Neurotransmitter receptors: Structure and function B. Bettler Institute of Physiology Basel March 30, 2010
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
Metabotropic receptors: - Structure and synaptic functions- New insights (structure/signaling/pharmacology)
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
ExcitationInhibition
IPSPs no actionpotentialSleep
Normal balance between excitation and inhibition
InhibitionExcitation
Excitation
ExcitationInhibition
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
Ionotropic receptors: - Structure and synaptic functions- Pathology
Metabotropic receptors: - Structure and synaptic functions- New insights (structure/signaling/pharmacology)
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
Ionotropic receptors: - Structure and synaptic functions- Pathology
Metabotropic receptors: - Structure and synaptic functions- New insights (structure/signaling/pharmacology)
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