 compared

with the wild-type KATP by unpaired Student's t-test.

41

activity rapidly runs down in the absence of Mg-nucleotides, complicating the assessment of

both inhibitory nucleotide sensitivity and intrinsic open probability of the channel. In the patient,

the mutation is present in only one allele and since the active channels are tetramers, expressed

KATP channels in native cells will therefore be expected to consist of a mixture of both

Cys176Ser and wild-type Kir6.1 or Kir6.2 subunits. To take advantage of this fact and

additionally test the effect of heterozygosity, we co-expressed a 1:1 mixture of wild-type or

mutant Kir6.1 cDNAs with wild-type Kir6.2 and SUR2A cDNAs (Figure 3.4).

These mixtures generated measurable fluxes in tracer studies and stable currents in

excised patches. From these measurements it is evident that enhanced channel activity in intact

cells expressing Cys176Ser (Figure 3.4) result from reduced ATP sensitivity (Figure 3.5B and

3.5C). We cannot formally rule out the possibility that altered trafficking and increased plasma

membrane channel density may also play a role, but such effects have not previously been

described for mutations in Kir channel pore regions. The findings indicate that the heterozygous

Cys176Ser mutation in Kir6.1 will over activate any native KATP channels in which it is present.

The results are very consistent with the effects of the exact equivalent, and well-studied

mutation, Cys166Ser in Kir6.2, which reduces ATP sensitivity of expressed channels by ∼50-

fold (Loussouarn et al., 2000; Tucker et al., 1998). Additional mutations at this residue in Kir6.2,

which also cause marked GOF, have been identified as causal in the most severe form of

neonatal diabetes that is associated with developmental delay and epilepsy (Gloyn et al., 2006).

3.3 Discussion

CS was first characterized as such by J.M. Cantú in 1982 (Cantu et al., 1982). A genetic cause

was recently reported (Harakalova et al., 2012; van Bon et al., 2012), with coding mutations

identified in the ABCC9 gene, in 25 out of 30 patients. The present study reveals that a

heterozygous mutation in KCNJ8, encoding the Kir6.1 pore-forming subunit of KATP channels,

can also cause CS. A recent study identified a different mutation in KCNJ8 (encoding Val65Met)

42

Figure 3.4- Increased channel activity in basal and stimulated conditions in

reconstituted heteromeric KATP channels containing Cys176Ser subunits.

A) Relative efflux of 86Rb+ as a function of time in basal conditions (left), in the

presence of potassium channel openers (center), or in metabolic inhibition (right) for

heteromeric Kir6.1/Kir6.2 or Kir6.1[Cys176Ser]/Kir6.2 plus SUR2A KATP channel-

expressing cells, and untransfected control cells (mean + s.e.m., from 3 experiments).

Data were fit with a double-exponential equation to obtain rate constants for KATP-

dependent efflux, k2 (see Methods). Lines show mean fitted equations. B) Rate

constants for KATP-dependent 86Rb+ efflux (k2) in basal conditions (black) and in

metabolic inhibition (MI) (gray) from COS cells expressing heteromeric Kir6.1/Kir6.2

or Kir6.1[Cys176Ser] (C176S)/Kir6.2 plus SUR2A KATP channels.

43

Figure 3.5- Reduced ATP-sensitivity of reconstituted heteromeric KATP channels

containing Kir6.1[Cys176Ser] subunits. A: Rate constants for KATP-

dependent86Rb+ efflux (k2) in basal conditions and in metabolic inhibition (MI) from COS

cells expressing heteromeric Kir6.1/Kir6.2 or Kir6.1[Cys176Ser] (C176S)/Kir6.2 plus

SUR2A KATP channels. B: Representative currents recorded from inside-out membrane

patches from COS cells expressing heteromeric Kir6.1/Kir6.2 or Kir6.1[Cys176Ser]

(C176S)/Kir6.2 plus SUR2A KATP channels at −50 mV in Kint solution (see Supplementary

information). Patches were exposed to differing [ATP] and baseline current was determined

by exposure to 5 mM ATP. C: Steady-state dependence of membrane current on [ATP]

(relative to current in zero ATP (Irel)) for wild-type and Cys176Ser-containing channels.

Data points represent the mean ± s.e.m. (n = 6–8 patches). The fitted lines correspond to

least squares fits of a Hill equation. *P < 0.01 compared with the wild-type KATP by

unpaired Student's t-test.

44

in another CS patient (Brownstein et al., 2013), although there was no functional characterization

of mutant channels. The marked GOF in expressed Kir6.1[Cys176Ser] channels (Figures 3.1

and 3.2), which mirrors the well-studied properties of Kir6.2 channels with mutations at the

equivalent Cys166 residue (Gloyn et al., 2006; Loussouarn et al., 2000; Trapp et al., 1998;

Tucker et al., 1998), clearly establishes Cys176Ser as a severe GOF. Thus GOF mutations in

both ABCC9 and KCNJ8 cause essentially the same hallmark CS features in the affected patients

indicating that these features all result from GOF of KATP channels formed from these subunits.

This is a subtle but critical conclusion: previous studies (Aggarwal et al., 2010; Stoller et al.,

2010) have raised the possibility of Kir6-independent roles of SUR2 proteins and, in the absence

of the present findings, some or all of the features of CS could conceivably have arisen from

non-channel functions of the SUR2 protein.

The results underscore the key role of KATP channels in coupling cell membrane potential

with diverse tissue functions. Our results are consistent with the finding that vascular smooth

muscle contractility is markedly decreased in the presence of KATP channel openers (Flagg et al.,

2010) and in transgenic mice expressing mutant Kir6.1 subunits with reduced sensitivity to

inhibitory ATP (Li et al., 2013). These observations potentially explain some of the key findings

in CS, including patent ductus arteriosus, as was present in the patient reported here.

A novel finding is the puzzling combination of biochemical signs of absolute growth

hormone (GH) deficiency, yet postnatal growth with height within the normal range. Pituitary

GH secretion is mainly regulated by two hypothalamic neuropeptides. GHRH stimulates GH

release, whereas somatostatin inhibits GH secretion (Muller et al., 1999). In addition, a number

of GH-releasing peptides are able to modulate GH release. The rat pituitary expresses several Kir

channel alpha-subunits, including Kir6.1 (Wulfsen et al., 2000). GHRP-2, one member of the

45

GH-releasing neuropeptides, exerts its GH secretory effect through a reduction in potassium

current (Xu et al., 2002). It is therefore tempting to speculate that GOF mutations in pituitary

KATP channels exert an opposite effect on GHRH stimulation, leading to resistance to

hypothalamic induction of GH release. On the other hand, GH insufficiency yet normal growth

can only be explained by a concurrent stimulation of long bone growth. Previous animal models

indicated that a significant part of postnatal growth depends on local growth stimulation in the

growth plate rather than on the effect of circulating IGF-1 (Ohlsson et al., 2009; Yakar et al.,

1999), although understanding of molecular mechanisms at the growth plate leading to long bone

growth is incomplete. It is not yet clear whether ABCC9 mutation patients exhibit similar

deficiencies, and if not, this finding may be a KCNJ8-specific outcome, or may be unrelated to

CS.

A common KATP pathway can account for direct overlap of CS features resulting from

mutations in ABCC9 and KCNJ8, as well as with features resulting from overexposure to

minoxidil and other KATP channel openers (Avatapalle et al., 2012; Kaler et al., 1987; Nguyen

and Marks, 2003; Shanti et al., 2009). However, this does not provide an obvious explanation for

many CS features. For instance, macrocephaly and characteristic facial features, as well as

skeletal abnormalities are present in both ABCC9 and KCNJ8 patients. However, while KATP

channels generated by these subunits have been reported in human chondrocytes (Mobasheri et

al., 2012; Rufino et al., 2013) and osteoblasts (Kawase et al., 1996), their role in maturation and

proliferation remains unknown, and the cellular pathway involved is not obvious. Interestingly,

minoxidil has been reported to induce pseudoacromegalic features in the absence of elevated GH

or IGF-1 (Nguyen and Marks, 2003). Similarly, epicanthal folds and deep plantar creases might

result from KATP gain-of-function, but the underlying cause is unknown. Finally, pericardial

46

effusion, polydramnios and lymphoedemia, potentially all related problems of membrane barrier

breakdown, are unexplained. SUR2/Kir6.1 KATP channels have been reported in epithelial cells

(Bardou et al., 2009), but again, cellular pathways are unexplained.

Finally, we would note that several other studies have reported a different mutation

(p.Ser422Leu) in the Kir6.1 protein to be associated with ‘early repolarization syndrome’ (ERS),

characterized by abnormalities in the J-point of the electrocardiogram (Delaney et al., 2012;

Haissaguerre et al., 2009). Recent studies have reported that this p.Ser422Leu mutation enhances

channel activity (Medeiros-Domingo et al., 2010), by reducing ATP-sensitivity (Barajas-

Martinez et al., 2012). If so, there is a clear inconsistency: neither J-wave abnormalities nor other

arrhythmias have been reported in CS patients, and none of the CS features have yet been

reported for ERS patients. Our own data (Figure 3.2) indicate that, in recombinant COS cells,

p.Ser422Leu does not affect Kir6.1-dependent KATP channel activity, consistent with the recent

recognition that this mutation may actually represent a common variant (~4%) in Ashkenazim

subjects, and not clearly associated with ERS (Veeramah et al., 2014).

47

CHAPTER f: DISEASE CAUSING SLIDE HELIX RESIDUE MUTATION IN KIR6.1 AND

KIR6.2

4.1 Introduction

While all previous reports of CS-associated mutations are in the ABCC9 gene, a CS patient with

a KCNJ8 mutation, encoding V64M in the Kir6.1 subunit, was described by Brownstein et al. in

2013, although no functional support for this mutation causing any change in channel activity

was provided. Interestingly, a previously identified mutation at the equivalent position in Kir6.2

(V64L), was reported to have no effect on KATP channel a. Here, I directly compare these two

published mutations: valine to methionine and valine to leucine in the equivalent positions in

Kir6.1 (V65) and Kir6.2 (V64), co-expressed with SUR1 or SUR2A. Using macroscopic

rubidium (86Rb+) efflux assays, I show that, compared to WT channels, both Kir6.1-V65M and

Kir6.2-V64M enhance KATP activity, but channels formed with Kir6.1-V65L and Kir6.2-V64L

are unaffected. Additionally, Kir6.2-V64M-based channels decrease ATP sensitivity and PIP2

activation, but V64L-based channels do not. Taken together, these data provide functional

support for the notion that CS-associated Kir6.1-V65M mutation leads to overactive KATP

channels. More specifically, the mechanism of this GOF is a result of the valine-to-methionine

substitution increasing channel Po and thereby indirectly decreasing ATP inhibition. These

findings are consistent with a causal involvement of this mutation in CS.

4.2 Results

Kir6.1-V65M, but not Kir6.1-V65L, forms overactive KATP channels paired with SUR1 or SUR2

To examine KATP channel activity in intact cells, I performed rubidium (86Rb+) efflux assays (see

Methods) under three metabolic conditions: basal; metabolic inhibition (MI), which prevents

48

ATP production; or in the presence of SURX-specific potassium channel openers diazoxide

(SUR1) or pinacidil (SUR2). Kir6.1-WT, Kir6.1-V65M and Kir6.1-V65L were each

homomerically expressed with SUR1 (Figure 4.1). Under basal and diazoxide conditions Kir6.1-

V65M based channels exhibited significantly higher 86Rb+ efflux rate compared to WT channels,

while Kir6.1-V65L channels efflux rates were comparable to WT (Figures 4.1A and 4.1B).

Maximal activation, estimated under MI conditions, comparable for Kir6.1-V65M, V65L and

Kir6.1-WT based channels, implying no differences in expressed channel densities (Figure

4.1C).

When paired with SUR2A, basal fluxes through Kir6.1-WT, Kir6.1-V65M and Kir6.1-V65L

were undistinguishable (Figure 4.2A), presumably due to lower activation of SUR2 by MgATP

or MgADP compared to SUR1(Masia et al., 2005). However, when KATP- specific fluxes were

activated in pinacidil or MI conditions, Kir6.1-V65M but not Kir6.1-V65L fluxes were

significantly increased compared to WT channels (Figure 4.2B and 4.2C). These trends are

consistent with Kir6.1-V65M causing a GOF, but Kir6.1-V65L having WT-level KATP channel

activity when paired with SUR1 or SUR2 (Figures 4.1 and 4.2).

Kir6.2-V64 mutated to methionine or leucine produces comparable functional changes to Kir6.1-

V65M or L mutations

Kir6.1 (V65) and Kir6.2 (V64) are localized within an identical sequence region in the slide

helix (Inagaki et al., 1995c). Therefore, it is likely the role this valine residue plays in gating is

conserved between each channel. In the basal condition, whether expressed with SUR1 or SUR2,

Kir6.2-V64M formed channels with increased efflux rates compared to wild type channels, but

Kir6.2-V64L-based channels exhibited comparable activity to WT (Figure 4.3A and 4.4A).

49

Figure 4.1- Homomeric Kir6.1-V65M–but not Kir6.1-V65L–expressed with SUR1

increases KATP channel activity under basal conditions and in the presence of diazoxide

in intact cells. Cumulative 86Rb+ efflux, as a function of time, was measured in GFP-

transfected control cells (black symbols), and in cells transiently expressing SUR1 plus

Kir6.1-WT (white triangles), Kir6.1-V65M (blue squares) or Kir6.1-V65L (light grey

circles). Experiments were done in basal conditions (A), in the presence of the K+ channel

opener diazoxide (B), or metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C).

The data represents the means + S.E.M. of 8-18 experiments. Flux data from each condition

was fit with a double-exponential equation to obtain the rate constants for KATP-dependent

efflux, k2, where lines show mean fitted relationships. Graphical representation of the

determined k2 rate constants are shown on the right of the corresponding cumulative efflux

over time. *p< 0.05 as compared to WT (unpaired Student’s t test).

50

Figure 4.2- Homomeric Kir6.1-V65M (but not Kir6.1-V65L) expressed with SUR2A

increases KATP channel activity under Metabolic Inhibition (MI) and in the presence of

pinacidil in intact cells. Cumulative 86Rb+ efflux, as a function of time, was measured in GFP-

transfected control cells (black symbols), and in cells transiently expressing SUR2A with Kir6.1-

WT (white triangles), Kir6.1-V65M (blue squares) or Kir6.1-V65L (light grey circles).

Experiments were done under basal conditions (A), in the presence of the K+ channel opener

pinacidil (B), or metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C). The data

represents the means + S.E.M. of 3 experiments. Flux data from each condition was fit with a

double-exponential equation to obtain the rate constants for KATP-dependent efflux, k2 (right),

where lines show mean fitted relationships. Graphical representation of the determined k2 rate

constants are shown on the right of the corresponding cumulative efflux over time. * p< 0.05 as

compared to WT (unpaired Student’s t test).

51

Figure 4.3- Homomeric Kir6.2-V64M–but not Kir6.2-V64L– expressed with SUR1

increases KATP channel activity in basal conditions and in the presence of Diazoxide in

intact cells. The cumulative 86Rb+ efflux, as a function of time, was measured in GFP-

transfected control cells (black symbols), and in cells transiently expressing SUR1 with Kir6.2-

WT (white triangles), Kir6.2-V64M (navy squares) or Kir6.2-V64L (dark grey circles).

Experiments were done in basal conditions (A), in the presence of the K+ channel opener

diazoxide (B), or metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C). The data

represents the means + S.E.M. of 3-5 experiments. Flux data from each condition was fit with a

double-exponential equation to obtain the rate constants for KATP-dependent efflux, k2 (right),

where lines show mean fitted relationships. Graphical representation of the determined k2 rate

constants are shown on the right of the corresponding cumulative efflux over time. * p< 0.05 as


52

Figure 4.4- Homomeric Kir6.2-V64M–but not Kir6.2-V64L–with SUR2A

increases KATP channel activity in basal conditions and in the presence of pinacidil

in intact cells. The cumulative 86Rb+ efflux, as a function of time, was measured in

GFP-transfected control cells (black symbols), and in cells transiently expressing

SUR2A with Kir6.2-WT (white triangles), Kir6.2-V64M (navy squares) or Kir6.2-

V64L (dark grey circles). Experiments were done in basal conditions (A), in the

presence of the K+ channel opener pinacidil (B), or metabolic inhibitors (MI)

oligomycin and 2-deoxy-d-glucose (C). The data represents the means + S.E.M. of 3-6

experiments. Flux data from each condition was fit with a double-exponential equation

to obtain the rate constants for KATP-dependent efflux, k2 (right), where lines show

mean fitted relationships. Graphical representation of the determined k2 rate constants

are shown on the right of the corresponding cumulative efflux over time. * p< 0.05 as


53

These trends were also seen in the presence of diazoxide or pinacidil (Figure 4.3B and 4.4B). In

MI Kir6.2 WT, Kir6.2-V64M and Kir6.2-V64L reached the same maximum with comparable

efflux rates, whether paired with SUR1 or SUR2, indicating that these mutations do not alter the

expression of KATP channels (Figure 4.3C and 4.4C). Overall, compared to WT channels, the

valine-to-methionine substitution in both Kir6.1 and Kir6.2 results in a gain of function in KATP

activity, but the valine-to-leucine mutation exhibits comparable channel activity (Figures 4.1-

4.4).

Kir6.2-V64M overactivity results from decreased ATP sensitivity

The 86Rb+ efflux experiments demonstrate that the behavior of valine-to-methionine and valine-

to-leucine substitutions are consistent whether expressed in Kir6.1 or Kir6.2, but do not reveal

underlying molecular mechanism(s). Unlike Kir6.2, Kir6.1-based channels quickly run down and

require activatory nucleotides to maintain channel activity, making assessment of nucleotide

sensitivity experimentally challenging (Babenko et al., 2000). To assess the details of increased

channel activity, inside-out excised patch-clamp experiments on Kir6.2-WT-, Kir6.2-V64M- and

V64L-based channels paired with SUR2A were carried out. Intrinsic sensitivity to ATP

inhibition (in zero Mg2+) was similar for WT (Ki = 25 μM) and Kir6.2-V64L (Ki = 16 μM)

channels. However, KATP channels expressing Kir6.2-V64M exhibited a significant right shift in

the [ATP]-response (Ki = 289 μM) (Fig. 4.5B). A decreased sensitivity to intracellular ATP

could account for the increased activity of KATP channels under basal conditions in the intact cell

(Figure 4.4A). PIP2 potentiation experiments were carried out on wild type and Kir6.2-V64M-

based channels to assess ATP-independent open state stability (Enkvetchakul and Nichols, 2003)

(Figure 4.6B). As seen in Figure 4.6C, wild type channels have a relative open probability of

54

Figure 4.5- Gain of function in Kir6.2-V64M due to decreased ATP-sensitivity.

Representative excised patch current recordings from COSm6 cells co-expressing SUR2A-

WT with Kir6.2-WT, Kir6.2-V64M or Kir6.2-V64L (A). Membrane potential was held at -

50mV, and currents were recorded continuously on-cell and in inside-out excised patches

exposed to KINT in the absence or in the presence of 0.01, 0.1 or 1 mM ATP. Dose-

response data (mean ± SEM 10 patches each) were fit to equation 1 to estimate the ATP

concentration for half-maximal inhibition Ki: WT (24.7 µM), Kir6.2-V64M (16 µM), and

Kir6.2-V64L (289.3 µM) (B).

55

Figure 4.6- Increased open state stability in Kir6.2-V64M-based channels underlies decreased

ATP sensitivity. Representative KATP current traces for SUR2A-WT expressed with Kir6.2-WT (A)

or Kir6.2-V64M (B) following excision and in the presence of 10 mM ATP, 1mM ATP, or 5 µg/µL-

PIP2. The relative Po for individual patches from WT (white diamonds) and V64M (navy blue

squares) was determined as a ratio of the maximum steady-state current upon excision, in the absence

of nucleotides, over the maximum current measured in PIP2. The average Po (black bars) is the mean

of 8 individual patches for each construct ± SEM. The relative Po for WT channels is 0.73 ± 0.08

(WT) and 1.05 ± 0.07 for Kir6.2-V64Mbased channels. *p< 0.05 as compared to WT (unpaired

Student’s t test).

56

0.73±0.08 where as Kir6.2-V64M channels have a significantly increased Po of 1.05±0.07,

indicating that the valine-to-methionine substitutions also affect channel PO.

4.3 Discussion

Cantu Syndrome, a complex multi-organ disease, was first described in 1982(Cantu et al.,

1982). Recently, several studies have demonstrated that the majority of CS patients have

a mutation in ABCC9 (Harakalova et al., 2012; van Bon et al., 2012). In addition, two patients

with clinically defined CS have been identified with coding mutations in KCNJ8 (Brownstein et

al., 2013; Cooper et al., 2014), and two of these studies, together with the present study, confirm

that the disease therefore results from gain of function in KATP channel activity that is generated

by the SUR2 and Kir6.1 subunits encoded by these genes.

Effects of equivalent residue mutations on KATP channel activity

In a previously reported case of neonatal diabetes, a mutation at the equivalent position in Kir6.2

(V64L) was reported along with an additional mutation at F60Y. When these mutations were

expressed separately and channel activity was assessed, Kir6.2-V64L was determined to have no

effect on KATP activity, and GOF in KATP activity was solely a result of the increased KATP

channel activity from the F60Y mutation (Mannikko et al., 2009). Similarly, Kir6.2-V64A does

not perturb KATP activity (Li et al., 2013). In the present study I have confirmed this finding, and

shown that mutation of the equivalent valine to leucine in either Kir6.1 or Kir6.2 is without

effect on recombinant channel activity. Conversely, mutation to methionine in either Kir6.1 or

Kir6.2 generates a net gain of function. The inability of either leucine or alanine substitutions at

this position to alter KATP activity might suggest that the bulkiness of the mutated residue at this

position is relevant to if and how it alters KATP channel activity.

57

Mechanism of increased KATP channel activity through Kir6.1-V65M and Kir6.2-V64M-based

channels

Given the experimental difficulty of recording Kir6.1/SUR2 currents (Cooper et al., 2014),

Kir6.2-V64M and V64L were instead paired with SUR2A to determine nucleotide sensitivity of

KATP channels formed with each mutation. In excised patches, the ATP-sensitivity of Kir6.2-

V64M based channels, but not Kir6.2-V64L, was significantly reduced, compared to Kir6.2-WT

channels. This result is consistent with the overall gain of function in channel activity in Kir6.2-

V64M and Kir6.1-V65M being a result of decreased ATP-sensitivity (Figure 4.5). As has

previously been described, mutations in Kir6.2 can reduce ATP sensitivity by two distinct

mechanisms: 1) by directly altering ATP binding and/or 2) by increasing ATP-independent open

probability, thus indirectly reducing ATP sensitivity (Koster et al., 2008a). By measuring the

relative PIP2 activation of Kir6.2-V64M compared to Kir6.2-WT-based channels, we have

shown that the Po is increased in the absence of ATP (Figure 4.6). This result is consistent with

this mutation lying outside the ATP binding pocket, and thereby altering ATP sensitivity

indirectly.

58

CHAPTER g: DIFFERENTIAL MECHANISMS OF CANTU SYNDROME-ASSOCIATED

GAIN-OF-FUNCTION MUTATIONS IN THE ABCC9 (SUR2) SUBUNIT OF THE KATP

CHANNEL

5.1 Introduction

The functional consequences of multiple uncharacterized CS-mutations remain unclear. In this

paper, we have focused on determining the functional consequences of 3 documented human CS-

associated ABCC9 mutations: human P432L, A478V and C1043Y. The mutations were

engineered in the equivalent position in rat SUR2A (P429L, A475V and C1039Y) and each was

co-expressed with mouse Kir6.2. Using macroscopic rubidium (86Rb+) efflux experiments, we

show that KATP channels formed with P429L, A475V or C1039Y mutants enhance KATP activity

compared to WT channels. We used inside-out patch-clamp electrophysiology to measure

channel sensitivity to ATP-inhibition and MgADP-activation. For P429L and A475V mutants,

sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was

significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition

by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate

that these three Cantu Syndrome mutations all lead to overactive KATP channels, but at least two

mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced

MgADP activation.

5.2 Methods

All rubidium efflux experiments were done by Monica Sala-Rabanal. Sequence alignment and

homology model by Sun Joo Lee.

59

Homology Modeling

Models of human SUR2A (Figure 5.1) were built using Modeller v9.8 from the template of the

multidrug ABC transporter Sav1866 (2ONJ, to model the ‘open’ conformation) and

heterodimeric ABC transporter TM287-TM288 (3QF4, to model the ‘closed’ conformation). The

TM0 domain and L0 loop (1-281) were omitted due to lack of sequence homology to any

proteins of known structure. Two extended loops unique to SUR2A were also omitted: one

connecting TM1 and NBD1 (614-672) and a second connecting NBD1 and TM2 (920-966). A

multiple sequence alignment (MSA) was carried out using ClustalW2 between SUR2 and the

two template sequences. Due to low sequence identity, other proteins in the human ABCC family

(ABCC1, 2, 3, 4, 5, 6, 8 and 10) were included in the alignment for TM1 and NBD1. Highly

conserved sequence identity enabled a reliable sequence alignment of TMD2 and NBD2 by

MSA between SUR2 and SUR1, 2ONJ, and 3QF4.

5.3 Results

Homomeric mutant channels are overactive in various metabolic conditions

Cantu syndrome-associated mutations have been found throughout the coding sequence (Figure

5.1). For the present study, we focused on two previously unexamined mutations (human P432L

and C1043Y, corresponding to rat P429L and C1039Y, respectively), located in the TMD1 and

TMD2 segments, and A478 (corresponding to rat A475V), also located in the TMD1 region. To

examine KATP channel activity in intact cells, we performed 86Rb+ efflux experiments under four

different conditions: basal; metabolic inhibition (MI); in the presence of pinacidil (PIN); and MI

and pinacidil combined (MI + PIN). As shown in Figures 5.2 and 5.3, homomeric expression of

60

Figure 5.1- Cantu Syndrome mutations in SUR2 Alignment of SUR2 (ABCC9) with the

multidrug ABC transporter Sav1866 (2ONJ) and heterodimeric ABC transporter TM287-TM288

(3QF4), upon which we built homology models. Key structural domains TMD1,2 and NBD1,2)

are indicated, as well as predicted alpha helical (pink) and beta strand (green) segments (A).

Homology models of ‘open’ (B) and ‘closed’ (C) conformations of the SUR2A protein, which are

based on Staphylococcus aureus Sav1866, a bacterial homolog of the human ABC transporter

Mdr1 and hetero-dimeric ABC transporter TM287-TM288 (TM287/288) from Thermotoga

maritime, respectively. Published CS mutations from previous reports are shown in green (open)

and light pink (closed).

61

Figure 5.2- Increased channel activity in intact cells expressing homomeric P429L, A475V or

C1039Y-containing KATP channels. 86Rb+ efflux, as a function of time, was measured in GFP-

transfected control cells (dashed), and in cells transiently expressing reconstituted Kir6.2-based KATP

channels with WT, P429L, A475V or C1039Y SUR2 subunits in homomeric configuration, in basal

conditions (A), in the presence of metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (B), or in

the K+ channel opener pinacidil (C), or in the presence of both MI and pinacidil (D). The data represent

means + S.E.M. of 6-10 experiments. Flux data were fit with equation 1 (GFP) to obtain the rate

constant k1 or equation 2 to obtain the rate constants for KATP-dependent efflux, k2 (Figure 5.3), where

lines show mean fitted relationships.

62

Figure 5.3- KATP conductance is increased in basal and stimulated conditions in

intact cells expressing homomeric P429L and A475V-based KATP channels. Rate

constants for KATP-dependent 86Rb+ efflux (k1 in gray, proportional to non-specific K+

conductance and k2, proportional to KATP specific K+ conductance), were calculated

from data shown in Figure 5.2. *p< 0.05 as compared to WT (unpaired Student’s t test).

63

SUR2A-P429L, A475V or C1039Y channels, results in significantly higher basal 86Rb+ efflux

rates compared to WT (Figure 5.2A and Figure 5.3A). P429L and A475V-based channels, but

not those composed of C1039Y, also showed significantly higher rate of efflux compared to WT

channels under MI conditions and in the presence of pinacidil (Figures 5.2B-C and 5.3B-C),

consistent with the gain of function(GOF) observed in the basal condition. Maximal efflux rates

(estimated using simultaneous exposure to MI and pinacidil) of P429L and A475V were

comparable to SUR2A-WT, implying similar channel densities at the cell surface (Figure 5.2D

and 5.3D). C1039Y showed significantly lower absolute fluxes in all stimulatory conditions

(Figures 5.2B-D and 5.3B-D), implying a lower channel density at the membrane (Figure 5.4).

The ratio of KATP-dependent (k2) rate constants in basal, MI and pinacidil to the maximal

KATP-dependent rate constant (k2 in MI+PIN) should reflect the relative activation of each

channel in these specific conditions. In the basal condition, the ratio was higher for all three

mutations, compared to WT, markedly so for C1039Y (Figure 5.4A). In pinacidil alone, while

only P429L and A475V reach significance, the ratio of active channels in all three mutations are

also increased (Figure 5.4C). Together, the results suggest that each mutant will result in higher

channel activity under any stimulatory conditions, particularly for C1039Y.

C1039Y (hC1043Y) overactivity results from decreased ATP-sensitivity via increased Po

86Rb+ flux assays provide evidence for overactivity in CS mutants, but do not provide any

indication of underlying molecular mechanisms. We assessed the details of channel properties in

inside-out excised patch-clamp electrophysiology experiments. Intrinsic sensitivity to ATP

inhibition (in zero Mg2+), was similar for WT, P429L and A475V channels (Ki = 7-9 μM).

64

Figure 5.4- Relative KATP conductance is markedly increased in basal and stimulated

conditions in intact cells expressing homomeric C1039Y KATP channels. The ratio of

the rate constants for KATP-dependent 86Rb+ efflux (k2) in basal and pinacidil- or MI-

stimulated conditions to the maximal activation (in MI+Pinacidil) is plotted for WT and

mutant channels. *p< 0.05 as compared to WT (unpaired Student’s t test).

65

However, KATP channels expressing C1039Y exhibited a significant right shift in ATP-sensitivity

(Ki = 21.3 µM)(Figure 5.5B). A diminished sensitivity to intracellular ATP may account, at

least in part, for the increased activity of C1039Y-based KATP channels in basal conditions in the

intact cell (Figures. 5.2A and 5.3A). Consistent with reduced channel density, the maximal

current in zero ATP was significantly lower in C1039Y channels than WT or the P429L and

A475V mutant channels (Figure 5.5C).

In PIP2 activation experiments, WT and C1039Y channels lost ATP sensitivity the more the

patch was exposed to PIP2. Simultaneously, the maximum currents for WT channels increased

while the maximum current in C1039Y channels activity remained the same (Figure 5.6). The

ratio of the maximum current in zero ATP over max current in PIP2 for WT patches (0.70 ± 0.11)

was significantly lower than that of C1039Y channel patches (1.26 ± 0.21), which implies

increased intrinsic activity from C1039Y channels.

Overactivity in P429L (hP432L) and A475V (hA478V) results from increased MgADP activation

We investigated the current response to intracellular MgADP in the presence of 0.1 mM ATP

(Figure 5.7A). MgADP-dependent activation was estimated as the ratio between the steady-state

activated current in MgADP+ATP and the maximal current in zero ATP, immediately after

excision (Figure 5.7B). In P429L, the relative current in MgADP was markedly higher than WT

for P429L and A475V channels. Using this analysis, the current was also statistically higher for

C1039Y channels. However, this analysis fails to account for the intrinsically lower sensitivity of

C1039Y channels to ATP inhibition. An alternative estimation for the stimulatory action of Mg-

66

Figure 5.5- ATP-sensitivity is decreased in C1039Y channels. Representative excised patch

clamp recordings from COSm6 cells co-expressing Kir6.2 and WT or CS mutant SUR2 subunits

P429L, A475V or C1039Y (A). Membrane potential was held at -50mV, and negative currents

(plotted as upward deflections) were recorded continuously on-cell and in inside-out excised

patches exposed to KINT in the absence or in the presence of 0.01, 0.1 or 1 mM ATP; arrowheads

mark the point of excision. Dose-response data (mean ± SEM from 8-11 patches) was fit with

equation 3 to estimate the ATP concentration for half-maximal inhibition Ki: WT (9 µM), P429L

(9 µM), A475V (7 µM), and C1039Y (21 µM) (B). The maximum patch current determined

immediately following patch excision (mean ± SEM from 8-11 patches) (C).

67

Figure 5.6- C1039Y channels decreased ATP-sensitivity is due to increased Po

Representative KATP current traces following excision and in the presence of 1 mM ATP or 5

µg/µL-PIP2 as indicated(A). Relative Po determined as a ratio of the maximum steady-state

current in the patch upon excision, in the absence of nucleotides, over the maximum current

measured in PIP2. Individual patch data represented by symbols (n = 7-10); bars are the

means ± SEM respectively, where the relative Po for WT= 0.70 ± 0.11 and 1.26 ± 0.21 for

C1039Y (B).. *p< 0.05 as compared to WT (unpaired Student’s t test).

68

Figure 5.7- P429L and A475V show enhanced MgADP activation Representative KATP current

traces following excision and in the presence of nucleotides as indicated (A). Relative MgADP

activation in individual patch data represented by symbols (n = 11-24); bars are the means ± SEM

respectively (B). *p< 0.05 as compared to WT (unpaired Student’s t test). Insert shows the mean

current measured in patches in the presence of 0.1 mM Mg-free ATP (from Figure 5.5B) subtracted

from the mean value of steady-state current measured in patches in the presence of MgADP (from

Figure 5.7A). Error bars represent the propagated error from both experiments half-time of activation

by MgADP (C).

69

nucleotides is the ratio of current in MgADP+ATP to the current in Mg-free ATP alone (Figure

5.7B, inset). This analysis implies no difference in the MgADP activation of C1039Y and WT

channels. In addition, while the MgADP activation for P429L and A475V channels is more rapid

than WT, activation of C1039Y channel is even slower than WT (Figure 5.7C).

Gain of function is reduced in heteromeric mutant channels

All documented CS patients with mutations in ABCC9 are heterozygous. To mimic the predicted

heteromeric composition of channels in such patients we also expressed each mutation in a 1:1

ratio with SUR2A-WT plus Kir6.2-WT, and assessed channel activity by 86Rb+ efflux

experiments (Figures 5.8 and 5.9). In all conditions P429L, A475V and C1039Y heteromeric

channels display no significant increases in the rates of 86Rb+ efflux, compared to WT channels

(Figures 5.8A and 5.9A). However, when normalized to the maximal efflux rates, channels with

heteromeric expression of C1039Y are still considerably more active than WT. Additionally, in

the heteromeric state, maximal C1039Y channel fluxes were markedly higher than in the

homomeric state (Figure 5.9D). Taken together, these results imply a partial rescue of the

surface expression of C1039Y subunits by WT subunits (Figure 5.7B-D and 5.8B-D).

Altered response to glibenclamide in C1039Y channels

Some NDM patients with GOF mutations in Kir6.2 or SUR1 have successfully switched from

insulin therapy to KATP channel inhibitors, such as glibenclamide (Zung et al., 2004). However, a

correlation between increased channel activity and the diminished effectiveness of such drugs

has been noted (Koster et al., 2005b). Therefore to test the effectiveness of glibenclamide on

overactive P429L, A475V or C1039Y channels, expressed both homomerically and

heteromerically, 86Rb+ efflux experiments were carried out in the presence of MI+ 10 µM

70

Figure 5.8- Channel activity in cells expressing heteromeric P429L, A475V or C1039Y-

based KATP channels. 86Rb+ efflux, as a function of time, was measured in GFP-transfected

control cells (dashed), and in cells transiently expressing reconstituted Kir6.2-based KATP

channels with WT or 1:1 mixtures of WT and P429L, A475V or C1039Y mutant SUR2

subunits, in basal conditions (A), in the presence of metabolic inhibitors (MI) oligomycin and

2-deoxy-d-glucose (B), or in the K+ channel opener pinacidil (C), or in the presence of both

MI and pinacidil (D). The data represent means + S.E.M. of 6-10 experiments. Flux data

were fit with equation 1 (GFP) to obtain the rate constant k1 or equation 2 to obtain the rate

constants for KATP-dependent efflux, k2 (Figure 5.9), where lines show mean fitted

relationships.

71

Figure 5.9- KATP conductance normalized in the basal condition and in stimulated

conditions for heteromeric P429L, A475V or C1039Y-based KATP channels. The rate

constants for non-specific efflux (k1 represented by gray area) and KATP-dependent 86Rb+

efflux (k2), proportional to KATP specific K+ conductance, were estimated from data shown

in Figure 5.8. *p< 0.05 as compared to WT (unpaired Student’s t test).

72

glibenclamide (Fig. 5.10). The time course of 86Rb+ effluxes shows an unusual behavior in that

although initial fluxes are markedly lower than in MI without glibenclamide, the inhibition is not

maintained through the time course of the assay. It is well understood that the inhibitory action

of glibenclamide is a complex function of the metabolic conditions, and decreases under

conditions of metabolic inhibition (Findlay, 1993; Koster et al., 1999). To account for this

behavior, the efflux data in glibenclamide were again fit by equation 2, where negative values for

k-2 result in the rate of efflux actually increasing with time for A475V and P429L channels. From

this analysis, the ratio of the k2 in MI+glibenclamide and k2 in MI alone approximates the

relative glibenclamide sensitivity (Figure 5.10C). While the sensitivity of A475V and P429L

channels was not different from WT, C1039Y channels are notably less sensitive. Qualitatively

similar results were obtained with channels expressed in heteromeric mixture with WT subunits

(Figures 5.9B,D). As discussed below, this is consistent with similar findings for SUR1

mutations that results in increased activity of the resultant KATP channels.

5.3 Discussion

Distinct mechanisms of KATP GOF in Cantu Syndrome ABCC9 mutations

Cantu Syndrome (CS) is a rare disease characterized by complex vascular and skeletal

anomalies, the underlying cellular and molecular mechanisms of which we are only now

beginning to understand (Nichols et al., 2013). The majority of genotyped CS patients are

heterozygous for mutations in the genes encoding ABCC9 (SUR2) (Harakalova et al., 2012; van

Bon et al., 2012) in most cases, and KCNJ8 (Kir6.1) (Brownstein et al., 2013; Cooper et al.,

2014) in others. A select few of these mutations, to date, have been shown to form overactive

KATP channels (Cooper et al., 2014; Harakalova et al., 2012), but most remain unexplored. Here,

we characterized three CS-associated ABCC9 mutations, namely P429L (hP432L), A475V

73

Figure 5.10- Decreased sensitivity to glibenclamide inhibition in C1039Y channels. 86Rb+ efflux, as a

function of time, was measured in GFP-transfected control cells, and in cells transiently expressing KATP

channels composed of Kir6.2 plus WT or mutant SUR2 subunits (A), as well as in cells expressing 1:1

mixture of WT and mutant SUR2 subunits (B). Experiments were performed in MI+ glibenclamide. The

data represent the means + S.E.M. of 3-6 experiments. Data were fit with equation 2 to obtain rate constants

for KATP-dependent efflux (k2). k2.in glibenclamide + MI was divided by k2 in MI for each condition(C,D).

74

(hA478V) and C1039Y (hC1043Y), that localize to distinct regions of the SUR2 protein

(Figure 5.1). Each mutation leads to KATP channel gain of function (GOF) (Figures 5.2-5.7),

consistent with previous findings on isolated CS-associated ABCC9 mutations (Harakalova et al.,

2012).

In the only previous assessment of CS-associated ABCC9 mutations, Harakalova et al.

measured channel sensitivity to ATP in the presence of Mg2+. Mg-nucleotides have complex

activatory/inhibitory effects on KATP channels: MgATP can both inhibit the Kir6.X subunit and

be hydrolyzed to activatory MgADP at the SUR subunits (Nichols, 2006). Therefore, such an

analysis cannot separate mutant effects on ATP inhibition from those on Mg-nucleotide

activation. By separately assessing channel activity in response to Mg-free ATP and to MgADP

we show that CS-associated ABCC9 mutations lead to increased channel activity via different

mechanisms, as has also been observed with NDM-associated ABCC8 mutations (de Wet et al.,

2008; de Wet et al., 2007; Masia et al., 2007; Zhou et al., 2010). Specifically, we demonstrate

two mechanisms of increased channel activity, where P429L and A475V channels results from

increased MgADP activation and C1039Y channels results predominantly from decreased ATP

sensitivity.

KATP mutations in SUR1 have been shown to decrease ATP-sensitivity (Takagi et al.,

2013; Tarasov et al., 2008). There are at least two mechanisms to decrease ATP sensitivity in

KATP channels including decreased ATP binding or increased open probability (Po) (Denton and

Jacobson, 2012). Considering the SUR2-C1039Y mutation is not likely to disrupt binding of

ATP to the Kir6.X subunit we tested for changes in channel Po. The simple gating model,

Ci↔C↔O, Ci represents closed channels inhibited by ATP, C represents intrinsic closing and O

represents open channels. PIP2 activates KATP currents by increasing the intrinsic Po, or in

75

reference to a simple gating model, PIP2 forces channels to stay in the open state, preventing

transition from C to Ci by ATP binding (Shyng and Nichols, 1998). Therefore, the ability of PIP2

to decrease ATP sensitivity, but not increase the maximum currents from C1039Y channels

imply the Po of these channels is near 1 (relative Po = 1.26 ± 0.21) which is significantly higher

than the Po for WT channels (relative Po = 0.70 ± 0.11) (Figure 5.6), and thus ultimately results

in decreased ATP sensitivity (Figure 5.5).

In addition to increasing channel activity, homomeric expression of C1039Y appears to

decrease expression of channels in the membrane. A similar dual effect of GOF mutations in

both Kir6.2 (Lin et al., 2013) and SUR1 (Zhou et al., 2010) has previously been shown. In these

previous examples, decreased surface expression was demonstrated by western blot analysis.

Significant rescue of channel activity in heteromeric C1039Y/WT channels is consistent with the

patterns from such mutations (Figures 5.8 and 5.9), suggesting that in addition to reducing

ATP-sensitivity of expressed channels, C1039Y also decreases KATP surface expression.

Implications for tissue pathology in in Cantu Syndrome

Kir6.1 GOF mutations have also been found in two Cantu Syndrome patients (Brownstein et al.,

2013; Cooper et al., 2014), confirming Kir6.1/SUR2 as the key subunit combination generating

the overactive channels. Given the experimental difficulty of recording Kir6.1/SUR2 currents

(Cooper et al., 2014), we used Kir6.2+SUR2A channels in the present study. ABCC9 encodes

two splice variants: SUR2A and SUR2B, which vary only in the last 40 amino acids. All CS-

associated SUR2 mutations identified to date are located within the core of the SUR2 protein,

such that these mutations will be present in both SUR2A and SUR2B. SUR2A is predominant in

skeletal muscle and heart, while SUR2B is expressed in the vasculature. Kir6.1/SUR2B may be

76

the more dramatically affected combination, since vascular phenotypes seem to predominate in

CS patients (Nichols et al., 2013). Conversely, neither skeletal muscle weakness nor shortening

of the QT interval on the ECG have been noted, as might be expected if Kir6.2/SUR2A were

markedly activated.

The severity of disease varies between Cantu Syndrome patients such that not all reported

features are exhibited by all patients. Direct comparison of the phenotypes of patients carrying

the A475V and C1039Y equivalent mutations (Harakalova et al., 2012) revealed hypertrichosis,

macrosomia, macrocephaly, coarse features, cardiac anomalies, and umbilical hernia to be

common to both. However, pulmonary hypertension, cardiomegaly, osteopenia,

hyperextensibility and lymphedema were only reported for the A475V equivalent patient. The

reduced sensitivity to ATP inhibition, due to the C1039Y mutation, results in greater relative

basal channel activity than the enhanced Mg-nucleotide activation, due to A475V. However, a

less severe disease phenotype for C1039Y suggests that reduced expression of this mutation may

also be a consequence in vivo, as has also been reported for SUR1 NDM mutations (Zhou et al.,

2010).

Implications for therapeutic intervention in Cantu Syndrome

Finally, sulfonylureas, which inhibit KATP channels via interaction with the SUR subunits, are

effective treatments for NDM patients with ABCC8-activating mutations (Babenko et al., 2006),

and are promising potential therapies for Cantu Syndrome (Nichols, 2013 #7510). However,

GOF mutations in Kir6.2 (Koster et al., 2005a) and SUR1 (Takagi et al., 2013) frequently also

decrease sensitivity to sulfonylureas, which may result in a lack of sulfonylurea therapeutic

efficacy in patients with these mutations. Assessment of 86Rb+ efflux in glibenclamide

77

demonstrates that WT, P429L and A475V channels show complex time dependence of efflux

(Figure 5.10). Each required a very negative k-2 rate constant, which is indicative of channel

activation versus inactivation. We interpret this activation to be reflective of a loss of

glibenclamide inhibition over time. Divergent from the others, the k-2 for C1039Y has a very

small positive value, which is consistent with some channel inactivation. Similarly, k2 values in

MI+glibenclamide WT, P429L and A475V channel activity drops to less than 10% in MI alone

while C1039Y activity drops to ~50%. Taken together this would indicate that the C1039Y

channel is glibenclamide insensitive. However, in general, SUR2 sensitivity to sulfonylureas is

much lower than SUR1-sensitivity (Dorschner et al., 1999). Therefore, given the further

reduction in drug sensitivity of GOF mutants, the introduction of sulfonylureas to treat CS

patients is likely to require high doses, which would result in undesired inhibition of SUR1-

based KATP channels. SUR1 inhibition can lead to hypoglycemic effects, which will need to be

carefully considered. Such concerns may ultimately make necessary the development of Kir6.1

or SUR2 specific inhibitors to provide a safer and more effective treatment options for CS

patients.

78

CHAPTER j: DISCUSSION

GOF in KATP channel underlies CS

In 2012, genetic analysis of 2 cohorts of CS patients demonstrated that the majority have

mutations in ABBC9, which encodes the SUR2 protein (Harakalova et al., 2012; van Bon et al.,

2012). Initial studies of a few CS-SUR2 mutations expressed with Kir6.2 demonstrated increased

KATP channel activity (Harakalova et al., 2012). My analysis of new, previously uncharacterized

CS-associated SUR2 mutations confirms this effect. In 2013, two additional CS patients were

reported (Brownstein et al., 2013; Cooper et al., 2014) who presented with the hallmark features

of CS, but were negative for a SUR2 mutation. Instead, both carry a mutation in KCNJ8 (Kir6.1-

V65M and C176S). I also demonstrate that each of these CS-Kir6.1 mutations (V64M and

C176S) also increase KATP channel activity. Therefore, having demonstrated that both SUR2 and

Kir6.1 CS-mutations increase KATP channel activity, I conclude that gain of function (GOF) in

KATP channel activity underlies CS. The previous data showing that multiple SUR2 mutations

result in a GOF in channel activity was strong evidence to support increased KATP channel

activity underlying CS. However, the demonstration that CS- associated mutations in Kir6.1 also

increase KATP channel activity was crucial in confirming this, as there are reports of SUR2

involvement in KATP channel independent activity (Aggarwal et al., 2010; Stoller et al., 2010).

Molecular basis of KATP channel gain of function

Gain of Function (GOF) in channel activity can arise from increases in channel activity and/or

from increased channel expression. The majority of Neonatal Diabetes Mellitus (NDM) patients

carry mutations in Kir6.2 or SUR1 that increase KATP channel activity (Babenko et al., 2006;

Gloyn et al., 2004). Several mechanisms by which NDM mutations enhance KATP channel

79

activity have been described (Denton and Jacobson, 2012). For example, Kir6.2 NDM mutations

have been shown to enhance channel open probability (Po) (Mannikko et al., 2009) or to decrease

ATP binding (Proks et al., 2004). SUR1 NDM mutations have been shown to enhance KATP

channel activity by enhancing ATPase activity (de Wet et al., 2008), by allosteric changes that

indirectly decrease ATP sensitivity (Babenko et al., 2006), or by increased MgADP activation

(Masia et al., 2007). However, no molecular details of CS-causing Kir6.1 or SUR2 mutation

mechanisms have previously been described.

In this project, I have sought to address this question. To do this, I examined nucleotide

sensitivity for SUR2 mutations in inside out patch clamp experiments. For Kir6.1 mutations,

nucleotide sensitivity was also assessed and, by estimating PIP2 activation, I also determined

relative Po. CS-associated Kir6.1 and SUR2 GOF mutations result from several distinct

mechanisms. I have, for example, demonstrated that SUR2 mutations can disrupt response to

ATP or MgADP. The SUR2-C1039Y mutation results in GOF, by indirectly decreasing

sensitivity to ATP inhibition without affecting MgADP activation. Conversely, both SUR-P429L

and A475V increase channel activity by enhancing MgADP activation. A third mechanism, in

both Kir6.1 mutations (Kir6.1-V65M and C176S), enhances Po, thereby indirectly decreasing

channel sensitivity to ATP inhibition. Additionally, because one of the SUR2 mutations

(C1039Y) unexpectedly demonstrates decreased trafficking, surface expression may also

confound disease presentation.

Kir6.1/SUR2 expression and CS symptoms

At the tissue level, the effects of NDM and CS mutations depend on the tissues where the

channels are expressed, and on how sensitive each tissue is to KATP channel activity. Kir6.1 is

expressed ubiquitously and has been confirmed in the vasculature (Inagaki et al., 1995c). SUR2

80

can be alternatively spliced into two major isoforms: SUR2A and SUR2B (Isomoto et al., 1996).

The splice variant SUR2A is expressed in the heart and skeletal muscle (Chutkow et al., 1996).

In parallel to Kir6.1, the splice variant SUR2B is also expressed ubiquitously and confirmed in

the vasculature (Chutkow et al., 1996; Inagaki et al., 1995c).Conversely, Kir6.2 and SUR1 are

expressed primarily in pancreatic beta cells, brain, heart and skeletal muscle (Inagaki et al.,

1995a).

There have been several reports of tissues expressing both Kir6.1 and Kir6.2 (Insuk et al.,

2003; Teramoto et al., 2009). However, to date, there appears to be no overlap in symptoms of

CS and NDM patients experience. CS patients generally present with cardiomegaly,

hypertrichosis, macrocephaly, osteochrondyslpasia, and macrosomia (Grange 2006, van Bon

2012, and Harakalova 2012). Conversely, NDM patients experience hyperglycemia and

hypoinsulinemia along with neurological complications in the most severe cases. This lack of

overlap in symptoms implies that where expression of pore-forming subunits overlaps, the

predominance of non-mutated pore-forming subunits might be protective.

Potential for CS genotype/phenotype correlation

NDM presents with varying severity, from Transient NDM (TNDM), to Permanent NDM

(PNDM), to developmental delay, epilepsy and neonatal diabetes (DEND) (Naylor et al., 2011).

The severity of the disease correlates with the molecular severity of the Kir6.2 or SUR2

mutations (Flanagan et al., 2009; Hattersley and Ashcroft, 2005). TNDM patients have

hyperglycemia and hypoinsulinemia that is present at birth, but is not permanent. The Kir6.2 or

SUR1 mutations associated with this form of NDM have weak shifts in ATP sensitivity or

enhancement of MgADP activation (Gloyn et al., 2005; Proks et al., 2004). PNDM patients have

81

hyperglycemia and hypoinsulinemia that also appears within the first 6 months of life, but

persists throughout life. Compared to TNDM mutations, the Kir6.2 and SUR1 mutations in this

form of NDM typically have larger shifts in ATP sensitivity or enhancement of MgADP

activation (Gloyn et al., 2004; Proks et al., 2004). Along with the diabetes, DEND patients can

have developmental delay and epilepsy. Intermediate DEND (iDEND) is a form of DEND with

milder developmental delay and no epilepsy. Kir6.2 and SUR1 mutations causing isDEND and

DEND have the most severe shifts in ATP sensitivity or enhancement of MgADP activation

compared with all other forms of this disease (Proks et al., 2004; Proks et al., 2006; Proks et al.,

2005). Is there a range of symptoms in CS? CS patients generally present with cardiomegaly,

hypertrichosis, macrocephaly, osteochrondyslpasia, and macrosomia (Grange 2006, van Bon

2012, and Harakalova 2012).There are also symptoms that are only found in small subsets of

patients, including patent ductus arteriosus (PDA), pericardial effusions, and valvular

abnormalities. This raises the question: do CS mutations correlate with symptomatic

presentation?

In considering this question with just the patients with Kir6.1 or SUR2 mutations which I

have functionally described (Table 6.1), the major common symptoms were present in all. The

mutations carried by patients who have all the major symptoms with the addition of at least one

of the rare symptoms included: Kir6.1-V65M, C176S or SUR2-A475V.Based on 86Rb+ efflux

experiments (Figures 3.1A and 3.2A, Figure 4.1, and Figures 5.2A and 5.3A) these mutations

result in the largest fluxes in the basal and pinacidil stimulated conditions. Of the other SUR2

mutations considered, A475V has one of the largest increases in relative 86Rb+ efflux under basal

conditions. Both the CS-Kir6.1 patients carrying the Kir6.1-V65M and Kir6.1-C176S mutations

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Table 6.1- Cantu Syndrome Mutations and Clinical Symptoms.

Kir6.1-

V65M

Kir6.1-

C176S

SUR2-

P429L

SUR2-

A75V

SUR2-

A75V

SUR2-

C1039Y

Gender M M F F M F

Inherited (Y/N) N N N Y unk N

Alive (Y/N) Y Y Y Y Y Y

Coarse face + + + + + +

congential

hypertichosis + + + + + +

Macrosomia

at birth + + + + + +

Macrocephaly unk + unk + + +

developmental

delay mild + - + - -

cardiomegaly + + + - + -

structural

cardiac

anaomalies + + - + + +

pulmonary

hypertension + - - + - -

pericardial

effusion - - - - - unk

hypertrophic

and/or dilated

cardiomyopathy + + + - unk -

lymphedema - - - + -

PDA unk + - unk unk unk

The + symbols indicates clinical symptoms present in patients, - symbol indicates clinical

symptoms that are absent in the patients. The unk designation indicates symptoms not test and/or

reported in the patients. Common CS symptoms are indicated in black and symptoms present only

in small subsets of CS patients are indicated in red.

83

have similar symptoms and the mutant channels displayed comparable rates of 86Rb+ efflux in

the basal condition (Figures 4.1 and 4.2). Taken together, potential genotype/phenotype

correlations exist, but additional studies in a larger set of CS Kir6.1 and SUR2 mutations will

need to be done to accompany the clinical findings reported.

Potential for treating CS

There is currently no specific treatment for CS. Sulfonylureas bind directly to the SUR subunits

of KATP channel to inhibit KATP channel activity (Ashfield et al., 1999).With the understanding

that the molecular basis of CS is GOF in KATP channel activity, use of a sulfonylurea drugs, such

as glyburide, a KATP channel-specific inhibitor that is currently used to treat diabetes (Feldman,

1985), may be a treatment option. However, a potential detrimental side effect to using

glyburide, or other sulfonylurea drugs, as a CS treatment option, is that KATP channels containing

SUR1 are typically ~100fold more sensitive to these drugs than KATP channels containing SUR2

(Gribble et al., 1998). Even SUR2 mutations with reduced sulfonylurea sensitivity may be

capable of being inhibited partially by these drugs, but perhaps require higher doses. However, in

addition to the efficacy of these drugs to inhibit SUR2 based channels, they will also bind and

inhibit SUR1-based channels from increasing insulin secretion, which can result in

hypoglycemia. Thus, along with the potential benefits of administering sulfonylureas to CS

patients, there would also be a risk of patients becoming hypoglycemic which, if left unmanaged,

can be lethal.

An alternate option to sulfonylureas would be inhibitors that target KATP channels

composed of Kir6.1+SUR2A specifically. Currently, there is a reportedly Kir6.1-specific

inhibitor, U-37883 or PNU-37883A. Electrophysiology experiments, in a heterologous

expression system, demonstrate that PNU-37883A preferentially inhibits ~80% of Kir6.1 based

84

channels at 100µM, but also partially inhibits ~15% of Kir6.2-based channels (Surah-Narwal et

al., 1999). While this Kir6.1 selective inhibitor is not be perfect, it may be a good starting point

for the development of a specific CS treatment.

Is there a Kir6.1/SUR2 loss of function (LOF) disease?

Kir6.2 and SUR1 mutations which result in a loss of function (LOF) in KATP channel activity

underlie Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI) also known as congenital

hyperinsulinemia (Darendeliler et al., 2002). PHHI is characterized by increased plasma levels of

insulin in hypoglycemic conditions along with decreased levels of ketone bodies and free fatty

acids (Aguilar-Bryan and Bryan, 1999), i.e., the opposite of NDM. CS has now been identified

as the result of Kir6.1/SUR2 GOF, but there is currently no known LOF disease caused by

Kir6.1/SUR2 mutations. Sudden Infant Death Syndrome (SIDS), is characterized by the sudden

and usually unexplained death of an infant (Dawes, 1968). Two Kir6.1 mutations (in-frame

deletion E332del and a missense mutation V346I) identified in babies with SIDS results in LOF

in KATP channel activity, when expressed with SUR2 (Tester et al., 2011). Though there are no

reported SIDS patients to date with SUR2 mutations, multiple SIDS babies with a Kir6.1-LOF

mutation make SIDS a strong candidate Kir6.1/SUR2 LOF disease.

The tissues impacted by GOF in Kir6.1 and SUR2 most commonly include the heart, hair,

vasculature and bone (Nichols et al., 2013). So what would be the result of LOF in those same

tissues? Most CS patients have enlarged hearts (Grange et al., 2014) and, thus, in a disease linked

with a LOF of KATP channel activity, a small heart might be anticipated. The opposite of the

failure of the ductus arteriosus to close, which is seen in CS, would be premature closure. When

considering the hair of LOF patients, the converse of excessive hair growth, i.e., reduced or no

hair would be potential symptoms. In the vasculature, CS patients have low blood pressure, and

85

the opposite of that, which may be present in LOF patients, would be hypertension. Finally, KATP

channels have been reported in mesenchymal cells, which differentiate along an osteogenic

lineage to form bone. Considering these various symptoms in different target tissues, CS may

therefore, provide insights into Kir6.1/SUR2 LOF disease(s)

86

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