GABA is the major inhibitory neurotransmitter in the brain. A variety of compounds, including benzodiazepines, barbiturates and ethanol potentiate GABA responses. In order to better understand the molecular basis of ligand-gated ion channel function and to identify the sites within the GABA receptor at which ethanol may act, we have established systems for the expression of cloned receptor-channel complexes in Xenopus oocytes injected with receptor-specific mRNA and in cultured cells transfected with receptor-specific cDNAs. Molecular cloning of the GABAA receptor has confirmed the existence of at least six alpha, three beta, two gamma, one delta, and one rho subunits. 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 GABAA receptor. Incubation of Xenopus oocytes microinjected with alpha1, beta1 and gamma2 subunit-specific mRNAs with tunicamycin inhibits the expression of the GABA receptor. Site-directed mutagenesis is being used to determine the contribution of each putative glycosylation site to functional receptor expression. These studies will 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 influence the binding of benzodiazepines. We have chosen the alpha6 subunit as a model because the presence of this subunit in a functional receptor complex is associated with high affinity binding for the benzodiazepine, RO15-4513 (a putative alcohol antagonist), but not for other benzodiazepines or beta-carbolines. Site-directed mutagenesis has been used to construct mutations in the alpha1 and alpha6 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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Intramural Research (Z01)
Project #
1Z01AA000484-02
Application #
3802005
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
National Institute on Alcohol Abuse and Alcoholism
Department
Type
DUNS #
City
State
Country
United States
Zip Code