glutamate receptor ion channels: structure, regulation, and function
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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
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.