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

Structure Ionotropic Glutamate Receptors AMPA-RNMDA-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

P P P P Mg2+ Cytoplasmic Glu Cytoplasmic P P P K+K+ Na + Ca 2+ Zn 2+ site glycosylation site H + site Polyamine 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 B NMDA receptor blocked by Mg2+ Glutamate locks into receptor Ca2+ flows through NMDA receptor Ca2+ Glutamate Glutamate release / Depolarization

NR2 subunit determines the functional properties of NMDAR Monyer et al. (1994), Neuron, 12,

Inhibition of NMDAR-EPSC (%) NVP-AAM077 Ro NVP-AAM077 NVP firstRo first C 100 ms 50 pA 25 pA ms 25 pA AB 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

hr EPSP % 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 Recording 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. Action Potential/current, EPSP/EPSC, IPSP/IPSC

Cuts ultra-thin ( µm) brain slices for electrophysiologi cal and imaging studies.

Cleaning procedure Patch procedure Visualized Patch

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 100pA 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 Yu et al. Journal of Neurochemistry. 2008

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 NMDAR Monyer et al. (1994), Neuron, 12,

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 Ro can’t block the induction of LTP 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 NMDA 20 ms NMDA AMPA 20 pA NMDA AMPA

Long-Term Potentiation control

Bidirectional synaptic plasticity in the hippocampus

AMPA receptors NMDA receptors AMPA receptor-containing secretory vesicles AMPA receptor-containing clathrin-coated vesicles ? Degradation?Synthesis Pre-synaptic terminal Post-synaptic neuron

Bidirectional hippocampal synaptic plasticity Hypothesis:

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

Possible Signal Cascade Involved in the Expression of LTP and LTD PSD-95 SynGAP CaMKII Ras-GDP Ras-GTP Ca 2+ MEK RasGRF2 ERK1/2 P LTP RasGRF1 CREB P PI3K AKT LTD p38 Cell Death Cell Survival Mitochondria VGCC AMPAR NR1/NR2A NR1/NR2A/NR2B NR1/NR2B AMPAR P Rap-GDP Rap-GTP

(Stress) Behaviour Synaptic Plasticity (LTP/LTD) Learning Memory output 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.

** ＃＃ ** Vehicle Curcumin Fluoxetine CCP Yu et al. Prog Neuropsychopharmacol Biol Psychiatry Pre-treatment with CPP (a competitive NMDA receptor antagonist) decreased anti-depressant effects of curcumin and fluoxetine.

** Vehicle Curcumin NVP-AAM Yu et al. Prog Neuropsychopharmacol Biol Psychiatry Pre-treatment with NVP-AAM077 (a GluN2A-prefering antagonist) can’t prevent the anti-depressant effects of curcumin.

** ＃＃ Vehicle Curcumin Ro Yu et al. Prog Neuropsychopharmacol Biol Psychiatry Pre-treatment with Ro (a specific GluN2B antagonist) prevent the anti-depressant effects of curcumin.