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) (GA BA(A)) receptor complex is actually unknown. In the present communicat ion we report a novel approach to characterize the minimal molecular c onformation of the native GABA(A) receptor complex. This novel approac h is based on the combination of subunit specific antibodies and speci fic H-3-labeled ligands in immunoprecipitation experiments. We have de termined the presence of beta(1/2) and gamma(2) subunits in the Type I GABA(A) receptor complex, from rat cerebral cortex and hippocampus, b y 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 r esults indicated that both yz and beta(2/3) were the most abundant sub units associated to either Type I or total benzodiazepine receptors fr om both cortex and hippocampus. Between 70-80% of Type I or total benz odiazepine binding activity was immunoprecipitated by either antibody. In addition, we have also investigated the coexistence of both subuni ts as part of the same population of Type I GABA(A) receptor complex b y cross-immunoprecipitation experiments with 62-3G1 and anti-gamma(2). The results indicated that, in cerebral cortex, both gamma(2) and bet a(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 sub stantial proportion (30% in cortex or 10-20% in hippocampus) of Type I receptor was not immunoprecipitated by either antibody. Therefore, th ese population of GABA(A) receptor complex with Type I pharmacology ma y be constructed by association of subunits others than gamma(2) or be ta(2/3).