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  • 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

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