Quantitative characterization of alpha 6 and alpha 1 alpha 6 subunit-containing native gamma-aminobutyric acidA receptors of adult rat cerebellum demonstrates two alpha subunits per receptor oligomer.
gamma-Aminobutyric acidA (GABAA) 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 GABAA receptor polypeptides. Further fractionation of this receptor subpopulation by anti-GABAA 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 GABAA 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 GABAA receptors.
Pollard
S
S
Department of Pharmaceutical Chemistry, School of Pharmacy, University of London, United Kingdom.
Thompson
C L
CL
Stephenson
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology; D001704:Biopolymers; D015153:Blotting, Western; D002417:Cattle; D002531:Cerebellum / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D007140:Immunoglobulin Fab Fragments; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D001704:Biopolymers; D007140:Immunoglobulin Fab Fragments; D011963:Receptors, GABA-A; D001569:Benzodiazepines