Glutamate Receptor Ion Channels: Structure, Regulation, and Function

1. Glutamate Receptor Ion Channels: Structure, Regulation, and Function Department of Physiology, Shandong University School of Medicine （Shu Yan Yu）

2. glutamate receptor (GluR) is the most important excitatory transmitter in the CNS

4. Ionotropic Glutamate Receptors AMPA KA NMDA

5. NMDA receptor: NR1/NR2A; NR1/NR2B; NR1/NR2A/2B AMPA receptor: GluR1/GluR2; GluR2/GluR3

6. StructureIonotropic Glutamate Receptors AMPA-R NMDA-R

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

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

11. GluA2 subunit protein

12. GluA2 subunit protein

13. Na+ 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

14. After 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

15. NR2subunit determines the functional properties of NMDAR Monyer et al. (1994), Neuron, 12, 529-540

16. A 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

17. Monyer et al. 2012

18. Function Role in Synaptic Function and Plasticity

19. Two important types of synaptic plasticity： Long-term potentiation (LTP) ; Long-term depression (LTD) ; • They are two potential mechanism that underlie learning and memory

20. Bidirectional synaptic plasticity in the hippocampus

21. hippocampus tetanus 1 hr 200 EPSP % 100

22. Amygdala SAH et al. Physiol Rev. Vol 83. P813

23. Fear conditioning 一朝被蛇咬，十年怕井绳

24. 1. The method for recording:

25. Brain Slice Recording

26. Whole-cell Patch Recording 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.

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

28. Visualized Patch Patch procedure Cleaning procedure

29. Blind 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

30. whole cell patch clamp recording were used to record evoked EPSC/IPSC or EPSP/IPSP in coronal slice.

31. 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

32. 2. Results: （一）Role of NMDA Receptors in LTP/LTD induction

33. HFS induction of LTP is NMDAR-dependent, APV (NMDA-R antagonist)blocked the induction of LTP Yu et al. Journal of Neurochemistry. 2008

34. APV blocked the induction of LTD by Pairing protocol Yu et al. Journal of Neurochemistry. 2008

35. Why one receptor leads to two Bidirectional synaptic plasticity --- LTP and LTD ? Many Hypothesis

36. NR2subunit determines the functional properties of NMDAR Monyer et al. (1994), Neuron, 12, 529-540

37. 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

38. NR2B antagonist Ro25-6981 can’t block the induction of LTP Dalton et al. Neuropharmacology 2012

39. NR2A antagonist NVP can’t block the induction of LTD Dalton et al. Neuropharmacology 2012

40. NR2A/2B antagonistas a pharmacological tool to investigate the physiological role of LTP/LTD

41. （二）Role of AMPA Receptors in LTP/LTD induction AMPA-R NMDA-R

42. NMDA AMPA Glu NMDA 20 pA AMPA AMPA NMDA 20 ms

44. Long-Term Potentiation control

45. Bidirectionalsynaptic plasticity in the hippocampus

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

47. Hypothesis: Bidirectional hippocampal synaptic plasticity

49. TeTxprevent the expression of LTP in LA Yu et al. Journal of Neurochemistry. 2008

50. GluR2-3Yprevent the LTD expression in LA Yu et al. Journal of Neurochemistry. 2008