SUBUNIT COMPOSITION AND QUANTITATIVE IMPORTANCE OF HETEROOLIGOMERIC RECEPTORS - GABA(A) RECEPTORS CONTAINING ALPHA(6) SUBUNITS

In cerebellum, GABA(A) receptors containing a, subunits are expressed exclusively in granule cells. The number of alpha(6) receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on alpha(6) subunit-specific ant ibodies indicated that 45% of GABA(A) receptors in cerebellar extracts contained alpha(6) subunits. Western blot analysis demonstrated that alpha(1), beta(1), beta(2), beta(3), gamma(2), and delta subunits co-p urified with alpha(6) subunits, suggesting the existence of multiple a lpha(6) receptor subtypes. These subtypes were identified using a new method based on the one-by-one immunochromatographic elimination of re ceptors containing the co-purifying subunits in parallel or subsequent experiments. By quantification and Western blot analysis of alpha(6) receptors remaining in the extract, the proportion of alpha(6) recepto rs containing the eliminated subunit could be calculated and the subun it composition of the remaining receptors could be determined. Results obtained indicated that alpha(6) receptors in cerebellum are composed predominantly of alpha(6) beta(x) gamma(2) (32%), alpha(1) alpha(6) b eta(x) gamma(2) (37%), alpha(6) beta(x) delta (14%), or alpha(1) alpha (6) beta(x) delta (15%) subunits. Other experiments indicated that 10% , 51%, or 21% of alpha(6) receptors contained homogeneous beta(1), bet a(2), or beta(3) subunits, respectively, whereas two different beta su bunits were present in 18% of all alpha(6) receptors. The method prese nted can be used to resolve the total number, subunit composition, and abundancy of GABA(A) receptor subtypes in the brain and can also be a pplied to the investigation of other hetero-oligomeric receptors.