GABA is the major inhibitory neurotransmitter in the brain and interacts with the GABA-A receptor. A variety of compounds, including benzodiazepines, barbiturates and ethanol potentiate GABA responses. Molecular cloning of the GABA-A receptor has confirmed the existence of at least six alpha, three beta, two gamma, and one rho subunits expressed in the brain and eye. The stoichiometry of these subunits in the native receptor is not known; neither are the structural determinants that influence ligand binding or maintain the chloride ion selectivity of the channel. We have undertaken a series of studies to determine the role of glycosylation in the functional expression of the GABA-A receptor. Incubation of Xenopus oocytes microinjected with alpha-1, beta-1 and gamma-2 subunit-specific mRNAs with tunicamycin inhibits are expression of the GABA receptor. Site -directed mutagenesis is being used to determine which glycosylation sites are required for functional assembly of the GABA receptor and whether glycosylation influences the binding of ligands. WE have also begun a characterization of the amino acids on the alpha subunit of the GABA receptor that influences the binding of benzodiazepines. We have chosen the alpha-6 subunit as a model because the presence of this alpha subunit in a functional receptor complex is associated with high affinity binding for the benzodiazepines, R015-4513 (a putative alcohol antagonist), but not for other benzodiazepines of beta-carbolines. Site-directed mutagenesis has been used to construct mutation in the alpha-1 and alpha-6 subunits in a region previously determined to be required for benzodiazepine binding. The properties of the mutant GABA receptors are being assessed in Xenopus oocytes and cultured cells.
