Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABA sub(A) Receptors Containing alpha sub(6) Subunits

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