Chemical signaling in the brain relies on rapid opening and closing of ligand-gated ion channels (LGICs) in the membranes of nerve cells. Members of the pentameric LGIC superfamily include nicotinic acetylcholine receptors (nAChR), serotonin-type-3 receptors (5HT3R), gamma-amino butyric acid type A receptors (GABAAR) and glycine receptors. Defects in these channels lead to a variety of neurological diseases and psychiatric disorders and a number of therapeutic drugs, including muscle relaxants, sedative-hypnotics, anti- convulsants, anxiolytics, intravenous and volatile anesthetics, anti-emetics, drugs for nicotine addiction and drugs to treat Alzheimer's disease target these channels. For these receptors, binding of neurotransmitter in the extracellular ligand-binding domain triggers opening of an intrinsic ion channel more than 50? away in the transmembrane domain of the receptor. Although we know a fair amount about the structure of these receptors, the mechanisms by which the binding of neurotransmitter triggers channel opening are still under debate and our understanding of the protein motions underlying this process limited. The general plan of this proposal is to investigate the binding-to-gating motions in the prokaryotic pLGIC homologs from Gloeobacter violaceus (GLIC) using site-directed spin labels and electron paramagnetic resonance spectroscopy (SDSL- EPR) and to test these motions in the GABAAR using an array of biochemical and electrophysiological approaches including voltage clamping, mutant cycle analysis, cysteine cross-linking, disulfide trapping and structural modeling. We will focus on three key regions: the extracellular binding domain (EBD), the gating interface and the transmembrane channel domain (TCD). These studies will build on our previous work and will provide new insights into how neurotransmitters activate pLGICs and how allosteric drugs modulate their activity. A deeper understanding of how these channels work at a molecular level will improve our ability to predict the actions of drugs and ligands that act on these channels, design safer and more effective drugs, develop better therapeutic strategies, and understand the etiology of disease-causing mutations.

Public Health Relevance

Ligand-gated ion channels are proteins that reside in the membranes of all nerve cells. These proteins form channels through the membrane to allow neurons to signal one another at synapses, and thus regulate information flow throughout the brain. Defects in these channels lead to wide variety of neurological diseases and psychiatric conditions and they are the targets of a large number of clinically used drugs. We cannot hope to predict the actions of a drug, design safer and more effective drugs, develop better therapeutic strategies or predict the outcome of a disease-causing mutation without knowledge of how these channels work at a molecular level. The successful completion of this project will advance our understanding of how these important channels work.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-N (04))
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Silberberg, Shai D
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University of Wisconsin Madison
Schools of Medicine
United States
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Ghosh, Borna; Tsao, Tzu-Wei; Czajkowski, Cynthia (2017) A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation. Neuropharmacology 125:343-352
Ding, Yun; Dellisanti, Cosma D; Ko, Mi Hee et al. (2014) The endoplasmic reticulum-based acetyltransferases, ATase1 and ATase2, associate with the oligosaccharyltransferase to acetylate correctly folded polypeptides. J Biol Chem 289:32044-55
Laha, Kurt T; Ghosh, Borna; Czajkowski, Cynthia (2013) Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel. PLoS One 8:e80322
Dellisanti, Cosma D; Ghosh, Borna; Hanson, Susan M et al. (2013) Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions. PLoS Biol 11:e1001714
Ghosh, Borna; Satyshur, Kenneth A; Czajkowski, Cynthia (2013) Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities. J Biol Chem 288:17420-31
Venkatachalan, Srinivasan P; Czajkowski, Cynthia (2012) Structural link between ?-aminobutyric acid type A (GABAA) receptor agonist binding site and inner ?-sheet governs channel activation and allosteric drug modulation. J Biol Chem 287:6714-24
Morlock, Elaine V; Czajkowski, Cynthia (2011) Different residues in the GABAA receptor benzodiazepine binding pocket mediate benzodiazepine efficacy and binding. Mol Pharmacol 80:14-22
Hanson, Susan M; Czajkowski, Cynthia (2011) Disulphide trapping of the GABA(A) receptor reveals the importance of the coupling interface in the action of benzodiazepines. Br J Pharmacol 162:673-87
Sancar, Feyza; Czajkowski, Cynthia (2011) Allosteric modulators induce distinct movements at the GABA-binding site interface of the GABA-A receptor. Neuropharmacology 60:520-8
Wagoner, Kelly R; Czajkowski, Cynthia (2010) Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected. J Biol Chem 285:14187-94

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