QUANTITATIVE CHARACTERIZATION OF ALPHA-6 AND ALPHA-1-ALPHA-6 SUBUNIT-CONTAINING NATIVE GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS OF ADULT-RAT CEREBELLUM DEMONSTRATES 2 ALPHA-SUBUNITS PER RECEPTOR OLIGOMER

gamma-Aminobutyric acid(A) (GABA(A)) receptors were purified from adult rat cerebella by anti-alpha 6(1-16 Cys) antibody affinity chromatography, Immunoblots of the alpha 6 subunit-containing receptors showed the copurification of the alpha 1, beta 2/3, gamma 2, delta but not alpha 2 and alpha 3 GABA(A) receptor polypeptides. Further fractionation of this receptor subpopulation by anti-GABA(A) receptor subunit alpha 6(1-16 Cys) and anti-alpha 1(413-429) antibody affinity columns in series substantiated the coassociation of the alpha 1 and alpha 6 polypeptides, The percentage of coexistence of the two subunits was determined by quantitative immunoblotting, which found that 41 +/- 12% of alpha 6 subunit immunoreactivity is associated with the alpha 1 subunit, The ratios of the alpha 1:alpha 6 subunits in the double purified receptor preparations was found to be 1:1, thus determining directly for the first time subunit ratios within native GABA(A) receptors. The benzodiazepine pharmacology of the alpha 1 alpha 6 subunit-containing receptors was shown to be predominantly benzodiazepine insensitive by quantitative immunoprecipitation assays. These results are the first direct quantitative studies of subunit ratios within a population of native GABA(A) receptors.