The GABAA receptors are ligand-gated chloride ion channels formed as heteropentamers from 17 subunit possibilities. Of the thousands of potential combinations, only a few, 15-20 isoforms of such complexes occur in nature. The relationship between the subunit composition/ stoichiometry and functional characteristics is poorly defined. Our work seeks a) to analyze the structural basis of receptor heterogeneity; b) to determine what controls assembly of subunits into oligomers; c) to identify subunit specificity of biological regulatory mechanisms involving zinc, phosphorylation, and neurosteroids; d) to define functional domains within the subunits; and e) to test models of structure, using expression of recombinant receptor cDNAs in heterologous host cells. We employ traditional systems such as frog oocytes, and a new technology using Baculovirus shuttle vectors to test several subunit combinations and site-directed mutagenesis in insect cell line Sf9 expression. We also use mouse mutants to prove pharmacological biological roles of individual subunits. We will test the hypothesis that isoforms of GABAA receptors with different subunit composition have different biological functions/ regulatory mechanisms, and coincidentally pharmacological specificity. Using electrophysiology and binding assays, we seek to identify recombinant receptor subunit combinations that reconstitute functional subtypes observed in nature using binding, in vitro functional assays, an subunit-specific antibodies to isolate protein isoforms. For example, isoforms differ in their sensitivity to GABA, to modulatory drugs, to endogenous regulatory mechanisms including neurosteroids, zinc, and phosphorylation. We will also test the hypothesis that there is a subunit specificity and functional domain sequence specificity that controls assembly of subunits into functional membrane channel oligomers. We will test the hypotheses that a portion of the extracellular domain contains parts of the GABA and benzodiazepine binding sites on two beta- strands that surround an alpha-helix, a secondary structural element involved in channel gating; and that the binding sites for GABA, benzodiazepines, and anesthetics (barbiturates, steroids, and volatile agents) are all allosterically coupled to each other and to the structural element of the extracellular domain of the protein that mechanically moves the ion channel into open and shut conformations. This will involve some carefully chosen site-directed mutagenesis to identify amino acids involved in functional domains and test structural models deduced from our previous results. In addition, structural biochemistry on the receptor protein in Sf9 cell membranes is made possible by the overexpression of the genetically engineered system. Since the GABA A receptors are brain proteins that mediate the bulk of inhibitory communication in the brain, understanding of the molecular heterogeneity of GABA A receptors will be useful to knowledge of normal brain function as well as important disease processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS028772-09
Application #
2714489
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Program Officer
Jacobs, Margaret
Project Start
1990-08-01
Project End
1999-05-31
Budget Start
1998-06-01
Budget End
1999-05-31
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Olsen, Richard W; Sieghart, Werner (2009) GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology 56:141-8
Chen, Zi-Wei; Chang, Chang-Sheng S; Leil, Tarek A et al. (2005) GABAA receptor-associated protein regulates GABAA receptor cell-surface number in Xenopus laevis oocytes. Mol Pharmacol 68:152-9
Handforth, Adrian; Delorey, Timothy M; Homanics, Gregg E et al. (2005) Pharmacologic evidence for abnormal thalamocortical functioning in GABA receptor beta3 subunit-deficient mice, a model of Angelman syndrome. Epilepsia 46:1860-70
Leil, Tarek A; Chen, Zi-Wei; Chang, Chang-Sheng S et al. (2004) GABAA receptor-associated protein traffics GABAA receptors to the plasma membrane in neurons. J Neurosci 24:11429-38
Nymann-Andersen, Jesper; Sawyer, Gregory W; Olsen, Richard W (2002) Interaction between GABAA receptor subunit intracellular loops: implications for higher order complex formation. J Neurochem 83:1164-71
Nymann-Andersen, Jesper; Wang, Hongbing; Olsen, Richard W (2002) Biochemical identification of the binding domain in the GABA(A) receptor-associated protein (GABARAP) mediating dimer formation. Neuropharmacology 43:476-81
Engblom, A Christine; Carlson, Berit X; Olsen, Richard W et al. (2002) Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating. J Biol Chem 277:17438-47
Nymann-Andersen, Jesper; Wang, Hongbing; Chen, Li et al. (2002) Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors. J Neurochem 80:815-23
Elster, L; Kristiansen, U; Pickering, D S et al. (2001) Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2. Neurochem Int 38:581-92
Smith, G B; Olsen, R W (2000) Deduction of amino acid residues in the GABA(A) receptor alpha subunits photoaffinity labeled with the benzodiazepine flunitrazepam. Neuropharmacology 39:55-64

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