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.
Showing the most recent 10 out of 46 publications
