Glutamate Neurotransmission - The University of New Mexiconeurohsc/532/Glutamate... · Glutamate Neurotransmission Excitatory Amino Acid Neurotransmitters Neurochemistry MS 532 September

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 60’s - L-Glu and L-Asp are excitatory

Late 70’s - Na+ / ATP dependent transportersEarly 80’s - Selective agonists at iGluR’sMid 80’s - Selective antagonists at iGluR’sLate 80’s - Cloning of iGluR’s / iGluR modulatorsEarly 90’s - Cloning of mGluR’s / iGluR AB’sMid 90’s - mGluR agonists / mGluR AB’sLate 90’s - mGluR antagonistsEarly 00’s - 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”- activate“non-synaptic”GluRs ?- 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 are“asymetric”much 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-insensitive”binding 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 mGluR

II / III

Gq

GluSynapsin I

GAP-43:CaM

CaM

CaMKII

PP-2B

PresynapticTerminal

Actin

mGluR5

Gq/11

PLC-1PIP2

IP3

DAG

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

1500

2500

3500

0

1000

2000

3000

+ 100 M MPEP

0

CHPG-Potentiation of Evoked D-ASP Release

CHPG M0.5 2 3 5 20 30 50 200300 5001 10 100 1000

Pot

entia

tion

of E

voke

d [3 H

]-D-A

SP

Rel

ease

( S

2/S

1 )

0.25

0.75

1.25

1.75

2.25

0.00

0.50

1.00

1.50

2.00

+ 100 M MPEP

0

Glutamate Receptors on Glia

AMPA, KA, NMDA, mGluR Groups I & II

Glutamate effects on glia - EPSP’s ↑ iCa++

Modulate glial glutamate uptake

Modulate glial K+ permeability

Release neuroactive substancesGlutamateTrophic factors & Neurosteroids

Alter gene transcription in glia

Aspartate as a Neurotransmitter ?

• Synthesis ?• Storage ( Stored w/ Glu / varied ratios )• Release• Termination of Action ( Reuptake ? )• Receptor Actions

- AMPA / KA No- NMDA Yes

• Homocysteate also activates NMDA receptors

Putative Mechanisms for Therapeutic Applications

1. Glutamate reuptake inhibition ?

2. Glutamate agonists / antagonists ?

3. Allosteric modulation of receptors ?

4. Modulation of glutamate release ?

Putative Therapeutic Applications of AgentsAffecting Glutamatergic Neurotransmission

1. Neuroprotection iGluR antagonistsnegative allosteric modulators

Neuronal Cell Death after IschemiaNormal

Ischemia

Siegel: Fig. 32-4

Putative Therapeutic Applications of AgentsAffecting Glutamatergic Neurotransmission

1. Neuroprotection iGluR antagonistsnegative allosteric modulators

2. Anticonvulsants iGluR antagonistsNegative allosteric modulators

3. Analgesia mGluR Group I antagonistsmGluR Group II agonists

4. Substance Abuse mGluR5 antagonists

5. Schizophrenia Group II mGluR agonists

6. Cognition Enhancers iGluR positive allosteric modulatorsmGluR Group I positive modulators

Principal Synaptic Inputs to the Dentate Granule Cell

Targets for Intervention:

1. Receptors on Granule Cell Dendrites- Positive allosteric modulators of AMPA receptors- Positive allosteric modulators of NMDA receptors

2. Receptors on Entorhinal Cortical Nerve Terminals- Group II/III mGluR autoreceptors- Group I mGluR receptors- Heterologous neurotransmitter receptors

DentateGranule

Cell(Glu)

EntorhinalCorticalNeuron(Glu)

Basket CellInterneuron

(GABA)

MedialSeptalNeuron(ACh)

Receptors on Glutamate Nerve Terminals

Increase Glutamate Release

Group I mGluRs (1 & 5)

7 and 4/2 containing NCRsSerotonin 5HT3 receptors

Decrease Glutamate Release

Group II mGluRs ( 2 & 3)Group III mGluRs (4, 6, 7, 8)

Histamine H3 receptorsSerotonin 5HT4 and 5HT6 receptors

PresynapticTerminal

Glu Glu Glu

Histamine H3 ReceptorsInhibit adenylate cyclaseDecrease glutamate release

Regulation of Glutamate Release

H3 mGluRII / III

HistamineABT-239

mGluR Group II (2 & 3)Inhibit adenylate cyclaseDecrease glutamate release

LY 341495

log [ ABT-239 ] (nanomolar)3 30 300 300010 100 1000 10000

Evo

ked

[3 H]-D

-Asp

arta

te R

elea

se (%

)0.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0

Frontal Cortical SlicesWhole Hippocampal Slices

0

Fraction Intervals ( minutes )0 6 12 18 24 30 36 42 48 54 60

Frac

tiona

l [3 H

]-D-A

SP

Rel

ease

( %

)

1.5

2.0

2.5

3.0

1.5

2.0

2.5

3.0Control ABT-239 (100 nM)

S1 S2

Effect of ABT-239 on electrically-evoked [3H]-D-asparate release