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Ligand-Gated Ion Channels Genevieve Bell, Erminia Fardone, Kirill Korshunov Membrane Biophysics Fall 2014 Slide 2 Primary Secondary Tertiary Quaternary Slide 3 Slide 4 Slide 5 Slide 6 Slide 7 L + R Na+ Slide 8 Na+ LRNa+ Slide 9 Na+ LR O Na+Na+ Slide 10 Na+ LR O D Na+Na+ Na+ Slide 11 Slide 12 Slide 13 LR O D Slide 14 100 M Zinc 50 M AMPA 100 M Zinc 50 M AMPA 20 pA 50 pA 5 s Slide 15 Slide 16 Slide 17 Control Slide 18 Control Zinc Slide 19 Threshold for action potential Control Zinc Slide 20 Control Zinc Slide 21 Slide 22 Introduction Phosphorylated Ligand Gated Ion Channels (pLGICs) include: nAChRs GABA A Rs GlyRs 5-HT3Rs Best known for mediating fast neurotansmission in nervous system Slide 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 Slide 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 -helices form concentric rings around a central pore, directly lined by 5 M2 helices Slide 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 Slide 26 Receptor Phosphorylation Slide 27 Biological Function Slide 28 Chronic Inflammatory Pain The 3-glycine receptor (3 GlyR) is prominent in the spinal cord Lamina I and II nociceptive neurons Phosphorylation at serine346 attributed to chronic inflammation 3-Glycine receptor Slide 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 Slide 30 Zeilofer HU., 2005 Slide 31 Other Disorders Implicated in Phosphorylation of pLGICs Alcoholism Implicated in GABA A Rs PKC inhibits GABA A Rs IPSCs Nicotine addiction Implicated in 42 nAChRs Phosphorylation/de- phosphorylation lead to receptor desensitization at the Ser368 residue Continuous nicotinic exposure leads to permanent receptor desensitization Slide 32 Myasthenia gravis Implicated in muscle AChRs PKA phosphorylates and subunits PKC phosphorylates and subunits PTK phosphorylates , , and subunits Slide 33 Summary Nociception sensitization occurs in 3 GlyRs, caused by phosphorylation of the ser346 residue This is a possible mechanism for chronic inflammation Alcoholism is attributed to GABA A R phosphorylation Nicotine addiction is attributed to desensitization of 42 nAChRs Continuous nicotinic exposure leads to permanent channel desensitization Phosphorylation of different muscle AChRs subunits could lead to myasthenia gravis Slide 34 Possible medical application Slide 35 Inappropriate phosphorilation Neurological disorders Global allosteric conformational change Chronic pain: PKA-mediated phosphorilation 3 GlyRs inhibit current and causes a conformational change of the gly-binding site. Alcholism: Protein phosphorilation can casue an increase in ethanol sensitivity of 2- containing GABA A Rs. Slide 36 Conformational changes in pLGICs can be targeted by drugs to treat several diseases. Slide 37 GABA A Receptor and Subunits Shape Synaptic Currents via Different Mechanisms Christine Dixon, Pankaj Sah, Joseph W. Lynch, and Angelo Keramidas Queensland Brain Institute, University of Queensland, Australia Slide 38 Introduction GABA A Receptors -Mediate the majority of inhibitory neurotransmission in the mammalian brain -Pentamers : Consist of two , two , and a subunit -6 different subunits -4 different subunits -3 different subunits -GABA A R that contain a variety of subunits are expressed throughout the brain Slide 39 Slide 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 - subunit = Key determinant of the functional properties of GABA A R Slide 41 The Amygdala Plays a key role in processing fear Dysfunction associated with anxiety- related disorders Disorders are typically managed via benzodiazepines Enhances the action of GABA at GABA A R containing 2 subunits Acts indiscriminately on GABA A R throughout the brain, producing side effects such as tolerance and sedation Benzodiazepines Slide 42 1 and 2 subunits are expressed throughout the CNS, while the 2 and 1 subunits have restricted distribution : Amygdala Forebrain Cerebellum Hypothalamus Pallidum Substantia Nigra Properties of receptors containing 1 and 2 subunits and their impact on synaptic currents are very well known, in contrast nothing is known regarding the impact of 1 containing GABA A R on inhibitory synaptic current Slide 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 Slide 44 Slide 45 Slide 46 Slide 47 Slide 48 Slide 49 Effects of Zn 2+ on wild and mutant neuronal 7 nicotinic receptors E. Palma, L. Maggi, and F. Eusebi PNAS 1998 Slide 50 Introduction 7 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 7 ( L247T 7) exhibits spontaneous inward currents in the absence of ACh. Zn 2+ is also largely found in the hippocampus and retina. How does Zn 2+ affect 7 nAChRs? Influence on the spontaneous currents? 7-nicotinic acetylcholine receptor Slide 51 Methods and Materials Model organism = Xenopus oocytes. cDNA (for either WT 7 or L247T 7) was injected into the nuclei of stage 6 oocyte. Electrophysiology Two-four days after cDNA injection Voltage-clamp ACh applied at 3 min intervals Zn 2+ from ZnCl 2 and Zn 2+ acetate nAChR blockers methyllycaconitine (MLA) and - bungarotoxin (Bgt) Slide 52 Zincs effect on WT 7 ACh current (I ACh ) Slide 53 Zn 2+ Blocks WT 7 I ACh [Zn 2+ ] = 10 nM to 10 mM [ACh] = 150 M Slide 54 Zn 2+ Blocks WT 7 I ACh Voltage- Independently [Zn 2+ ] = 30 M [ACh] = 150 M [Zn 2+ ] = 20 M Slide 55 L247T 7 and the zinc current (I Zn ) Slide 56 Zn 2+ Induces Currents in L247T 7 [Zn 2+ ] = 1 mM [ACh] = 0.2 M [MLA] = 1 M [BuTx ] = 100 nM Slide 57 The Dual Role of Zn 2+ on L247T 7 [Zn 2+ ] = 10 fM to 10 mM Slide 58 I Zn on L247T 7 Mimics I ACh on WT 7 [Zn 2+ ] = 10 nM @ +45 mV @ -100 mV I ACh on WT 7 Slide 59 L247T 7 has one Zn 2+ -Gated Channel Pore [Zn 2+ ] = 10 nM @ - 63 mV Slide 60 Zn 2+ -induced Modulation of I ACh in L247T 7 Slide 61 Zn 2+ Modulation of I ACh in L247T 7 For A + C: [Zn 2+ ] = 1 mM For B: [Zn 2+ ] = 10 mM Slide 62 Conclusion WT 7 Pretreatment (20-30 s) of Zn 2+ blocks I ACh Blockage increases with [Zn 2+ ] Blockage is voltage-independent L247T 7 Zn 2+ produces its own current (I Zn ) Zn 2+ acts as an agonist at low concentrations (10 fM- 10 nM) Acts as an antagonist at higher concentrations (10