Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABAAReceptors Containing α6Subunits

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Abstract
In cerebellum, GABAA receptors containing α6 subunits are expressed exclusively in granule cells. The number of α6 receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on α6 subunit-specific antibodies indicated that 45% of GABAA receptors in cerebellar extracts contained α6 subunits. Western blot analysis demonstrated that α1, β1, β2, β3, γ2, and δ subunits co-purified with α6 subunits, suggesting the existence of multiple α6 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 α6 receptors remaining in the extract, the proportion of α6 receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that α6 receptors in cerebellum are composed predominantly of α6βxγ2(32%), α1α6βxγ2(37%), α6βxδ (14%), or α1α6βxδ (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of α6 receptors contained homogeneous β1, β2, or β3subunits, respectively, whereas two different β subunits were present in 18% of all α6 receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABAA receptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors.

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