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Neurotransmitter and Receptors

Synaptic transmission: The release of neurotransmitter by a presynaptic

cell and the detection and response of the neurotransmitter by the

postsynaptic cell

Objective of these lectures: to learn the specific mechanisms of the

principal neurotransmitters, to introduce basic neuropharmacology.

Touch on physiological role now, explore in later lectures in more depth.

Classification of neurotransmission

Fast neurotransmissionNeurotransmitter directly activates ligand-gated ion channel receptor

Neuromodulation

Neurotransmitter binds to G-protein coupled receptor to activate a

chemical signaling cascade

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Outline

Survey of neurotransmitter structures

Fast neurotransmission: glutamate, GABA, glycine, acetylcholineMetabolism and vesicular transport

Reuptake and degradation

Receptor systems

Pharmacology: agonists and antagonists

Synaptic integration

Neuromodulation: catecholamines, serotonin, histamine, neuropeptides

Overview of G-protein signaling

Metabolism and vesicular transport

Reuptake and degradationReceptor systems, coupling, downstream targets

Pharmacology: agonists and antagonists

Unconventional neurotransmitters

endocannabinoids, NO

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Survey of the major neurotransmitters

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HowareGABAreceptorsregulated?

Fast neurotransmission, simplified

Neurotransmitter

Ionotropic receptor

Change in membrane potential

Ion (Na+, Ca++, Cl-)

Ion (Na+, Ca++, Cl-)

Metabolism and vesicular

transport

Reuptake and degradation

Receptor systems

Pharmacology: agonists

and antagonists for studyand therapy

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Glutamate fast neurotransmission

Synthesis, packaging, reuptake, degradation

(error - should

be EAAT)

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Molecular diversity of glutamate

receptors:

3 types, based on sensitivity topharmacological agents: AMPA, kainate, N-

methyl d-aspartate (NMDA)

AMPA: homotetramers or heterotetramers

assembled from Glu R1-4 subunits

NMDA: heterotetramers that contain an NR1

subunit, and a subunit from the NR2 family

Kainate: heterotetramers containing subunits

from the KA1,2 family, and from the GluR5-7

family

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AMPA receptor structure

(NMDA, kainate receptors have the same principal structural features)

Also

called

GluR-1

through 4

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Expression patterns of AMPA receptor subunits in the brain

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AMPA receptor functional diversityMixing and matching of subunits (see GABA receptors for examples)

Further diversity generated by alternative splicing, editing

Flip and flop splice forms

desensitize at differentrates, both have rapid

onset kinetics

(gluR2 homomers shown)

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Kandel, Schwartz, Jessel (2000) Principles of Neural Science 4ed

Ion selectivity is modulated

by RNA editing

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NMDA receptors

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NMDA receptors

show slow onset and

decay kinetics

Some synapses have

both glutamate

receptor types, andproduce a two-

component synaptic

current

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NMDA receptors are strongly rectifying because of Mg++block

Coincidence detector in learning and memory

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NMDA receptors are calcium permeable

This property is particularly significant because calcium is a

second messenger that plays many important regulatory roles

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Pharmacology

AMPA

agonists: AMPA, glutamateantagonists: CNQX, NBQX

Kainate

agonists: kainic acid, glutamateantagonist: CNQX

NMDA

agonists: glutamate, aspartate, NMDA

antagonists: D-APV, D-AP5, MK-801, Ketamine,Phencyclidine, (Mg++)

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Glutamate receptors are physically tethered at synapses and

associated with signaling molecules

AMPA receptors interact with GRIP, SAP-97 and others

Synaptic strength and Ca++permeability of glutamate postsynaptic

complexes is a major determinant of synaptic plasticity, and probably

underlies learning and memory - stay tuned

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Inhibitory GABA

presynaptic neuron

excitatory

presynaptic

neuron

Postsynaptic

neuron

inhibitory

Inhibitory neurotrans-

mission prevents

excitation of the post-

synaptic neuron

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Whereas glutamate is the principal excitatory neurotransmitter,

GABA is the principal inhibitory neurotransmitter in the brain

A typical GABA

presynaptic terminal

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GABA synthesis

Biosynthetic enzyme: GAD65

, GAD67

GAD65more highly enriched in nerve terminals, therefore might be more

important for neurotransmission

GAD requires pyridoxal phosphate as cofactor (might be regulated by

GABA and ATP)

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GABA release, reuptake

Vesicular release is the major mechanism

Uptake is mediated by plasma membrane transporters

GAT-1, GAT-2, GAT-3, BGT-1

GAT1-3 in brain, BGT-1 in kidney but may also be in brain

1 GABA

2 Na+

1 Cl- GAT

out in

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Degradation

GABA aminotransferase

(aka GABA transaminase or

GABA T)

Astrocytes and neurons, mitochondrial

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a-ketoglutarate glutamate

Summary of GABA synthesis, release, reuptake, degradation

1. GABA is formed by removal of

carboxyl group of glutamate, by

the enzyme GAD2. GABA is packaged into synaptic

vesicles by VIAAT and released

by depolarization

3. GABA may be taken up by

nerve terminal by GAT proteins

for repackaging into synapticvesicles

4. GABA may be taken up by glial

cells, where it undergoes

reconversion to glutamate

(amine group is transferred to a-

ketoglutarate, generatingglutamate and succinic

semialdehyde)

5. Glutamate is transported back

into nerve terminal, where it

serves as precursor for new

GABA synthesis

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GABA receptors:

Fast GABA transmission mediated mainly by GABAA

receptors, which are ligand-activated chloride channels.

Some fast GABA transmission mediated by so-called GABAC

receptors, which are a closely-related sub-family of GABAA

receptors

GABA also utilizes a metabotropic receptor called the

GABABreceptor, described in Neuromodulation section.

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CCCM2 M3 M4M1

C C

N

BDI

BDII

Pentameric structure of GABAAreceptors

M2M2

M2M2

M2

GABAAreceptors belong to the ligand-gated ion channelsuperfamily, which also includes nicotinic acetylcholine

receptors, glycine receptors, and the 5-HT3serotonin receptor.

Fine structure and function of this receptor class will be

covered in more detail in the acetylcholine section, upcoming.

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2

2

a1

GABAAreceptors areheteromultimers

subunits

-Alpha (1-6)-Beta (1-4)

-Gamma (1-4)

-delta, epsilon, pi, theta

-Rho (1-3) - make upthe GABACreceptor

Potentially thousands of

different subunit combin-

ations, or subtypes. Which

really occur in the brain?

About 12 subtypes are

prevalent

Bowery et al. 2002

Pharmacological Reviews 54:

247-264

McKernan and Whiting (1996)

TINS 19:139-143

Wh i h i ifi f hi di i ?

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What is the significance of this receptor diversity?

Different subunit combinations (receptor subtypes) confer

different functional properties.

Those properties allow the receptors to do different jobs

a122

EC50=13 M, fastdesensitization

G GGG

a62

EC50=0.27 M, nodesensitization

GABA

terminal

Postsynaptic

membrane

Low GABA sensitivity of and fast desensitization a122are suited for phasic

activity and high GABA concentrations found right at the synapse. High

GABA sensitivity and lack of desensitization allows a62to detect GABA that

spills over from the active synaptic zone

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Phasic synaptic

GABA response

Extrasynaptic

tonic response

Inhibitor of all GABAA

receptors, eliminates

both phasic and tonic

responses, showing that

they are both GABA

currents

Recording from cerebellar granule cells, showing both

synaptic and extrasynaptic GABA responses

Extrasynaptic tonic currents are dependent

on the presence of an intact a6subunit

gene

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GABAAreceptor tethering at the synapse

Several proteins that are important for GABAAreceptortethering have been proposed, principally gephyrin,

but the tethering mechanism is not well characterized.

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GABAAreceptor pharmacology

Antagonists:

Bicucculine competitive

SR95531 (gabazine) competitivePicrotoxin mixed competitive, non-competitive

Penicillin G open channel block

Pentelenetetrazole (PTZ) open channel block

Pregnenolone sulfate non-competitive

Agonist:

Muscimol

Barbiturates, neurosteroids (high concentrations)

Enhancers:

BenzodiazepinesBarbiturates, neurosteroids (low concentrations)

GABAAreceptor antagonists are important research tools, but not

clinically useful. GABAAreceptor enhancement, but not direct

agonism, is useful therapeutically in neurology.

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Glycine neurotransmission

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Glycine neurotransmission: receptors

Glycine is a neurotransmitter in its own rightDistinct from NMDA receptor co-agonist role

Ionotropic receptor, ligand-gated ion channel

superfamily receptors, homologous to GABAA

receptorsa1-4, subunits - ahomomers in early development, aheteromers in

adults

Major spinal cord inhibitory transmitter

Retinal, brainstem as well

No allosteric regulators used as drugsStrychnine is a competitive antagonist

Human mutations in glyR found in startle disease,

hyperekplexia, Jumping Frenchman disease

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Acetylcholine neurotransmission

1. Acetylcholine synthesized from

choline and acetyl CoA by choline

acetyltransferase (ChAT)2. ACh loaded into synaptic vesicles

by VAchT

3. Released ACh broken down by

acetylcholinesterase (notable

difference from other neuro-

transmitters discussed so far)

4. Choline taken up by presynaptic

terminal as precursor to further

ACh synthesis

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Nicontinic acetylcholine receptorsFast ACh neurotransmission utilizes ligand-gated ion channel superfamily

receptors sensitive to nicotine, hence called nicotinic ACh receptors

Muscle nAChRs: 2a, , , subunits in the ratio of 2a:::

Neuronal nAChRs: 3a:2or a7homomers

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nAChR characteristics

Non-selective cation channels,

therefore excitatory

Muscle receptors localized at end

plates, postsynaptic to the motor

neurons, cause muscle excitation

(see Control of Movement lectures)

Neuronal receptors localized on

presynaptic terminals, modulate the

release of other neurotransmitters

Agonists: Nicotine

Antagonists: a-bungarotoxin, -

tubocurarine (muscle)

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Electron micrograph of nicotinic acetylcholine receptor

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Structure determined by cryo-EM to 4 Angstroms. Helix arrangement correct

Miyazawa et al. 2003

Nature 423:949-955

Structure in greater detail

Molecular interactions underlying LGIC superfamily receptor

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Side view Top view,

closed

Top view,

open

Helices rotate

Kinked M2 helix

Molecular interactions underlying LGIC superfamily receptor

activation (i.e. GABAA, glycine, nAChR)

Kash et al. (2003)

Nature 421:272-275

Lee and Sine (2005)

Nature 438, 243-247

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Synaptic physiology and integration

Textbook p.107-117

EPC ACh(V E )

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EPC = gACh(Vm-Erev)

Since EPCs reverse at about 0 mV, ACh channels must be equally

permeable to Na+and K+

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GABA neurotransmission will drive membrane potential

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GABA neurotransmission will drive membrane potential

toward the Cl- reversal potential

GABA d l i ll d di th di ti f th

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GABA can depolarize cells depending on the direction of the

chloride gradient (i.e. EClmay be suprathreshold)

S ti f t ti b t ti l ll

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Summation of postsynaptic membrane potentials allows

multiple synaptic inputs to be integrated