Quantitative Immunoprecipitation Studies with Anti-?-Aminobutyric Acid_A Receptor ?2 1?15 Cys Antibodies

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma-2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma-2 subunit of the gamma-aminobutyric acid(A) (GABA(A)) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma-2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [H-3]flunitrazepam- and [H-3]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [H-3]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha-1 324-341 antibodies carried out in parallel with anti-gamma-2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma-2 subunit within native GABA(A) receptors. These results substantiate the association of the gamma-2 polypeptide with native GABA(A) receptors.