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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS034727-16A1
Application #
8373037
Study Section
Special Emphasis Panel (ZRG1-MDCN-N (04))
Program Officer
Silberberg, Shai D
Project Start
1996-12-01
Project End
2017-02-28
Budget Start
2013-04-01
Budget End
2014-02-28
Support Year
16
Fiscal Year
2013
Total Cost
$511,989
Indirect Cost
$163,420
Name
University of Wisconsin Madison
Department
Neurosciences
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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
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
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
Boileau, Andrew J; Pearce, Robert A; Czajkowski, Cynthia (2010) The short splice variant of the gamma 2 subunit acts as an external modulator of GABA(A) receptor function. J Neurosci 30:4895-903
Hanson, Susan M; Czajkowski, Cynthia (2008) Structural mechanisms underlying benzodiazepine modulation of the GABA(A) receptor. J Neurosci 28:3490-9
Mercado, Jose; Czajkowski, Cynthia (2008) Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions. J Biol Chem 283:15250-7
Hanson, Susan M; Morlock, Elaine V; Satyshur, Kenneth A et al. (2008) Structural requirements for eszopiclone and zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) receptor are different. J Med Chem 51:7243-52
Sharkey, L M; Czajkowski, C (2008) Individually monitoring ligand-induced changes in the structure of the GABAA receptor at benzodiazepine binding site and non-binding-site interfaces. Mol Pharmacol 74:203-12
Venkatachalan, Srinivasan P; Czajkowski, Cynthia (2008) A conserved salt bridge critical for GABA(A) receptor function and loop C dynamics. Proc Natl Acad Sci U S A 105:13604-9

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