01-04 Intro to Receptors_nAChR-4pp

MCDB 126B/226BMolecular Pharmacology of Receptors

and Signaling

Dr. Carol [email protected] hours: Mon 10-11am & Wed 2-3pm, 5175 Bio2 (office inside

lab)

TA: Kelsey [email protected]

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

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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.

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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


DNA binding domains

Kinase-linked receptors

N

C


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

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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


G-protein-coupled receptors

G enzyme

ligand

G

ions

change in excitability second messengers

otherprotein phosphorylationCa++ release

cellular effects


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Nuclear receptors

ligand

gene transcription

cellular effects

proteinsynthesisnucleus


Kinase-linked receptors (Receptor Kinase)

ligand

gene transcription

cellular effects

proteinsynthesis

nucleus

proteinphosphorylation


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

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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

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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

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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

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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

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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

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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

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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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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

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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

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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

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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

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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


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

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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


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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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01-04 Intro to Receptors_nAChR-4pp

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