Quantitative characterization of α6 and α1α6 subunit-containing native γ-aminobutyric acidA receptors of adult rat cerebellum demonstrates two α subunits per receptor oligomer

Abstract

γ-Aminobutyric acidA (GABAA) receptors were purified from adult rat cerebella by anti-α6(1-16 Cys) antibody affinity chromatography. Imniunoblots of the α6 subunit-containing receptors showed the copurification of the α1, β2/3, γ2, δ but not α2 and α3 GABAA receptor polypeptides. Further fractionation of this receptor subpopulation by anti-GABAA receptor subunit α6(1-16 Cys) and anti-α1(413-429) antibody affinity columns in series substantiated the coassociation of the α1 and α6 polypeptides. The percentage of coexistence of the two subunits was determined by quantitative immunoblotting, which found that 41 ± 12% of α6 subunit immunoreactivity is associated with the α1 subunit. The ratios of the α1:α6 subunits in the double purified receptor preparations was found to be 1:1, thus determining directly for the first time subunit ratios within native GABAA receptors. The benzodiazepine pharmacology of the α1α6 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 GABAA receptors.