MOLECULAR CHARACTERIZATION OF TYPE-I GABA(A) RECEPTOR COMPLEX FROM RAT CEREBRAL-CORTEX AND HIPPOCAMPUS

The molecular composition of the native gamma-aminobutyric acid(A) (GABA(A)) receptor complex is actually unknown. In the present communication we report a novel approach to characterize the minimal molecular conformation of the native GABA(A) receptor complex. This novel approach is based on the combination of subunit specific antibodies and specific H-3-labeled ligands in immunoprecipitation experiments. We have determined the presence of beta(1/2) and gamma(2) subunits in the Type I GABA(A) receptor complex, from rat cerebral cortex and hippocampus, by using two antibodies, the monoclonal 62-3G1 (specific for beta(2/3)) and the polyclonal anti-gamma(2) (to the large intracellular loop of the gamma(2) short form) together with the Type I-specific ligand [H-3]zolpidem. The association of gamma(2) and beta(2/3) subunits with the GABA(A) receptor complex was also tested using [H-3]flumazenil. The results indicated that both yz and beta(2/3) were the most abundant subunits associated to either Type I or total benzodiazepine receptors from both cortex and hippocampus. Between 70-80% of Type I or total benzodiazepine binding activity was immunoprecipitated by either antibody. In addition, we have also investigated the coexistence of both subunits as part of the same population of Type I GABA(A) receptor complex by cross-immunoprecipitation experiments with 62-3G1 and anti-gamma(2). The results indicated that, in cerebral cortex, both gamma(2) and beta(2/3) subunits were part of the same population of Type I receptors. In hippocampus, an additional 20% of Type I receptors displayed either gamma(2) or beta(2/3) but not both subunits. On the other hand, a substantial proportion (30% in cortex or 10-20% in hippocampus) of Type I receptor was not immunoprecipitated by either antibody. Therefore, these population of GABA(A) receptor complex with Type I pharmacology may be constructed by association of subunits others than gamma(2) or beta(2/3).