GABA/A 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 detailed molecular structures of the GABA and BZD binding sites are unknown. The long-term goal of our research program is to understand the function of the GABA/A 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 and determine the secondary protein structures which contribute to forming these sites. We are utilizing the substituted cysteine accessibility method in combination with mutagenesis, affinity labeling and cross-linking experiments to achieve these aims. The combination of these approaches is quite powerful and can provide details about the molecular structure of these binding sites which are not obtainable by mutagenesis and photoaffinity labeling techniques alone. If the structure of the GABA and BZD binding sites were known in detail, it is possible that whole new 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- substitutions by site-directed mutagenesis of wild-type amino acid residues, heterologous functional expression of the mutants, and the probing of the substituted cysteines with sulfhydryl-specific (SH-) reagents. If a cysteine-substituted residue is part of a binding site, reaction with a SH-reagent will alter binding irreversibly, and site-selective ligands will protect the engineered cysteine from covalent modification. Direct detection of a modified cysteine can be accomplished by using the SH- reagent, MTSEA-Biotin, in combination with avidin-bead precipitation and Western blotting techniques. By examining the pattern of accessibility of consecutively engineered cysteines to reaction with SH-reagents, the secondary structure of these residues can be determined; e.g. alpha-helix or beta-strand. Elaborations of this approach allow us to determine the charge-selectivity of the binding sites and to determine the distance between pairs of engineered cysteines. Thus, detailed structural maps of the GABA and BZD binding sites can be obtained, and the 3-dimensional structure of the sites can be potentially modeled. In the absence of an X- ray crystal structure, this biochemical approach will provide, at a molecular level, the most detailed structural picture of the GABA/A receptor available.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS034727-07
Application #
6625491
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (01))
Program Officer
Stewart, Randall
Project Start
2000-02-01
Project End
2003-11-30
Budget Start
2002-12-01
Budget End
2003-11-30
Support Year
7
Fiscal Year
2003
Total Cost
$273,600
Indirect Cost
Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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

Showing the most recent 10 out of 35 publications