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

Ionotropic Glutamate Receptors

AMPA

NMDA

KA

NMDA receptor:

NR1/NR2A; NR1/NR2B;

NR1/NR2A/2B

AMPA receptor:

GluR1/GluR2; GluR2/GluR3

StructureIonotropic 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

GluA2 subunit protein

P

P

P

P

Mg2+

Cytoplasmic

21 34

Glu

Cytoplasmic

PPP

K+

Na+

Ca2+

Zn2+ site

glycosylationsite

H+ sitePolyamine site

glycine coagonist site

redox site

S S

MK-801,PCP site

phosphorylation site

N

C

Scaffolding/Signalling Proteins

NR1 NR2

Before Stimulation

During Stimulation

After Stimulation

Mg2+ block relieved_ _ _ _ _ _

+ ++ + +++ ++ + ++

_ _ _ _ _ _

Neuron A Neuron A Neuron A

Neuron B Neuron B Neuron B

NMDA receptor

blocked by Mg2+

Glutamate locks into receptor

Ca2+ flows throughNMDA receptor

Ca2+

Glutamate

Glutamate release / Depolarization

NR2 subunit determines the functional properties of

NMDAR

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

Inh

ibit

ion

of

NM

DA

R-E

PS

C (

%)

NVP-

AAM077 Ro25-

6981 Ro25-

6981 NVP-

AAM077

NVP first Ro first

0

20

40

60

80

100NVP-AAM077Ro25-6981

C100 ms

50 pA

25

pA

1

2

3

100 ms25 pA

25

pA

1

2

3

A B

Yu et al. Neuroscience. 2010

Monyer et al. 2012

Function

Role in Synaptic Function and Plasticity

Two important types of synaptic 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

100

200 1 hr

EP

SP

%

tetanus

hippocampus

Amygdala

SAH et al. Physiol Rev. Vol 83. P813

Fear conditioning

一朝被蛇咬，十年怕井绳

1. The method for recording:

Whole-cell Patch RecordingAdvantage: . 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.

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

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

Cleaning procedure Patch procedure

Visualized Patch

1. Approaching 2. Attaching 3. Seal formation 4. Rupture of membrane

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

Blind Patch

whole cell patch clamp recording

were used to record evoked

EPSC/IPSC or EPSP/IPSP in coronal

slice.

E(I)PSP vs E(I)PSC

EPSP ------ Excitatory Post Synaptic Potential

IPSP ------ Inhibitory Post Synaptic Potential

Measured By Current Clamp

Measured By Voltage Clamp

0.5

mV

50 ms

EPSC ------ Excitatory Post Synaptic Current

IPSC ------ Inhibitory Post Synaptic Current

10

0p

A

50ms

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

2. Results:

HFS induction of LTP is NMDAR-dependent, APV (NMDA-R antagonist) blocked the induction of LTP

Control APV

40 mS

10

0 p

A

40 mS

10

0 p

A

1

2

1

2

ControlAPV

-10 0 10 20 30 40 50 60

Time (min)

0.6

1.0

1.4

1.8

2.2

Nor

mal

ized

EP

SC

Am

plitu

de

ControlAPVControlAPV

-10 0 10 20 30 40 50 60

Time (min)

0.6

1.0

1.4

1.8

2.2

Nor

mal

ized

EP

SC

Am

plitu

de

HFS

1 2

Yu et al. Journal of Neurochemistry. 2008

APV blocked the induction of LTD by Pairing

protocol Control APV

40 mS

10

0 pA

40 mS

10

0 pA

1

2

1

2

-10 0 10 20 30 40 50 600.2

0.4

0.6

0.8

1.0

1.2

ControlAPVControlAPV

Time (min)

Nor

mal

ized

EP

SC

Am

plitu

de

Pairing

12

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

NMDAR

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

NR2A antagonist NVP block the induction of LTP

00.2

0.40.6

0.81

1.21.41.61.8

2

0 10 20 30 40 50 60Time (min)

EP

SC

Am

plit

ude

(100

%)

NVP 0.4 uMControl

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

Time (min)

EP

SC

Am

plitu

de (

100%

)

R o25-6981 3uM

C ontro lR o25-6981 0 .5uM

Dalton et al. Neuropharmacology 2012

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

（二）Role of AMPA Receptors in LTP/LTD induction

NMDA-RAMPA-R

Glu

AMPA NMDA20 ms

NMDA

AMPA

20pA

NMDA

AMPA

Long-TermPotentiation

control

Bidirectional synaptic plasticity in the hippocampus

AMPA receptors

NMDA receptors

AMPA receptor-containingsecretory vesicles

AMPA receptor-containingclathrin-coated vesicles

?

Degradation?Synthesis

Pre-synaptic terminal

Post-synaptic neuron

Bidirectional hippocampal synaptic plasticity

Hypothesis:

A

-10 0 10 20 30 40 50 60

0.6

1.0

1.4

1.8

2.2

ControlTeTxControlTeTx

No

rma

lize

d E

PS

C A

mp

litud

e

Time (min)

Control TeTx

40 mS

10

0 p

A

40 mS

10

0 p

A

Pairing

1

2

1

2

1 2

TeTx prevent the expression of LTP in LA

Yu et al. Journal of Neurochemistry. 2008

Control GluR2-3YControl GluR2-3Y

-10 0 10 20 30 40 50 600.2

0.4

0.6

0.8

1.0

1.2

ControlGluR2-3Y

No

rma

lize

d E

PS

C A

mp

litud

e

Time (min)

-10 0 10 20 30 40 50 600.2

0.4

0.6

0.8

1.0

1.2

ControlGluR2-3YControlGluR2-3Y

No

rma

lize

d E

PS

C A

mp

litud

e

Time (min)

Pairing

1

2

1

2

12

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

0.00.20.40.60.81.01.21.41.6

Nor

mal

ized

Sur

face

Exp

ress

ion *

*

Yu et al. Journal of Neurochemistry. 2008

Possible Signal Cascade Involved in the Expression of LTP and LTD

PSD-95SynGAP

CaMKII

Ras-GDP

Ras-GTP

Ca2+

Ca2+

Ca 2+

Ca 2+

Ca2+

Ca2+

Ca2+

Ca2

+

MEKRasGRF2

ERK1/2P

LTP

Ras

GR

F1

CREB

P

PI3K

AKT LTDp38

Cell DeathCell Survival

Mitochondria

VGCC

AMPAR

NR1/NR2ANR1/NR2A/NR2B

NR1/NR2B

AMPAR

P

Rap

-GD

P

Rap

-GT

P

(Stress)Behaviour

Synaptic Plasticity(LTP/LTD)

Learning Memoryoutput

Behaviour Results

The forced swim test

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.

Imm

obil

ity

tim

e (s

)

0

50

100

150

200

****

＃＃

**

Vehicle

Curcumin

Fluoxetine

CCP

+ + +

++

+

+ + +

+

+--

-

--

- -

--

- -

--

Yu et al. Prog Neuropsychopharmacol Biol Psychiatry. 2013

Pre-treatment with CPP (a competitive NMDA receptor antagonist)

decreased anti-depressant effects of curcumin and fluoxetine.

Imm

obili

ty t

ime

(s)

0

50

100

150

200

** **

Vehicle

Curcumin

NVP-AAM077

+ +

+

+

+ +

+

-

-

--

-

Yu et al. Prog Neuropsychopharmacol Biol Psychiatry. 2013

Pre-treatment with NVP-AAM077 (a GluN2A-prefering antagonist) can’t

prevent the anti-depressant effects of curcumin.

Imm

obil

ity

tim

e (s

)

0

50

100

150

200

**

＃＃

Vehicle

Curcumin

Ro25-6981

+ +

+

+

+ +

+

-

-

--

-

Yu et al. Prog Neuropsychopharmacol Biol Psychiatry. 2013

Pre-treatment with Ro25-6981 (a specific GluN2B antagonist) prevent

the anti-depressant effects of curcumin.