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Glutamate Receptor Ion Channels: Structure, Regulation, and Function. Department of Physiology, Shandong University School of Medicine ( Shu Yan Yu ). glutamate receptor (GluR) is the most important excitatory transmitter in the CNS. Ionotropic Glutamate Receptors. AMPA. KA. NMDA.

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Glutamate receptor ion channels structure regulation and function

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

Department of Physiology, Shandong University

School of Medicine

(Shu Yan Yu)


Glutamate receptor glur is the most important excitatory transmitter in the cns
glutamate receptor (GluR) Function is the most important excitatory transmitter in the CNS


Ionotropic Glutamate Receptors Function

AMPA

KA

NMDA


  • NMDA receptor: Function

    NR1/NR2A; NR1/NR2B; NR1/NR2A/2B

    AMPA receptor:

    GluR1/GluR2; GluR2/GluR3


Structure ionotropic glutamate receptors
Structure FunctionIonotropic Glutamate Receptors

AMPA-R

NMDA-R


Binding sites for agonists, antagonists, and modulators in the ligand binding domain(LBD), amino terminal domain (ATD), and transmembrane domain (TMD)


Transmembrane topology (A) and crystal structure of the agonist-binding domain (B–D) of the GluA2 subunit protein


GluA2 subunit protein agonist-binding domain (B–D) of the GluA2 subunit protein


GluA2 subunit protein agonist-binding domain (B–D) of the GluA2 subunit protein


Na agonist-binding domain (B–D) of the GluA2 subunit protein+

Ca2+

redox site

glycosylation

site

S

S

N

H+ site

Polyamine

site

Glu

glycine coagonist site

Zn2+ site

MK-801,

PCP site

Mg2+

4

3

1

2

Cytoplasmic

Cytoplasmic

C

Scaffolding/

Signalling Proteins

P

P

P

K+

phosphorylation site

P

P

P

P

NR2

NR1


After agonist-binding domain (B–D) of the GluA2 subunit protein

Stimulation

During

Stimulation

Before

Stimulation

Neuron A

Neuron A

Neuron A

Glutamate

Glutamate locks

into receptor

Ca2+

Mg2+ block

relieved

_ _ _

_ _ _

+

+

+

+

+

+

_ _ _

_ _ _

+

+

+

+

+

+

NMDA receptor

blocked by Mg2+

Ca2+ flows through

NMDA receptor

Neuron B

Neuron B

Neuron B

Glutamate release / Depolarization


Nr2 subunit determines the functional properties of nmdar
NR2 agonist-binding domain (B–D) of the GluA2 subunit proteinsubunit determines the functional properties of NMDAR

Monyer et al. (1994), Neuron, 12, 529-540


A agonist-binding domain (B–D) of the GluA2 subunit protein

B

3

3

25 pA

25 pA

2

2

1

1

50 pA

100 ms

25 pA

C

100 ms

Inhibition of NMDAR-EPSC (%)

NVP first

Ro first

NVP-AAM077

NVP-AAM077

Ro25-6981

Ro25-6981

Yu et al. Neuroscience. 2010


Monyer et al. 2012 agonist-binding domain (B–D) of the GluA2 subunit protein


Function agonist-binding domain (B–D) of the GluA2 subunit protein

Role in Synaptic Function and Plasticity


  • Two important types of agonist-binding domain (B–D) of the GluA2 subunit proteinsynaptic plasticity:

    Long-term potentiation (LTP) ;

    Long-term depression (LTD) ;

  • They are two potential mechanism that underlie

    learning and memory


Bidirectional synaptic plasticity in the hippocampus
Bidirectional synaptic plasticity in the agonist-binding domain (B–D) of the GluA2 subunit proteinhippocampus


hippocampus agonist-binding domain (B–D) of the GluA2 subunit protein

tetanus

1 hr

200

EPSP %

100


Amygdala
Amygdala agonist-binding domain (B–D) of the GluA2 subunit protein

SAH et al. Physiol Rev. Vol 83. P813


Fear conditioning
Fear conditioning agonist-binding domain (B–D) of the GluA2 subunit protein

一朝被蛇咬,十年怕井绳


1 the method for recording
1. The method for recording: agonist-binding domain (B–D) of the GluA2 subunit protein


Brain Slice Recording agonist-binding domain (B–D) of the GluA2 subunit protein


Whole-cell Patch Recording agonist-binding domain (B–D) of the GluA2 subunit protein

Action Potential/current, EPSP/EPSC, IPSP/IPSC

Advantage:

. Single cell recording

. Record currents through multiple channels at once

. Can do both current clamp and voltage clamp

. lower access resistance & easier to clamp

. Bigger response

. Easy to apply compound intracellularly & modify intracellular component & pathway

. Using membrane impermeable drug can distinguish post/pre-synaptic effect

Disadvantage:

. Dilute cytoplasmic components ("dialyzing“ the cell's contents)

. Hard to get stable & long last recording

There is a "grace period" at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements before the cell has been dialyzed.


Vibrating Blade Microtome agonist-binding domain (B–D) of the GluA2 subunit protein

Cuts ultra-thin (100-400 µm) brain slices for electrophysiological and imaging studies.


Visualized Patch agonist-binding domain (B–D) of the GluA2 subunit protein

Patch procedure

Cleaning procedure


Blind Patch agonist-binding domain (B–D) of the GluA2 subunit protein

A small repetitive current or voltage pulse is applied to the electrode at relatively high frequency (e.g., 10 Hz) and the voltage or current response is monitored with anoscilloscope



100pA

50ms

E(I)PSP vs E(I)PSC

0.5 mV

50 ms

EPSP ------ Excitatory Post Synaptic Potential

IPSP ------ Inhibitory Post Synaptic Potential

Measured By Current Clamp

EPSC ------ Excitatory Post Synaptic Current

IPSC ------ Inhibitory Post Synaptic Current

Measured By Voltage Clamp


2 results
2. Results:

(一)Role of NMDA Receptors in LTP/LTD induction


Hfs induction of ltp is nmdar dependent apv nmda r antagonist blocked the induction of ltp
HFS induction of LTP is NMDAR-dependent, APV (NMDA-R antagonist)blocked the induction of LTP

Yu et al. Journal of Neurochemistry. 2008


Apv blocked the induction of ltd by pairing protocol
APV blocked the induction of LTD by Pairing protocol

Yu et al. Journal of Neurochemistry. 2008


Why one receptor leads to two Bidirectional synaptic plasticity ---

LTP and LTD ?

Many Hypothesis


Nr2 subunit determines the functional properties of nmdar1
NR2 subunit determines the functional properties of NMDAR

Monyer et al. (1994), Neuron, 12, 529-540


Nr2a antagonist nvp block the induction of ltp
NR2A antagonist NVP block the induction of LTP

NVP-AAM077 (0.4uM, NVP: NR2A antagonist) block the induction of LTP.

Dalton et al. Neuropharmacology 2012


Nr2b antagonist ro25 6981 can t block the induction of ltp
NR2B antagonist Ro25-6981 can’t block the induction of LTP

Dalton et al. Neuropharmacology 2012


Nr2a antagonist nvp can t block the induction of ltd
NR2A antagonist NVP can’t block the induction of LTD

Dalton et al. Neuropharmacology 2012


Nr2a 2b antagonist as a pharmacological tool to investigate the physiological role of ltp ltd
NR2A/2B antagonist as a pharmacological tool to investigate the physiological role of LTP/LTD


Role of ampa receptors in ltp ltd induction
二)Role of AMPA Receptors in LTP/LTD induction

AMPA-R

NMDA-R


NMDA

AMPA

Glu

NMDA

20

pA

AMPA

AMPA NMDA

20 ms


Long-Term

Potentiation

control


Bidirectional synaptic plasticity in the hippocampus1
Bidirectional synaptic plasticity in the hippocampus


Pre-synaptic

terminal

NMDA receptors

AMPA receptors

?

AMPA receptor-containing

secretory vesicles

Post-synaptic

neuron

Synthesis

Degradation?

AMPA receptor-containing

clathrin-coated vesicles


Bidirectional hippocampal synaptic plasticity

Hypothesis:

Bidirectional hippocampal synaptic plasticity


TeTx prevent the expression of LTP in LA

Yu et al. Journal of Neurochemistry. 2008


GluR2-3Y prevent the LTD expression in LA

Yu et al. Journal of Neurochemistry. 2008


Increased expression of AMPA receptors in membrane surface of LA neurons during the induction of LTP

Yu et al. Journal of Neurochemistry. 2008


Ca of LA neurons during the induction of LTP 2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

PSD-95

Ras-GTP

p38

Ras-GDP

Possible Signal Cascade Involved in the Expression of LTP and LTD

NR1/NR2A/NR2B

NR1/NR2A

NR1/NR2B

VGCC

AMPAR

AMPAR

PI3K

Rap-GDP

LTP

CaMKII

RasGRF1

SynGAP

ERK1/2

LTD

AKT

P

Rap-GTP

P

RasGRF2

MEK

P

Mitochondria

CREB

Cell Death

Cell Survival


Synaptic Plasticity of LA neurons during the induction of LTP

(LTP/LTD)

Learning

Memory

Behaviour

(Stress)

output

Behaviour Results


The forced swim test
The forced swim test of LA neurons during the induction of LTP

  • The forced swim test is a predictive model widely used for assessing antidepressant efficacy.

  • The immobility time of animals in the cylinder is interpreted as representing despair or a depression-like state which can be shortened by repeated antidepressant treatment.


## of LA neurons during the induction of LTP

**

**

**

Vehicle

+

+

+

+

-

-

Curcumin

-

-

+

-

-

+

-

-

-

-

+

+

Fluoxetine

-

-

-

+

+

+

CCP

Pre-treatment with CPP (a competitive NMDA receptor antagonist) decreased anti-depressant effects of curcumin and fluoxetine.

Yu et al. Prog Neuropsychopharmacol Biol Psychiatry. 2013


** of LA neurons during the induction of LTP

**

+

+

-

+

Vehicle

Curcumin

-

-

+

+

-

+

-

+

NVP-AAM077

Pre-treatment with NVP-AAM077 (a GluN2A-prefering antagonist) can’t prevent the anti-depressant effects of curcumin.

Yu et al. Prog Neuropsychopharmacol Biol Psychiatry. 2013


## of LA neurons during the induction of LTP

**

+

-

Vehicle

+

+

Curcumin

-

-

+

+

+

-

+

-

Ro25-6981

Pre-treatment with Ro25-6981 (a specific GluN2B antagonist) prevent the anti-depressant effects of curcumin.

Yu et al. Prog Neuropsychopharmacol Biol Psychiatry. 2013


Thank you of LA neurons during the induction of LTP


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