B. Bettler Institute of Physiology - unibas.ch

Neurotransmitter receptors: Structure and function

B. BettlerInstitute of Physiology

BaselMarch 30, 2010

History

Ionotropic receptors: - Structure and synaptic functions- Pathology

Metabotropic receptors: - Structure and synaptic functions- New insights (structure/signaling/pharmacology)

Overview

“Electrical Synapses” “Chemical Synapses”

Camillo GolgiNobelprize 1906

Santiago Ramon y CajalNobelprize 1906

John EcclesNobelprize 1963

Bernard KatzNobelprize 1970

Electrical versus Chemical Synapses 2

bidirectional

no information processing atthe synapse

Schmidt/Unsicker Fig 2-1

Electrical Synapses4

directional

information processing at synapse: excitation can be changed in inhibition

Schmidt/UnsickerFig 2-2

Chemical Synapses: Possibility for reversal of signal 11

presynaptic postsynaptic

Chemical Synapses: Inhibitory and excitatory postsynaptic ionchannels

Ca++

Na+

x

Cl-

K+

ionotropic

metabotropic

metabotropic

Ca++

Kandel Fig 10.1

17

History

Ionotropic receptors: - Structure and synaptic functions


Overview

Purves 3rd Fig. 1.7

excitatory

inhibitory

A fundamental principle: Excitatory and inhibitory synapses

Purves 3rd Fig 1.8

Frequency of actionpotentials in neurons of a reflex arc 19

Ca++

Na+

x

Cl-

Ca++

Na+

Cl-

Hyperpolarisation:Inhibition

Depolarisation:Excitability

Permeabilites of ions at excitatory and inhibitory synapses (1)

GABA, glycine

glutamate, acetylcholine, serotonin, ATP

Excitatory: postsynaptic depolarisation Na+, Ca2+

Inhibitory: postsynaptic hyperpolarisation (mostly) Cl-, K+

Purves Tab 2.1

Permeabilites of ions at excitatory and inhibitory synapses (2)

-91mV+60mV-82mV

+125mV

EquiPot

Cl--permeable ion channels are inhibitory even if they lead todepolarization

Purves 3rd Fig 5.19

Hyperpolarisation Depolarisation

Cl-influx Cl-efflux

6

IPSPs und EPSPs act simultaneously on individual neurons

∑ Erregung∑ Hemmung

Aktionspotentiale ↑

∑ Erregung ∑ Hemmung

Aktionspotentiale ↓

InhibitionExcitation

ExcitationInhibition

EPSPs actionpotentialLearning/Memory


IPSPs no actionpotentialSleep

Normal balance between excitation and inhibition

InhibitionExcitation

Excitation


Inhibition

epilepsy,anxiety, depression, insomnia,

spasticity

cognitive problems, loss of muscle tone,

coma, respiratory arrest

Abnormal balance between excitation and inhibition

Excitatory and inhibitory neurotransmitter receptors

Purves Fig 7.11

localisation (pre-, post-, extrasynaptic)

affinity for the neurotransmitter

kinetics

ion selectivity (Ca2+)

Cells express distinct subunits receptors with distinct properties

NR2CNR2A NR2BNR1

Example NMDA receptors: Overlapping mRNA distribution of receptor subunits

Klinke/Pape Fig 5.12

9

Kandel Fig 11.14

Structure of ionotropic neurotransmitter receptors

Molecular basis for the ion selectivity of ionotropic acetylcholine receptors

Kandel Fig 11.15

Ionotropic GABAA receptors: Site of action of the benzodiazepines

L-GlutamateGABA

IPSPs ↑sleep, anti-epileptic

AMPA NMDA

Ca2+ influx Mg2+ block

“no” Ca2+ influx

Ionotropic glutamate receptors

Kainate

Ca2+ influx

Auxiliary AMPA receptor subunits influence surface trafficking,pharmacology and kinetics of the receptor response

Nature 2000

Science 2010 Nature 2009

TARPs: transmembraneAMPA receptor regulatory proteins

TARPs influence pharmacology and kinetics of the AMPA receptor

Kato et al., TINS, in press

Purves 3rd Fig 6.7

NMDA receptors: Voltage-sensitive Mg2+ block

Einwärtsstrom

Auswärtsstrom

Purves 3rd Fig 6.7

Kinetics of AMPA and NMDA receptors:

+50 mV

+50 mV

+50 mV

EPSPKainate/AMPA > EPSPNMDA

KandelFig 12.7

NMDA receptors do not contribute much to EPSCs at hyperpolarized membrane potentials

Ca2

Glutamate

NMDA-R

AMPA-R

+

+

Mg+2

Ca2+-dependentprocesses

Na+

NMDA receptors act as coincidence detectors during synapticplasticity processes

presynaptic activity: glutamate release postsynaptic activity: depolarisation

Longterm potentiation (LTP) after tetanic stimulation

Purves 3rd Fig 24.6

After tetanusto pathway 1

Before tetanusto pathway 1

(1h) (1h)

LTP is specificfor tetanically stimulatedsynapse 1

1957 junge Mäuse mit Glutamat-Diät: neuronaler Zelltod Retina

1967 neuronaler Zelltod Gehirn

Olney: “Glutamat bewirkt neuronalen Zelltod durch langandauernde

exzitatorische synaptische Transmission”

NMDA Antagonisten blockieren neuronalen Zelltod

Kainat induziert neuronalen Zelltod (epileptischen Anfälle)

übermässige [Ca2+]i induziert Apoptose (programmierter Zelltod,

Proteasen werden aktiviert)

Neurodegenerative Prozesse: ExzitotoxizitätExcitotoxicity

Ischämie: O2, Glucose ATP (Glutamat uptake, Em, NMDAR, [Ca2+]i, Apoptose) Tote Neurone entlassen: [K+]e, [Glu]e

Hypoglykämie (Diabetes): Glucose

Epilepsie (status epilepticus)

“Chinese Food Syndrome”, MSG (Mono Sodium Glutamate), “Aromat”

Domoat/Kainat Vergiftungen (verdorbene Muscheln)

Exzitotoxische Prozesse finden statt bei:

AMPA kainate

Kainate und AMPA receptors are distinct

Ca2+ Ca2+

Nature 392, 1998

GluR6 is activated by kainate und domoate, butnot by AMPA

GluR6 subunit makes kainate receptorspermeable for Ca2+

(Ca2+)

GluR6 is predominantly expressed in the CA1 and CA3 regions,which are most susceptible to seizure-induced brain damage

Can GluR6 directly mediate excitotoxicty?

GluR6 mediates kainate-mediated excitotoxicity

Nature 392, 1998

GluR6 antagonists as anti-epileptic drugs in preclinical trials

History



Overview

Ionotropic

Metabotropic

Purves 3rd Fig 6.5+ Neuropeptides

Classical neurotransmitters activate ionotropic and metabotropic„G-protein coupled receptors“

- 6 families

- 7TM domains

- no sequence homologybetween families(evolutionary convergence)

- binding sites differ

Classification and diversity of GPCRs: Neurotransmitter receptorsbelong to different gene families

GPCRs activate G-proteins

Crystal structure of the human 2-adrenergic receptor bound to the partial inverse agonist carazolol

rhodopsin 2000 / 2-adrenergic 2007

Illustration of the central core of rhodopsin in its inactive and activeconformation viewed from the cytoplasm

Inactive Active

Change in TMIII/TMVI domain conformation unmasks G-protein binding site (C-term G) and activates the G-protein

Crystal structure of a heterotrimeric G-protein bound to a GPCR

Classical signaling pathways of GPCRs: How can they influenceto synaptic transmission?

GABABHeteroreceptors

Auto-receptors

G effector systems: Regulation of K+ and Ca2+ channels (1)

.

SpilloverGABA

.

.

.

..

.

..

.

.

.

..

.

.

.

.

.

.

.

.

..

.

..

..

.

..

. .

.

.

Activation of Kir3-type K+-channels

G effector systems: Regulation of K+- and Ca2+-channels (2)

Inhibition of PQ-type Ca2+-channels

1 m baclofen

Purves Fig 8.6

G effector systems: Phosphorylation of ion channels

G effector systems: Incorporation of additional ion channels atsynapses

Purves 3rd Fig 7.11

longterm effects

structural plasticity /synaptic plasticity- synapse ↑

- receptors ↑

History



Overview

Cloned GABAB receptor subunits bind GABA but do not activateeffector systems

130100

Mr (K) Cor

tex

Western blot

1a 1b

2a 3a 4a 5a 61b

Met 1a Met 1b

2 sushi domains

GABAB1 Gene

No efficient functional coupling of GABAB1a and GABAB1b to effector K+ / Ca2+ channelsand adenylate cyclase

Agonist afffinity differs between recombinant GABAB1a and GABAB1b proteins and native GABAB receptors

[125 I]

ant

i-myc

sur

face

bin

ding

(%)

1100

80

6040

20

20

myc

-GA

BAA1

myc

-GA

BAA3

Nm

yc-1

a

Cm

yc-1

a

myc-GABAA3 myc-1a

Couve et al., J. Biol. Chem., 1998

non-perm

perm

GABAB1a and GABAB1b are retained in the endoplasmaticreticulum

GABAB receptors only function as heterodimeric receptors

Nature 396, 1998

GABAB(1,2) receptors coupled to Kir3-type K+ channels in Xenopus oocytes

1a+2

1b+2

1a 1b 2

1a+1b

2

surface expression coupling to P/Q-, N-type Ca2+ channels negative coupling to adenylate cyclase

Heteromerization between GABAB(1) and GABAB(2) subunits is a prerequisite for receptor function in heterologous cells

Increasing number of reports demonstrating theexistence of heteromeric GPCRs

- 1998: 1st heteromeric GPCR- 2005: 35 heteromeric GPCRs

change in pharmacology (+ opioid, SSTR5+D2, M2+M3)

change in G-protein coupling selectivity (Go/i > Gs + opioid)

stabilization of receptor at cell surface (GABAB(1,2), + opioid)

increased agonist affinity (GABAB(1,2), SSTR5+D2)

Functional consequences of GPCR heterodimerization

Heteromeric GABAB(1,2) receptors display increased affinity foragonists but still do not match native pharmacology

Complete loss of GABAB responses in GABAB1 and GABAB2knockout mice: Core receptor subunits

WT

GABAB1-/-

GABAB2-/-

Anti-GABAB1 Anti-GABAB2

Schuler et al., Neuron, 2001Gassmann et al., J. Neurosci., 2004Fritschy et al., J. Comp. Neurol., 2004

Coupling to Kir3-type K+-channels in Xenopus oocytes

No pharmacological or functional differences between GABAB(1a, 2)and GABAB(1b,2) receptor subtypes in heterologous systems

GABAB(1a,2) GABAB(1b,2)

Cruz et al., Nature Neurosci., 2004

… but native GABAB responses differ in their pharmacological and kinetic properties

Possible explanations:

- effector channel subunit composition- phosphorylation of the receptor or effector- proteins that influence the G-protein activation/deactivation cycle (RGS)- unidentified auxiliary subunits (similar to the TARPs, cornichons etc).

“Regulator of G-protein signaling” (RGS) proteins accelerate GTP hydrolysis by Gα subunits and produce desensitization

RGS proteins are negative regulators of GPCR signaling

GEF: guanine nucleotide exchange factorGAP: GTPase-accelerating protein

Affinity purification of GABAB receptors reveals a high-molecular weight complex and lack of heterodimers

ab

Identification of four sequence-related auxiliary GABAB receptor Subunits using affinity purifcation/tandem MS

In press

GAS are tightly associated with high-molecular weight GABABreceptor complexes

GABAB2

GABAB1

anti-GAS4anti-GAS2

Differential but overlapping spatial distribution of GAS proteins in the adult mouse brain

GAS differentially alter baclofen-mediated Kir3 currentdesensitization in transfected CHO cells

+GAS2+GAS4

Native

GAS shorten the rise-time of baclofen-mediated Kir3 currentsin transfected CHO cells

baclofen

w/o GAS

w/o

GA

S

+GA

S1

+GA

S2

+GA

S3

+GA

S4

GAS differentially alter baclofen-mediated Cav2.2 currentinactivation

GAS1

GAS2

GAS alter baclofen-mediated Kir3 current kinetics in transfected hippocampal neurons

+GAS2

+GAS4Native

GAS2 knock-down alters baclofen-mediated Kir3 current kineticsin hippocampal neurons

Control shRNA

GAS2 shRNA

GAS4 knock-down/knock-out alters baclofen-mediated Kir3 current kinetics in hippocampal neurons

WT+GAS4 shRNAGAS4 KO mice

WT GAS4 shRNA

GAS4 KO

GAS increase agonist potency at GABAB receptors

+control+GAS1+GAS2

GAS do not alter agonist affinity at recombinant GABAB receptors:Additional auxiliary subunits?

[3H] CGP54626A radioligand Displacement

GAS1GAS2GAS4

Conclusions

Auxiliary receptor subunits not only exist for ion channels, but also for GPCRs

Auxiliary subunits alter kinetic and pharmacological propertiesof the receptor response (similar to auxiliary subunits of AMPA receptors

B. Bettler Institute of Physiology - unibas.ch

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