Neurotransmitter receptors: Structure and function

B. Bettler Institute of Physiology

Basel March 30, 2010 Overview

 History

 Ionotropic receptors: - Structure and synaptic functions - Pathology

 Metabotropic receptors: - Structure and synaptic functions - New insights (structure/signaling/pharmacology) Electrical versus Chemical Synapses 2

CamilloJohn Eccles Golgi SantiagoBernard Ramon Katz y Cajal NobelprizeNobelprize 1963 1906 Nobelprize 19701906

 “Electrical Synapses”  “Chemical Synapses” Electrical Synapses 4

 bidirectional

Schmidt/Unsicker Fig 2-1  no information processing at the synapse Chemical Synapses: Possibility for reversal of signal 11

Schmidt/Unsicker Fig 2-2  directional

 information processing at synapse: excitation can be changed in inhibition Chemical Synapses: Inhibitory and excitatory postsynaptic ion channels

metabotropic

++

x Ca K+

ionotropic + ++ Na Ca metabotropic - Cl presynaptic postsynaptic 17

Kandel Fig 10.1 Overview

 History

 Ionotropic receptors: - Structure and synaptic functions

 Metabotropic receptors: - Structure and synaptic functions - New insights (structure/signaling/pharmacology) A fundamental principle: Excitatory and inhibitory synapses

excitatory

inhibitory

Purves 3rd Fig. 1.7 Frequency of actionpotentials in neurons of a reflex arc 19

Purves 3rd Fig 1.8 Permeabilites of ions at excitatory and inhibitory synapses (1)

glutamate, acetylcholine, ++ serotonin, ATP Depolarisation: Ca Excitability  + Na ++

x Ca

+ Na - Cl - Cl Hyperpolarisation: Inhibition 

GABA, glycine Permeabilites of ions at excitatory and inhibitory synapses (2)

 Excitatory: postsynaptic depolarisation  Na+, Ca2+

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

Purves Tab 2.1

EquiPot -91mV +60mV -82mV +125mV Cl--permeable ion channels are inhibitory even if they lead to depolarization 6

Hyperpolarisation Depolarisation

Cl-influx Cl-efflux

Purves 3rd Fig 5.19 IPSPs und EPSPs act simultaneously on individual neurons

∑ Hemmung ∑ Erregung

Aktionspotentiale ↑

∑ Erregung ∑ Hemmung

Aktionspotentiale ↓ Normal balance between excitation and inhibition

Excitation Inhibition

n Ex Inhibitio citation n Inh Excitatio ibition

EPSPs  actionpotential IPSPs  no actionpotential Learning/Memory Sleep Abnormal balance between excitation and inhibition

Excitation Inhibition

n tio E ibi xc nh itat I ion

I ion nh at ibi cit tio Ex n epilepsy, cognitive problems, anxiety, depression, insomnia, loss of muscle tone, spasticity coma, respiratory arrest Excitatory and inhibitory neurotransmitter receptors

Purves Fig 7.11

 localisation (pre-, post-, extrasynaptic)  affinity for the neurotransmitter  kinetics  ion selectivity (Ca2+) Example NMDA receptors: Overlapping mRNA distribution of 9 receptor subunits

NR1 NR2A NR2B NR2C

Klinke/Pape Fig 5.12

Cells express distinct subunits  receptors with distinct properties Structure of ionotropic neurotransmitter receptors

Kandel Fig 11.14 Molecular basis for the ion selectivity of ionotropic acetylcholine receptors

Kandel Fig 11.15 Ionotropic GABAA receptors: Site of action of the benzodiazepines

L-Glu tamate GABA

IPSPs ↑ sleep, anti-epileptic Ionotropic glutamate receptors

AMPAKainate NMDA  “no” Ca2+ influx  Ca2+ influx  Ca2+ influx  Mg2+ block Auxiliary AMPA receptor subunits influence surface trafficking, pharmacology and kinetics of the receptor response

TARPs: transmembrane AMPA receptor regulatory proteins

Nature 2000

Science 2010 Nature 2009 TARPs influence pharmacology and kinetics of the AMPA receptor

Kato et al., TINS, in press NMDA receptors: Voltage-sensitive Mg2+ block

Auswärtsstrom

Einwärtsstrom

Purves 3rd Fig 6.7 Kinetics of AMPA and NMDA receptors: +50 mV

EPSPKainate/AMPA > EPSPNMDA

+50 mV

+50 mV

Purves 3rd Fig 6.7 NMDA receptors do not contribute much to EPSCs at hyperpolarized membrane potentials

Kandel Fig 12.7 NMDA receptors act as coincidence detectors during synaptic plasticity processes

2+ Ca

+ NMDA-R Mg 2

2+ Glutamate Ca -dependent processes

AMPA-R + Na+

 presynaptic activity: glutamate release  postsynaptic activity: depolarisation Longterm potentiation (LTP) after tetanic stimulation

LTP is specific for tetanically stimulated synapse 1

After tetanus (1h) to pathway 1 (1h)

Before tetanus to pathway 1 Purves 3rd Fig 24.6 ExcitotoxicityNeurodegenerative Prozesse: Exzitotoxizität

 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)

2+  übermässige [Ca ]i induziert Apoptose (programmierter Zelltod, Proteasen werden aktiviert) Exzitotoxische Prozesse finden statt bei:

 Ischämie: O2, Glucose 2+  ATP (Glutamat uptake, Em, NMDAR, [Ca ]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) Kainate und AMPA receptors are distinct

AMPA kainate

Nature 392, 1998

 GluR6 is activated by kainate und domoate, but not by AMPA  GluR6 subunit makes kainate receptors (Ca2+) Ca2+ Ca2+ permeable for Ca2+ Can GluR6 directly mediate excitotoxicty?

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

Nature 392, 1998 GluR6 antagonists as anti-epileptic drugs in preclinical trials Overview

 History

 Ionotropic receptors: - Structure and synaptic functions - Pathology

 Metabotropic receptors: - Structure and synaptic functions - New insights (structure/signaling/pharmacology) Classical neurotransmitters activate ionotropic and metabotropic „G-protein coupled receptors“

Metabotropic

Purves 3rd + Neuropeptides Fig 6.5

Ionotropic Classification and diversity of GPCRs: Neurotransmitter receptors belong to different gene families

-6 families

- 7TM domains

- no sequence homology between families (evolutionary convergence)

- binding sites differ 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 active conformation viewed from the cytoplasm

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

Inactive Active Crystal structure of a heterotrimeric G-protein bound to a GPCR Classical signaling pathways of GPCRs: How can they influence to synaptic transmission? G effector systems: Regulation of K+ and Ca2+ channels (1)

Heteroreceptors GABA . B ...... Auto- . Spillover receptors . GABA ...... G effector systems: Regulation of K+- and Ca2+-channels (2)

Activation of Kir3-type K+-channels

1 m baclofen

Inhibition of PQ-type Ca2+-channels G effector systems: Phosphorylation of ion channels

Purves Fig 8.6 G effector systems: Incorporation of additional ion channels at synapses

 longterm effects

 structural plasticity / synaptic plasticity - synapse ↑ - receptors ↑

Purves 3rd Fig 7.11 Overview

 History

 Ionotropic receptors: - Structure and synaptic functions - Pathology

 Metabotropic receptors: - Structure and synaptic functions - New insights (structure/signaling/pharmacology) Cloned GABAB receptor subunits bind GABA but do not activate effector systems

Western blot Cortex Mr (K) 1a 1b 130 100

GABAB1 Gene

Met 1a Met 1b

2a 3a 4a 5a1b 6

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

+ 2+ No efficient functional coupling of GABAB1a and GABAB1b to effector K / Ca channels and adenylate cyclase reticulum GABA perm perm non- Couve et al.,J.Biol.Chem., 1998 myc-GABA B1a and GABA A  3 B1b myc-1a r eandi h endoplasmatic inthe retained are

[125I] anti-myc surface binding (%) 1 1 20 40 60 80 00 20

myc-GABAA1

myc-GABAA3

Nmyc-1a

Cmyc-1a GABAB receptors only function as heterodimeric receptors

Nature 396, 1998 Heteromerization between GABAB(1) and GABAB(2) subunits is a prerequisite for receptor function in heterologous cells

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

2

1a 1b 2 1a 1b + + 2 2

 surface expression  coupling to P/Q-, N-type Ca2+ channels  negative coupling to adenylate cyclase Increasing number of reports demonstrating the existence of heteromeric GPCRs

- 1998: 1st heteromeric GPCR - 2005: 35 heteromeric GPCRs Functional consequences of GPCR heterodimerization

 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) Heteromeric GABAB(1,2) receptors display increased affinity for agonists but still do not match native pharmacology Complete loss of GABAB responses in GABAB1 and GABAB2 knockout mice: Core receptor subunits

Anti-GABAB1 Anti-GABAB2

WT

-/- GABAB1

GABA -/- B2 Schuler et al., Neuron, 2001 Gassmann et al., J. Neurosci., 2004 Fritschy et al., J. Comp. Neurol., 2004 No pharmacological or functional differences between GABAB(1a, 2) and GABAB(1b,2) receptor subtypes in heterologous systems

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

GABAB(1a,2) GABAB(1b,2) … but native GABAB responses differ in their pharmacological and kinetic properties

Cruz et al., Nature Neurosci., 2004

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 factor GAP: GTPase-accelerating protein Affinity purification of GABAB receptors reveals a high-molecular weight complex and lack of heterodimers

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

In press GAS are tightly associated with high-molecular weight GABAB receptor complexes

GABAB1

GABAB2

anti-GAS4 anti-GAS2 Differential but overlapping spatial distribution of GAS proteins in the adult mouse brain GAS differentially alter baclofen-mediated Kir3 current desensitization in transfected CHO cells

Native +GAS4 +GAS2 GAS shorten the rise-time of baclofen-mediated Kir3 currents in transfected CHO cells

baclofen

w/o GAS w/o GAS +GAS1 +GAS2 +GAS3 +GAS4 GAS differentially alter baclofen-mediated Cav2.2 current inactivation

GAS1

GAS2 GAS alter baclofen-mediated Kir3 current kinetics in transfected hippocampal neurons

+GAS4 Native

+GAS2 GAS2 knock-down alters baclofen-mediated Kir3 current kinetics in hippocampal neurons

Control shRNA GAS2 shRNA GAS4 knock-down/knock-out alters baclofen-mediated Kir3 current kinetics in hippocampal neurons

WT +GAS4 shRNA GAS4 KO mice

WT GAS4 GAS4 shRNA 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

GAS1 GAS2 GAS4 Conclusions

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

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