GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian CNS and are the site of action of benzodiazepines (BZDs). GABA analogues and BZDs are used in the treatment of a variety of neurological and psychiatric disorders, but the molecular structures of the GABA and BZD binding sites are unknown. The long-term goal of our researach program is to understand the function of the GABAA receptor in terms of its molecular structure. As a first step, we propose to identify and locate in the receptor structure the amino acid residues that form the GABA and BZD binding sites. An innovative approach, the substituted cysteine accessibility method, will be used to probe systematically the entire surface of the GABA and BZD binding site pockets of the GABAA receptor. This method can provide detailed molecular information about the structure of binding sites beyond that usually obtained using traitional mutagenesis or affinity labeling techniques. If the structure of the GABA and BZD binding sites were known in molecular detail, it is possible that whole neew classes of site-specific compounds would be discovered and existing compounds could be modified to exploit the physicochemical features of the binding site to yield higher affinity, more selective drugs. The substituted cysteine accessibility method is a combination of single, consecutive cysteine-substitution by site directed mutagenesis of wild-type amino acid residues, heterologous functional expression of the mutants, and the probing of the substituted cysteines with sulfhydry1-specific reagents. These reagents are small, charged, hydrophilic, and lipophobic, and thus reactonly at the water-accessible surface of the receptor. If binding to a cysteine substitution mutnt is irreversibly blocked by the sulfhydry1-specific reagents, and if this blockade of binding can be prevented by site-specific ligands, we would infer that the side chain of the corresponding wild-type residue lines the binding site pocket. The pattern of accessibility of consecutive engineered cysteines to reaction with the sulfhydry1-specific reagents reflects the secondary structure of these residues;e.g. alpha helix or beta sheet. Inmutants with abnormal function and binding, the accessibility of neighboring residues to reaction with the sulfhydry1-specific reagents will allow us to deduce the secondary structure of the region containing this residue,and thus determine whether or not the residue is exposedin the binding site. Thus, all residues which structurally form the binding site can be successfully mapped, and the 3-dimensional structure of the site can be potentially modeled. In the absence of an X-ray crystal structure, this biochemical aproach will provide, at a molecular level, the most detailed structural picture of the GABAA receptor yet available.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurological Sciences Subcommittee 1 (NLS)
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Baughman, Robert W
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University of Wisconsin Madison
Schools of Medicine
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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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