Glutamate Neurotransmission Excitatory Amino Acid Neurotransmitters
Neurochemistry MS 532September 11, 2014
Dr. Dan SavageBMSB 145A
[email protected]
Reference: Brady et al., Basic Neurochemistry 8th ed., pp 342-366
Lecture Outline
1. Historical Perspective
2. Distribution of glutamate neurons
3. Glutamate as a neurotransmitter
4. Ionotropic glutamate receptors
5. Metabotropic glutamate receptors
6. Regulation of glutamate receptors
7. Glutamate receptors on glia
8. Putative therapeutic applications of glutamatergic agents
Historical Perspective
Early 60s - L-Glu and L-Asp are excitatory
Late 70s - Na+ / ATP dependent transportersEarly 80s - Selective agonists at iGluRsMid 80s - Selective antagonists at iGluRsLate 80s - Cloning of iGluRs / iGluR modulatorsEarly 90s - Cloning of mGluRs / iGluR ABsMid 90s - mGluR agonists / mGluR ABsLate 90s - mGluR antagonistsEarly 00s - Selective mGluR agonists / mGluR modulators
Caudatenucleus
Thalamus
Cerebellar Cortex
HippocampalTrisynaptic Circuit
Adapted from Siegel and Sapru, Essential Neuroscience, 2006, Fig. 25.3
Glutamate Transmission in Brain
Dentate gyrus
CA3
CA1
Cerebral Cortex
80 - 90% of CNS neurons use GLU 80 - 90% of synapses are glutamatergic
~ 80% of energy utilization in brain is formembrane repolarization after GLU release
Glutamate as a Neurotransmitter
Synthesis
- Ubiquitous; amino acid in highest concentration in CNS
- Small proportion is transmitter specific
- No identifiable synthetic machinery
- Sources:Glucose via TCA cycle to KG GlutamateGlutamine via Phosphate-activated glutaminase
Figure 17-1
Glutamate as a Neurotransmitter Synthesis
Storage
- Small clear vesicles
Figure 17-4 A
Glutamate as a Neurotransmitter Synthesis Storage
- Small clear vesicles (~ 17 nm diameter)- SV Transporters (VGLUT 1 & 2)- Proton pump- SV GLU Conc.: ~ 60 to 250 mM
Co-localized substances:- Peptides (CCK, DYN, others)- Zinc (via ZNT3 transporter)
Glutamate as a Neurotransmitter
Synthesis
Storage
Release
Ca++ / voltage dependent release
N, P, Q type VSCCs
Glutamate as a Neurotransmitter Synthesis Storage Release Termination of Action - Reuptake
- High affinity (low M) Na+ / ATP dependent
- Five transporters, unlike NE / GABA family- EAAT 1 & 2 (glia) - EAAT 3 (neuronal)- Relatively high density compete with GluRs for GLU
- GTRAPS: Glutamate transporter-associated proteinsBind to and regulate GLU transporter affinity
- Recycling of glutamate
Recycling of Glutamate as a Neurotransmitter
EAAT1&2
EAAT3
~ 40% of synaptic GLU arises from the Glutamine Cycle
Glutamate Spillover- activatenon-synapticGluRs ?- activate mGluRs on GABA and
monoaminergic nerve terminals ?Synaptic [GLU] after vesicle release: ~low mM 13
Glutamate as a Neurotransmitter Synthesis Storage Release Termination of transmitter action Transmitter-specific receptors
- Ionotropic- Metabotropic
Ionotropic Glutamate Receptor Subtypes
AMPA (-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid)
NMDA (N-methyl-D-aspartate)
- Receptor localization
[3H]-FW Binding to AMPA Rs
[3H]-MK-801 Binding to NMDA Rs
Figure 17-4 A
GLU synapses areasymetricmuch higher postsynaptic density> 100 different proteins identified in PSDs
Figure 17-4 B(Group I)
Ionotropic Glutamate Receptor Subtypes
AMPA (-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid)
NMDA (N-methyl-D-aspartate)
- Receptor localization
- Agonist responses
Affinity for binding glutamate:AMPA - ~ 400 MNMDA - ~ 1M
Figure 17-10 B
Rapid decay of AMPA response: Lower affinity for GLU and a rapid desensitization of AMPA Rs
Slower decay of NMDA response: Higher affinity for GLU (slower dissociation kinetics)
Agonist & voltage-dependent activation of NMDA receptors
Also: NMDA requires binding of both glutamate and glycine to agonist recognition sites
Ionotropic Glutamate Receptor Subtypes
AMPA
NMDA
Kainate
Early electrophysiological studies could not differentiate between AMPA and KA receptors
Were often referred to as AMPA / KA or non-NMDA receptors
In the absence of selective antagonists, were identified by their differential sensitivities to cyclothiazine (AMPA Rs) and concanavalin A (KA Rs)
[3H]-VKA Binding to Kainate Rs
iGluR
nAChR
Glutamate-R
25
Figure 17-8 A
Figure 17-8 B
TransmitterBindingPocket(GluK2)
Compared to NCR subunits:- TM domains- N-terminal size- Cytoplasmic C-terminus
Hetero-oligomeric subunit combinations
the rule
Ionotropic glutamate receptor subunit types
nAChR
AMPAR
GluA1
GluA1
GluA2
GluA2
GluN1
NMDAR
Glutamate-gatedIon channels are
tetramersKainate-R
GluK2
GluK2
GluN1
GluN2
GluN2
GluK5
GluK528
NR1
NR1
NR2
NR2
GluN1
GluN1
GluN2
GluN2
The NMDA-R requires both glutamate and glycine (or D-serine) to be fully activated
Agonist: Glutamate
Glu
Glu
Co-agonist:Glycine or D-serine
strychnine-insensitivebinding sites Gly
Gly
GluN3s also have Gly recognition sites29
Sources of Glycine or D-Serine
1. CSF contains micromolar glycine concentrations (but glial transporters could decrease levels near NMDARs)
2. Astrocytes wrapped tightly around glutamatergic synapses can release saturating concentrations of D-serine (Panatier et al. Cell.125, 775-784, 2006)
30
Figure 17-7
Different Hetero-tetrameric Combinations of Subunits Confer Differential Function & Sensitivity
2. GluA2 subunit containing AMPA Rs less permeable to calcium.
3. Different NR2 subunits confer differential sensitivity to agents.
1. GluK subunit composition affects receptor affinity for KA.GluK1-3 containing: ~ 100 nMGluK4-5 containing: ~ 10 nM
Impermeable to Ca2+in most mature neurons
AMPAR
GluR1
GluR1
GluR2
GluR2
AMPAR
GluA1
GluA1
GluA2
GluA2
33
Presence of an arginine in M2 in GluA2 is responsible for reduced
Ca2+permeability of AMPARs
GluA2GluA1 GluA3GluA1
34
mRNA Splice Variants of AMPAR Subunits Confer Differential Function & Sensitivity
1. Flip / Flop Variants of GluA1-4 Subunits
2. Alters rates of desensitization to activation
3. Alters sensitivity to cyclothiazide, GYKI compounds, AMPAKines
NMDA Receptor Subunits :Differential Sensitivity to Various Agents
1. Glutamate: 2B > 2A > 2D > 2C
4. MK-801: 2A = 2B >> 2C = 2D
3. Mg++: 2A = 2B >> 2C =2D
5. Ifenprodil: 2B >>> 2A >> 2C, 2D
2. APV : 2A > 2B > 2D = 2C
6. Ethanol: 2A = 2B >> 2C, 2D?
mRNA Splice Variants of NMDAR Subunits Confer Differential Function & Sensitivity
1. NR1A-H splice variants- based on three alternative exon selections
2. Alters NMDA R sensitivity to: Hydrogen ion ( H+ channel opening ) Zinc ( biphasic regulation ) Polyamines ( biphasic regulation) Nitric oxide Glycine Neurosteroids
log [ Agent ] M0.0001 0.01 0.1 1 10 100 1000
NM
DA
-Sen
sitiv
e [3
H]-G
luta
mat
e B
indi
ng( f
emto
mol
es /
105
mm
2 )
2.0
2.5
3.0
3.5
4.0
4.5
2.0
2.5
3.0
3.5
4.0
4.5
Zinc
Pregnenolone
iGluR mGluR
Metabotropic Glutamate Receptors N- terminal confers agonist specificity Cytoplasmic Loops
Loops I and III highly conserved Loop II associated with effector coupling Loop IV:
G-Protein Coupling Scaffolding proteins (Homers) Phosphorylation sites
mGluR Allosterism - noncompetitive binding sites
mGluR Subtypes
Variable Kds forbinding glutamate:mGluR8 - ~ 2 nMmGluR7 - ~ 1 mM
Different Kds for G-protein coupling
Figure 17-4 B(Group I)
Three levels of Long-term Potentiation
Progressive activation of mechanisms for increasing cytoplasmic calcium:
LTP 1: NMDA receptorsLTP 2: mGluR receptorsLTP3 : VSCCs
From: Raymond , TINS, 2007
PresynapticTerminal
Glu Glu Glu
mGluR Group II (2 & 3) & Group III (4, 6, 7 & 8)Inhibit adenylate cyclaseDecrease glutamate release
mGluR Group I (1 & 5)Activate Phospholipase CIncrease glutamate release
mGluR Regulation of Glutamate Release
mGluRI mGluRII / III
Gq
GluSynapsin I
GAP-43:CaM
CaM
CaMKII
PP-2B
PresynapticTerminal
Actin
mGluR5
Gq/11
PLC-1PIP2
IP3DAG
PKCII/
GAP-43-P
Glu(RS)-2-Chloro-
5-hydroxyphenylglycine
( CHPG )
CHPG-Stimulated GAP-43 Phosphorylation
CHPG M0.5 2 3 5 20 30 50 200300 5001 10 100 1000
DP
Ms
32P
/ M
illig
ram
Pro
tein
500
