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MCDB 126B/226BMolecular Pharmacology of Receptors
and Signaling
Dr. Carol Vandenbergvandenbe@lifesci.ucsb.eduOffice hours: Mon 10-11am & Wed 2-3pm, 5175 Bio2 (office inside
lab)
TA: Kelsey Mollickkelsey.mollick@lifesci.ucsb.edu
Class web site:http://gauchospace.ucsb.eduPlease see me if you are an extension student & need to be added
to the web access list.
Honors Section
Are you interested in an honors section, and 1 extra unit of credit?
The honors section will meet Mondays at 12-1pm in 4164 Bio 2; the first organizational meeting is this Monday Jan 5.
The honors section involves reading, discussing and presenting current journal articles, together with graduate students. This quarter we will discuss molecular/cellular papers in neurobiology, cell biology & pharmacology.
This section would be in addition to your regular section for MCDB 126B.
Topics this Quarter: Nervous system/brain: excitatory & inhibitory neurotransmission,
learning & memory, excitotoxicity, anxiolytics, hypnotics,
Kidney: mechanism of salt & water reabsorption, electrolyte homeostasis
Control of blood pressure: diuretics, renin/angiotensin system
Heart: control of heart rate & cardiac contraction
Lungs: regulation of airways, asthma
Hormonal systems: hypothalamus & pituitary, oxytocin, vasopressin, growth hormone, others
Adrenal corticosteroids
Thyroid: regulation of metabolism
Pancreas: control of glucose, diabetes
Advances in Molecular Pharmacology
Identification & study of receptor proteins New and improved technologies Genomics
Study of signaling mechanisms
Study of molecular structure X-ray crystallographic structure Computational modeling & molecular dynamics studies Structure-function studies
Goal: Understanding of the receptor signaling mechanisms underlying hormonal and neurotransmitter function
Goal: Development of improved drugs based on knowledge of molecular structure and function
1
Todays Lecture Types of target proteins for drug binding
Ligand-gated ion channels (Ionotropic Receptors) Transporters G-protein-coupled receptors (Metabotropic receptors) Nuclear receptors Kinase-linked receptors (Receptor Kinases)
Signal transduction mechanisms How do different types of receptors transduce information?
Ionotropic receptors: Nicotinic acetylcholine receptor Physiology of electrical signaling: what makes a receptor
excitatory or inhibitory Distribution of Nicotinic ACh R
Cell Signaling Systems Process Information
Cell signaling systems receive input from the environment, process the information, and generate an appropriate output.
The process is conceptually similar in a single cell or a whole organism.
Cell Signaling
Signaling in a variety of different contexts reveals a common set of mechanisms and pathways that are the basis of diverse biological activities.
Integration of Cell Signaling and Gene Expression
Fast cell signaling pathways are integrated with slower gene expression networks to regulate cellular responses.
2
Challenges in Cellular Information Processing Cells must receive input from neighboring cells and the environment.
Cells are surrounded by a lipid bilayer membrane that is impermeable to many substances.
How do cells get input from the environment and transmit it into the cell?
What are the major types of hormone and neurotransmitter receptors?
Ligand-gated ion channels (Ionotropic receptors)
NC
X 4 or 5
Ligand binding domains
Channel lining
Transporters
N C
3
G-protein-coupled receptors
N
C
G-proteinbinding domain
Nuclear receptors
N
C
Ligand binding domains
DNA binding domains
Kinase-linked receptors
N
C
Ligand binding domains
catalytic domains
Receptors are composed of modular domains
Alteration in the binding or catalytic domains of receptors has resulted in superfamilies of biologically similar proteins that:
1) bind different ligands 2) differ in their functions
However the molecular mechanism of receptor function within each of the superfamilies is the same
4
How do receptors transduce their signals & what are the cellular effects? Alter membrane permeability & membrane potential
Alter membrane transport
Synthesize/degrade second messengers & activate/inhibit effector proteins
Enzymatically modify (e.g. phosphorylate) downstream target proteins
Regulate gene transcription
Ionotropic receptors (ligand-gated ion channels)
ligandions
Hyperpolarization or
depolarization
cellular effects
Signal Transduction:
Transporters
ions
Cotransport of ions & small molecules
cellular effects
ions
Changes in ion concentrations & osmolarity
Signal Transduction:
G-protein-coupled receptors
G enzyme
ligand
G
ions
change in excitability second messengers
otherprotein phosphorylationCa++ release
cellular effects
Signal Transduction:
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Nuclear receptors
ligand
gene transcription
cellular effects
proteinsynthesisnucleus
Signal Transduction:
Kinase-linked receptors (Receptor Kinase)
ligand
gene transcription
cellular effects
proteinsynthesis
nucleus
proteinphosphorylation
Signal Transduction:
How do these receptors transduce their signals & what are the cellular effects?
How are the signals turned on & off?
How fast are cellular signaling mechanisms?
How long do the effects last?
Are the signals amplified?
How do receptor pathways interact?
Different ways that the state of proteins can be changed serve as basis for cell signaling mechanisms.
E
How fast do receptors signal?
Minutes to
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HormonesNeurotransmitters
Endocrine
Autocrine Paracrine Neuroendocrine
Neurotransmitters & Hormones: local and long-distance communication
Molecular Neuropharmacology Brain function is far more complex than any other system in
the body
The human brain contains 1011 (=100 billion!) neurons, and 1015 synaptic connections that employ a variety of neurotransmitters
Much of what we know about the nervous system has come from studies using pharmacologic probes
Different levels of investigation Fundamental molecular & cellular building blocks Neural function and integration of neuronal systems Clinical and behavioral neuroscience Pharmacological intervention gives insight at each level
Neurological Disorders In the US, 50 million people suffer from damage to the nervous system National Institute for Neurological Disorders and Stroke (NINDS)
supports research on more than 600 neurological diseases
Neurogenetic - Huntingtons disease, Muscular dystrophy Developmental - cerebral palsy Degenerative - Alzheimers disease, Parkinsons disease Metabolic - Gauchers disease Cerebrovascular - stroke, vascular dementia Trauma - spinal cord, head injury Behavioral & cognitive syndromes Sleep disorders Convulsive - epilepsy Demyelinating - Multiple sclerosis Brain tumors
Pharmacological intervention:targeting nervous systems
Therapeutic interventions for neurodegeneration General and local anesthetic agents Antidepressant drugs Antipsychotic drugs Antiepileptic drugs Anxiolytic and hypnotic drugs Analgesic drugs CNS stimulants and psychotomimetic drugs Drugs to treat addiction
7
Many Types of Chemical Neurotransmitters for Communication between Cells
Transmitter Type Example
Biogenic amines Dopamine, Epinephrine, NorepinephrineSerotonin, Histamine
Amino acids -aminobutyric acid (GABA), GlycineGlutamate
Peptides Enkephalins, endorphins
Purines Adenosine, ATP
Gases Nitric oxide
Lipid-derived Endocannabinoids
Small molecules Acetylcholine (ACh)
Acetylcholine Receptors
Amanita muscaria
Ionotropic Receptor: Metabotropic Receptor:
Convergent evolution in ligand binding: same ligand recognized by unrelated receptors
Neurotransmitter Actions: excitatory or inhibitory
Excitatory channels
promote action potential firing depolarize membrane
towards threshold Na+, Ca2+ or nonspecific
cationic channels Many examples
Voltage-gated Na+ channels Voltage-gated Ca2+ channels Ligand-gated channels
nicotinic ACh R, Glutamate R
Nicotinic Acetylcholine R Glutamate R
Inhibitory channels
inhibit action potential firing hyperpolarize membrane or
maintain resting potential Cl- channels or K+ channels Many examples
Voltage-gated K+ channels ATP-gated some muscarinic Acetylcholine R Ligand-gated channels
GABAA R, Glycine R
GABAA R Glycine R
M
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What makes a receptor excitatory or inhibitory?
Ability to promote or inhibit the generation of action potentials
K+ K+Na+ Na+
Ca2+
Cl-Cl-Ca2+
cell
8
Principles of Synaptic Transmission
Neurotransmitter synthesized & stored in vesiclesHow does this differ for chemical and peptide neurotransmiters?
Neurotransmitter released into synaptic cleft Ca2+-dependent vesicle fusion and neurotransmitter exocytosis
Neurotransmitter binds to receptor in postsynaptic membrane to activate or inhibit postsynaptic cell
Neurotransmitter is removed from synaptic cleftEnzymatically degraded or taken up into neighboring cells
Acetylcholine biosynthesis
Choline + Acetyl coenzyme A
Acetylcholine
Choline + free Acetate
Acetylcholinesterase (AChE)
Choline acetyltransferase (CAT)
First substance identified as neurotransmitter
Cholinergic Synapse Choline is present in plasma (10 M), and is taken up into cholinergic
neurons by Choline transporter Choline + Acetyl-CoA form ACh
Choline Acetyltransferase (CAT) synthetic enzyme ACh is stored in vesicles via vesicular ACh transporter; utilizing vesicle
H+ gradient to take up ~10,000 molecules of ACh per vesicle Presynaptic action potential releases ACh into synaptic cleft via Ca2+-
dependent exocytosis ACh binds to receptor in postsynaptic membrane causing opening of ACh R
channel Na+ flows into the cell, K+ out to the synaptic cleft
Postsynaptic current causes excitatory postsynaptic potential that depolarizes the membrane and increases the excitability of the postsynaptic cell
Vesicular membrane is retrieved from plasma membrane ACh metabolized to Choline and free acetate in the synaptic cleft
Acetylcholine esterase (AChE) - metabolizing enzyme
9
Ligand-Gated (Ionotropic) Neurotransmitter Receptors:
Nicotinic Acetylcholine Receptor
Acetylcholine Receptors
Nicotinic receptor
Best studied ionotropicneurotransmitter R
Cationic channel
Generate excitatory postsynaptic responses
Neuromuscular Junction:downstream events
thebrain.mcgill.ca
Muscle action potential invades T-tubule
Dihydropyridine receptor in T-tubule is activated by voltage, and it interacts with ryanodine receptor in sarcoplasmic reticulum to release Ca2+ from sarcoplasmic reticulum
Ca2+ causes contraction of myofibril via actin & myosin
muscle cell membrane
Nicotinic ACh Rsare expressed in nervous system
Nicotinic ACh receptor role is to mediate fast excitatory synaptic transmission
Four sites with nicotinic AChreceptors: CNS: in the brain Somatic: located on skeletal
muscle at the neuromuscular junction
Autonomic Sympathetic: in ganglionic neuron
Autonomic Parasympathetic:in ganglionic neurons
10
Locations of Nicotinic ACh Receptors in Central Nervous System
Many regions
cortex: 4subtype
hippocampus: 7 subtype
Location of Nicotinic ACh Receptor in Peripheral Nervous Systems (Somatic)
SkeletalMuscle
Spinal Cord
Neuromuscular Junction
Nicotinic ACh Receptors at Neuromuscular Junction labeled with alpha-bungarotoxin
Kandel, Principals of Neural Science
Locations of Nicotinic ACh Receptors in Peripheral Nervous Systems (Autonomic)
Sympathetic Parasympathetic
11
Summary
5 Major types of Receptors in Hormonal & Neurotransmitter Systems
Ionotropic Receptors: Ligand-gated ion channels Excitatory (cation channel; depolarizies): Nicotinic ACh R, Glutamate R Inhibitory (anion channel; maintains resting potential): GABAA R, Glycine R
Principles of synaptic neurotransmission Neurotransmitter synthesis, vesicle storage, release, receptor binding,
degradation/uptake
Nicotinic AChR Localization Throughout the body as neuronal & muscle receptors CNS, Somatic: NMJ, Autonomic: sympathetic & parasympathetic ganglia
nAChRs affect almost every human function Movement, cognition, memory, breathing etc
Ligand-Receptor Binding
How can we determine receptor binding and activation properties?
Receptor number: How many receptors are there?
Binding: How well does a ligand (hormone/neurotransmitter/drug) bind to the receptor?
Efficacy: How well does a ligand initiate an effect via the receptor?
The strength of a binding interaction is defined by the equilibrium dissociation constant Kd
The affinity of an interaction is a measure of the intrinsic strength of the binding interaction. When affinity is high, then there is a high probability of finding molecules in a complex at equilibrium.
Physiological Kds vary over a wide range
12
Typical biological concentrations are closely matched to Kds
This enables the extent of binding to be easily regulated by small environmental changes
high
low
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high
low
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Receptor Occupancy Theory
Ability of drug to activate its target is graded, and depends on:
Affinity is the ability of the drug to bind to a receptor.
Efficacy is the relationship between receptor occupancy and the ability to initiate a response.
Response and binding curves
Response curves - whole animals, organs or tissues Understanding of drugs physiological activity Comparison of efficacy of different drugs
Binding (occupation) curves - purified receptor Comparison of different classes and analogs of drugs Allows molecular understanding of receptor binding Allows understanding of spare receptors
Measurement parameters of drug binding to receptors
Determination of number of receptors Compare receptor abundance in different tissues
Equilibrium ligand-receptor dissociation constant (Kd) Classification of receptors Comparison of drug actions Comparison of different receptors Required for receptor purification
13
How might we study receptor binding?
1. Obtain tissue
2. Solubilize receptor with mild detergent
3. Mix with a labeled ligand *L, and incubate to equilibrium
4. Separate bound *LR complex from *L
5. Quantify amount of [*LR]
6. Make several measurements of [*LR] at different [L]
Characteristics required of a protein to qualify it as a receptor
Saturability Finite number of receptors High affinity
Specificity
Reversibility Ligand is dissociable and recoverable
(different from substrate-enzyme)
Theory of drug-receptor interactions
LRk
kRL
1
1
][]][[
LRRL
kkKd
1
1
Ligand + Receptor [Ligand-Receptor] response
k1 = association constantk-1 = dissociation constant
At equilibrium, Rate of [LR] formation = rate of [LR] breakdown
Kd = equilibrium dissociation constant = dissociation rateassociation rate
affinity efficacy
Kd is related to rates of binding and dissociation
Interactions with the same affinity can have different rates of binding and dissociation
Red: fast ON rate and fast OFF rateGreen: slow ON rate and slow OFF rateSame level of binding at equilibrium = same Kd
14
Dose occupation curve[LR] vs. [L]:
[L]
[LR]
[Rt]
[Rt]/2
Kd
][]][[][LKd
RtLLR Log Dose occupation curve
Log [L]
[LR]
[LR] vs. Log [L]:
[Rt]
[Rt]/2
Log Kd
Double Reciprocal Plot
1/[LR] vs. 1/[L]: ][1
][1
][][1
RtLRtKd
LR
1/[L]
1/[LR]slope =Kd
[Rt]
1[Rt]
-1Kd
Scatchard Plot
[LR]/[L] vs. [LR]: KdRtLR
KdLLR ][ 1
][][
[LR]
[LR][L]
slope = -1Kd
[Rt]
[Rt]Kd
15
Practical aspects: What are the advantages and disadvantages of the plotting methods?
Linear or non-linear
Can data be plotted easily when there is a large range of [ligand]?
Can data be extrapolated easily when [ligand] is low?
What do the error bars look like?
NicotinicCholinergicSynapse &
its Pharmacology
Rang, Pharmacology, Fig 10.2
Pharmacology: nACh neurotransmission
Nicotinic ACh R agonist Acetylcholine: stimulates neuromuscular junction & postganglionic
autonomic neurons Nicotine: in CNS causes neural excitation, heart rate, blood
pressure, highly addictive, causes cardiovascular problems Varenicline (Chantix): partial agonist at 4subtype,
non-nicotine aid to smoking cessation Carbamylcholine Succinylcholine (suxamethonium): depolarizing muscle relaxant
Nicotinic ACh R competitive antagonists Tubocurarine, component of curare found in plants: used as arrow
poisons Pancuronium (reversible): used as muscle blocker together with
anesthetic during some surgery bungarotoxin (cobra & krait toxin), irreversible
Targeting NAChR for Smoking Cessation~10% of deaths worldwide attributed to smoking, largest preventable cause of death worldwide
~1/3 of all cancer deaths attributed to smoking: 90% lung cancer deaths, 80% bronchitis & emphysema, 17% of deaths from heart disease
Nicotine crosses blood-brain barrier to interact with specific NAChR in brain (4and 7 subtypes), which stimulates release of dopamine in nucleus accumbens, the reward region of the brain
Goal of smoking cessation therapeutics: Stimulate 4NAChR-mediated dopamine release to reduce nicotine craving
during abstinence Block 4NAChR during smoking to decrease nicotine reinforcement of
reward
Therapeutics: Nicotine replacement therapy Varenicline: 4NAChR partial agonist Bupropion: inhibits neuronal reuptake of dopamine, & weak 4NAChR
antagonist
16
Summary Methods to measure ligand-receptor binding
Dose-occupation curve
Log Dose-occupation curve
Double reciprocal plot
Scatchard plot
nAChRs and disease Neurodegeneration: Alzheimers disease
Nicotine addiction
Pharmacology of ACh neurotransmission Varenicline (Chantix): partial agonist for nAChR, 4subtype, smoking cessation Next lecture: Botulinum toxin (Botox): ACh release blocker, muscle relaxation
Next lecture: Donepezil (Aricept): AChE inhibitor, used for Alzheimers disease
Nicotinic Acetylcholine Receptors
Outline
Pharmacology of nAChR (continued)
Single-channel studies: How many agonists bind?
Efficacy: What distinguishes agonists/antagonists/partial agonists?
Cooperativity & Hill coefficient
Strategies for receptor isolation
Biochemistry of Nicotinic ACh Receptor
Inhibitor of Na+/Choline cotransporter Hemicholinium : blocks reuptake of choline into nerve terminal, thereby
blocking synthesis of ACh
Inhibitor of vesicular ACh transporter vesamicol : potential for mapping cholinergic innervations in brain in vivo
ACh release blocker Botulinum toxin: blocks release of ACh from presynaptic terminals on skeletal
muscle, paralyzes muscle
Pharmacology: nACh neurotransmission
Toxin from an anaerobic bacteria, poorly canned foods
Vesicles normally are released by formation of a SNARE fusion complex between SNAREs in vesicle and plasma membrane
Botox proteolytically cleaves & inactivates the SNARE proteins
Treat muscle spasm, chronic migraine, sweating, wrinkles
17
ACh releaser Black widow spider venom (-latrotoxin): promotes
massive release of ACh at neuromuscular junction by binding to presynaptic proteins, causing presynaptic calcium increases that trigger neurotransmitter release.
Acetylcholinesterase (AChE) inhibitors Physostigmine, neostigmine: reversibly inactivate AChE,
treatment of myasthenia gravis Donepezil (Aricept): treatment of Alzheimer's Disease Organophosphates (form covalent bond & irreversibly
inactivate AChE): insecticides, nerve gases
Pharmacology: nACh neurotransmission
Patch Clamp Methods to Record Currents through Ion Channels
Single Channel Recordings of Nicotinic ACh Receptor Channels
open
closed
Why do the channel openings appear in bursts? What does that tell us about how the receptor functions?
What is efficacy?
What distinguishes agonists, antagonists & partial agonists?
Example: Glycine ReceptorGlycine R response to Glycine,
a Full AgonistGlycine R response to Taurine,
a Partial Agonist
open
closed
open
closed
open
closed
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2L + R L2R L2R*affinity efficacy
Efficacy is the ability of a ligand to promote the conformational changes involved in receptor activation.
All can bind the resting inactive state R of the receptor, but agonists have greater ability to promote conformation change to the active R* state.
Partial agonist opens the channel fully, but it is open less of the time.
18
How many agonists bind? Hill Coefficient
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n = Hill coefficient
Steeper
One Ligand:
n Ligands: steeper slope
Nicotinic ACh R has a Hill coefficient of ~2, consistent with the idea that 2 agonists bind to open the channel.
Interpreting Hill Coefficients
Hill coefficent >1: Positive cooperativity in binding: once one ligand is bound to the receptor,
the affinity for binding additional ligands increases Positive cooperativity in function: more than one ligand is required to
activate the receptors function
Hill coefficient =1 non-cooperative binding: the affinity of a receptor for a ligand is not
dependent on whether another ligand is bound (or the receptor only binds one ligand molecule)
Hill coefficient
cDNA = complementary DNA
Many cDNAs
cDNA Library represents all themRNAs made by the tissue
(Note: ~25,000 genes in human genome)
Construction of cDNA Library Most Receptor Family cDNAs were Identified by Functional Expression Screening
Isolation of mRNA from tissue of interest
Injection of mRNA in frog oocytes and functional assay
Construction of a cDNA library from mRNA.
Division of the cDNAlibrary into different pools of clones.
Synthesis of in vitrotranscribed mRNA, injection into oocytes and functional assay
Subdivision of the pools as above to obtain a single cDNAclone
nAChR cDNA Cloning . Biochemical isolation of nAChRs
High expression of nAChRs in marine electric ray Torpedo Snake venom -bungarotoxin: Highly specific ligand of
nAChRs Make a column with -bungarotoxin linked to column matrix Solubilize Torpedo muscle in mild detergent, then incubate
with -bungarotoxin column. nAChRs will bind to column Wash away other proteins, elute nAChR with competitive
ligand Isolate pure nAChRs
Partial protein sequence analysis Generate oligonucleotide sequences Screen cDNA library Functional study to verify the clone
Biochemical study of the isolated nAChR protein SDS-polyacrylamide gel electrophoresis indicates
Torpedo muscle nAChR is a pentamer with 2subunit composition
electroplaques
Hall, Intro to Mol. Neurobiology
cDNA library screening with oligonucleotide probes
20
Molecular structural information:Why is it important?
Technologies/Methods bioinformatics data analysis Nuclear magnetic resonance (NMR): protein structure & dynamics X-ray crystallography: 2 A resolution Cryo EM
Knowing the structure of nAChRs, one can understand: How agonists and antagonists bind How nAChRs control ion flow How various protein domains affect function At the cellular level, interactions with other cellular components How to design ligands for therapeutic indications
Amino acid Hydrophobicity to Predict Transmembrane Segments of Receptors
~20 amino acids are required for a polypeptide to cross a membrane as an alpha helix.
nAChR hydropathy plot
www.jyi.org
Squire at el., FundamentalNeuroscience, Chapter 9
Hydrophobicity:
Nicotinic ACh Receptor, Glycine Receptor and GABAA Receptor have similar hydropathyprofiles
21
nACh, 5HT3, GABAA & Glycine Receptors form a superfamily of Ligand-gated Ion Channels (Ionotropic Receptors) based on amino acid similarity
Anion
CationACh
Glycine
GABA
5-HT3
Unifying properties of Cys-loop superfamily
s -- s N C
M1 M2 M3 M4
s -- s N C
s -- s N C
s -- s N C
s -- s N C
s -- s Receptor
Nicotinic(1 -subunit)Nicotinic(non- 1)GABAA(1)Glycine
5HT3
4 hydrophobic transmembrane segments
Conserved disulfide in N-terminal region
Nicotinic ACh Receptor
Freeze-fracture micrograph of Nicotinic-ACh Receptor at NMJ
CryoElectron Microscopy: Spray acetylcholine on
membranes with nAChR as sample is rapidly frozen in
nAChR subunitsmain types in mammalian tissues
Muscle type( (fetal)((adult)
Neuromuscular junction
Postsynaptic excitationNa+ & K+ permeable
Ganglion type(
Autonomic ganglia
Postsynaptic excitationNa+ & K+ permeable
CNS type(
Brain Pre and Postsynaptic excitationNa+ & K+ permeable
CNS type
Brain Pre and Postsynaptic excitationCa2+ permeable
Receptor Type Location Effects
10 genes, 4 genes, others:
Summary
2 agonists bind to open the nACh receptor (Hill coefficient)
Efficacy determined by ability of ligand to promote channel opening (increase open time during burst)
Several methods to isolate receptor cDNA: Expression cloning, protein purification, library screening, PCR, genome database searching
Superfamily of Cys-loop receptors: Nicotinic AChR, GABAA R and Glycine R
These receptors have pentameric subunit composition, each 4 transmembrane segments
Nicotinic ACh Receptor: Structure & Function of
Ligand-gated Ion Channels
23
Outline Chimeras: strategy for structure/function
studies
What determines whether channel is selective for cations or anions?
How does the pore open & close?
Where do agonists/antagonists bind?
How does agonist binding cause channel opening?
nAChR: How to gain insight into structure & function
www.jyi.org
Squire at el., FundamentalNeuroscience, Chapter 9
Where is the agonist binding region?Where is the pore?
Chimera studies
nAChR (7)
5HT3R
Chimera
m1 m2 m3 m4
m1 m2 m3 m4
m1 m2 m3 m4
Pharmacology: Pore:
ACh activates.TCurare, BT inhibit.
5HT activates.BT doesnt inhibit
Permeable to Ca2+
Blocked by Ca2+
? ?
Families of Ligand-gated Ion Channels (Ionotropic Receptors)
Anion
CationACh
Glycine
GABA
5-HT3
24
Chimeric Nicotinic AChR and 5HT3R:
Where is the agonist binding site?
Nature (1993) 366, 479.
nAChR Chimera 5HT3R
Chimera has the pharmacology of the nicotinic AChR 7 receptor channel
5HT
5HT
5HT
ACh ACh
ACh
C
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Chimeric Nicotinic AChR and 5HT3R:
Where is the channel pore?
Experiment: Measure inward Na+currents at various [Ca2+]
nAChR Chimera 5HT3R
Permeable to Ca2+ Blocked by Ca2+ Blocked by Ca2+
Chimera has the pore properties of the 5HT3 receptor channel
Where is the agonist binding region? Where is the pore?
Chimera studies
nAChR (7)
5HT3R
Chimera
m1 m2 m3 m4
m1 m2 m3 m4
m1 m2 m3 m4
Pharmacology: Pore:
ACh activates.TCurare, BT inhibit.
5HT activates.BT doesnt inhibit
Permeable to Ca2+
Blocked by Ca2+
? ?Blocked by Ca2+ACh activates.TCurare, BT inhibit.
Neurotransmitterbinds on N term
Pore properties conferred by C term
Nicotinic ACh Receptor
25
Ion Channel Selectivity:
What is the biochemical basis for selective permeability to specific ions?
Molecular Basis for Cation Selectivity of Nicotinic ACh Receptor:Rings of negatively charged amino acids (Glu & Asp)
Amino acids adjacent to TM2 region:
Conformational Changes in Opening & Closing N-ACh Receptor Channel:
Dilation of M2 transmembrane segments opens the pore (earlier models suggested rotation of TM2 shown below)
Where Are the Agonists Binding Pockets on Nicotinic ACh Receptor?
+
At interface between and adjacent subunit
26
Snail Acetylcholine Binding Protein:
An unusual soluble protein released from snail glial cells that decreases the response to cholinergic neurotransmission
Investigators studied cholinergic transmission from V neuron to L neuron in the absence or presence of glia
Smit et al (2001) Nature 411, 261Brecj et al (2001) Nature 411, 269
V&L neurons alone: V&L neurons with glia:
X-ray Crystal Structure of Snail Acetylcholine Binding Protein
Brecj et al (2001) Nature 411, 269
27
Pi - Cation Interaction
+Pi electron cloud above aromatic ring
Cation
Model of the N-AChR built based on Homology Modeling and Cryo-EM
Unwin (2005) J. Mol. Biol. 346, 967.
Extracellular domain: sheet Transmembrane domain:
helix
pp. 896
How does the extracellular ligand-binding domain communicate with the transmembrane pore domain?
Cytoplasmic
Extracellular
Gating Lever that Triggers Channel Opening Upon Agonist Binding
Lee & Sine (2005) Nature 438, 243-7
28
Conformationally Flexible Amino Acids often implicated at sites of Protein Movement
Glycine
Proline
side chain
M2 segments move to dilate the pore to open it
Cysteine Loopconserved among all members of this receptor class
Agonist binding siteat interface between 2 subunits
Proline at top of M2-M3 loopimplicated as the site of coupling between the binding domain & gating domain
Coupling between Agonist Binding and Channel Gating Shown for 2 Adjacent
Subunits of 5-HT3 Receptor
Dougherty lab:Lummis et al. (2005) Nature 438, 248-52
in
out
membrane
AChR Structure/Function SummaryAChR pore:
The M2 segments from each of the 5 subunits form the pore
Rings of negatively charged amino acid side chains from M2 line the pore: cation selective channel
Pore opens when M2 transmembrane segments move slightly to dilate the pore
Ligand binding site:
ACh-binding protein structure & mutagenesis studies show:
2 AChs bind at interfaces of and subunits ACh binding pocket surrounded by aromatic tyrosine &
tryptophans: electrons stabilize + charge on acetylcholine How does ligand binding open the pore?
Chimeric ACh binding protein/5-HT3R identifies the interface between the binding domain and the pore region: A network of amino acids link the extracellular ligand binding domain to the M2-M3 linker, and convey the conformational change of ligand binding into channel opening
29
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