Molecular characterization of type I GABAA receptor complex from rat cerebral cortex and hippocampus.
The molecular composition of the native gamma-aminobutyric acidA (GABAA) receptor complex is actually unknown. In the present communication we report a novel approach to characterize the minimal molecular conformation of the native GABAA receptor complex. This novel approach is based on the combination of subunit specific antibodies and specific 3H-labeled ligands in immunoprecipitation experiments. We have determined the presence of beta 2/3 and gamma 2 subunits in the Type I GABAA 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 [3H]zolpidem. The association of gamma 2 and beta 2/3 subunits with the GABAA receptor complex was also tested using [3H]flumazenil. The results indicated that both gamma 2 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 GABAA 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ruano
D
D
Departamento de Bioquimica, Bromatologia y Toxicologia, Facultad de Farmacia, Universidad de Sevilla, Spain.
Araujo
F
F
Machado
A
A
de Blas
A L
AL
Vitorica
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D002540:Cerebral Cortex / Q000737:chemistry*; D006624:Hippocampus / Q000737:chemistry*; D008566:Membranes / Q000378:metabolism; D011233:Precipitin Tests; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000737:chemistry*; D012995:Solubility
D000911:Antibodies, Monoclonal; D011963:Receptors, GABA-A