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 Excitation Inhibition anxiety, depression, insomnia, E epilepsy, x c i tat spasticity io n cognitive problems, I n loss of muscle tone, h coma, respiratory arrest ib it io n 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 A receptors: Site of action of the benzodiazepines Ionotropic GABA L-Gl utamate 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 GABAB1a and GABAB1b are retained in the endoplasmatic reticulum myc-GABAA3 myc-1a 120 non- 100 perm 80 60 40 20 I] anti-myc surface binding (%) 1 3 125 [ A perm A Cmyc-1a Nmyc-1a myc-GABA myc-GABA Couve et al., J. Biol. Chem., 1998 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