Ligand-Gated Ion Channels

1. Ligand-Gated Ion Channels Genevieve Bell, ErminiaFardone, Kirill Korshunov Membrane Biophysics – Fall 2014

2. Tertiary Primary Secondary Quaternary

7. Na+ L + R

8. Na+ Na+ L + R LR

9. Na+ Na+ O Na+ L + R LR

10. Na+ Na+ Na+ O Na+ L + R LR D

13. O L + R LR D

14. 100 µM Zinc 50 µM AMPA 20 pA 5 s 100 µM Zinc 50 µM AMPA 50 pA 5 s 50 µM AMPA 100 µM Zinc 50 pA 5 s

17. Control

18. Zinc Control

19. Threshold for action potential Zinc Control

20. Zinc Threshold for action potential Control

22. Introduction • Phosphorylated Ligand Gated Ion Channels (pLGICs) include: • nAChRs • GABAARs • GlyRs • 5-HT3Rs • Best known for mediating fast neurotansmission in nervous system

23. Introduction • Phosphorylation is well known to influence synaptic function by directly modulating pLGICs • Implicated in various disorders and can elicit a wide variety of effects • Understanding the structural basis of these effects  design of specifically targeted drugs to treat pathological receptor modification

24. pLGIC Architecture • Pentameric assemblies of identical or different subunits • 5 subunits together form a central water filled pore • Each subunit can be divided into three domains • Transmembrane α-helices form concentric rings around a central pore, directly lined by 5 M2 helices

25. M3-M4 Cytoplasmic Domain • M3-M4 domain is poorly conserved in length and AA sequence and therefore exhibits structural variation • Interactions between M3-M4 loops and other proteins or ions are known to modulate pLGIC activity, assembly, and trafficking • M3-M4 domain is the only region known to house phosphorylation sites

26. Receptor Phosphorylation

27. Biological Function

28. Chronic Inflammatory Pain • The α3-glycine receptor (α3 GlyR) is prominent in the spinal cord • Lamina I and II nociceptive neurons • Phosphorylation at serine346 attributed to chronic inflammation α3-Glycine receptor

29. Mechanism of Inflammation Sensitization • Prosteglandin 2 (PGE2) activates PGE2 receptor • PGE2 stimulates adenylyl cyclase to produce more cAMP • cAMP activate cAMP-dependent protein kinase A (PKA) • PKA phosphorylates ser346 residue, causing a block in the IPSCs produced by glycine • Ultimately, this leads to a sensitization in nociception in the spinal cord lamina I and II neurons

30. Zeilofer HU., 2005

31. Other Disorders Implicated in Phosphorylation of pLGICs • Alcoholism • Implicated in GABAARs • PKC inhibits GABAARs IPSCs • Nicotine addiction • Implicated in α4β2 nAChRs • Phosphorylation/de-phosphorylation lead to receptor desensitization at the Ser368 residue • Continuous nicotinic exposure leads to permanent receptor desensitization

32. Myasthenia gravis • Implicated in muscle AChRs • PKA phosphorylates γ and δ subunits • PKC phosphorylates α and δ subunits • PTK phosphorylates β, γ, and δ subunits

33. Summary • Nociception sensitization occurs in α3 GlyRs, caused by phosphorylation of the ser346 residue • This is a possible mechanism for chronic inflammation • Alcoholism is attributed to GABAAR phosphorylation • Nicotine addiction is attributed to desensitization of α4β2nAChRs • Continuous nicotinic exposure leads to permanent channel desensitization • Phosphorylation of different muscle AChRs subunits could lead to myasthenia gravis

34. Possible medical application

35. Inappropriate phosphorilation Global allosteric conformational change Neurological disorders Chronic pain: PKA-mediated phosphorilation α3 GlyRs inhibit current and causes a conformational change of the gly-binding site. Alcholism: Protein phosphorilation can casue an increase in ethanol sensitivity of γ2-containing GABAARs.

36. Conformational changes in pLGICs can be targeted by drugs to treat several diseases.

37. GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms Christine Dixon, Pankaj Sah, Joseph W. Lynch, and Angelo Keramidas Queensland Brain Institute, University of Queensland, Australia

38. Introduction GABAA Receptors • Mediate the majority of inhibitory neurotransmission in the mammalian brain • Pentamers : Consist of two α, two β, and a γ subunit • 6 different α subunits • 4 different β subunits • 3 different γ subunits • GABAA R that contain a variety of subunits are expressed throughout the brain

40. Inhibitory Postsynaptic Currents • - IPSCs at GABA-ergic are determined by: • The biophysical properties of postsynaptic receptors • How receptors are clustered at the postsynaptic membrane • - α subunit = Key determinant of the functional properties of GABAA R

41. The Amygdala • Plays a key role in processing fear • Dysfunction associated with anxiety-related disorders • Disorders are typically managed via benzodiazepines Benzodiazepines • Enhances the action of GABA at GABAA R containing γ2 subunits • Acts indiscriminately on GABAA R throughout the brain, producing side effects such as tolerance and sedation

42. α1 and γ2 subunits are expressed throughout the CNS, while the α2 and γ1 subunits have restricted distribution : • Amygdala • Forebrain • Cerebellum • Hypothalamus • Pallidum • Substantia Nigra Properties of receptors containing α1 and γ2 subunits and their impact on synaptic currents are very well known, in contrast nothing is known regarding the impact of γ1 containing GABAA R on inhibitory synaptic current

43. Experimental Procedures • Cell Culture and Molecular Biology • Subunits were transfected into HEK293 cells • Primary neuronal cell culture • Immunofluorescent Labeling • Electrophysiology • Patch-clamp: Outside-out and macropatch

49. Effects of Zn2+ on wild and mutant neuronal α7 nicotinic receptors E. Palma, L. Maggi, and F. Eusebi PNAS 1998

50. Introduction • α7 nAChR is a ligand-gated ion channel largely present in the hippocampus and the retina. • Receptor dysfunction linked to epileptic seizures and schizophrenia. • A mutated form of α7 (L247Tα7) exhibits spontaneous inward currents in the absence of ACh. • Zn2+is also largely found in the hippocampus and retina. • How does Zn2+ affect α7 nAChRs? • Influence on the spontaneous currents? α7-nicotinic acetylcholine receptor