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The Journal of Neuroscience, April 1, 1998, 18(7):2449-2457

Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABA_A Receptors Containing ₆ Subunits

Martin Jechlinger, Robert Pelz, Verena Tretter, Thomas Klausberger, and Werner Sieghart

Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria

In cerebellum, GABA_A receptors containing ₆ subunits are expressed exclusively in granule cells. The number of ₆ receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on ₆ subunit-specific antibodies indicated that 45% of GABA_A receptors in cerebellar extracts contained ₆ subunits. Western blot analysis demonstrated that ₁, ₁, ₂, ₃, ₂, and  subunits co-purified with ₆ subunits, suggesting the existence of multiple 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 ₆ receptors remaining in the extract, the proportion of ₆ receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that ₆ receptors in cerebellum are composed predominantly of _6x2 (32%), _16x2 (37%), _6x (14%), or _16x (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of ₆ receptors contained homogeneous ₁, ₂, or ₃ subunits, respectively, whereas two different  subunits were present in 18% of all ₆ receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABA_A receptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors.

Key words: GABA_A receptor; composition, ₆ subunit; granule cell; cerebellum; antibodies; immunoaffinity chromatography; immunoprecipitation; [³H]muscimol; [³H]Ro 15-4513; binding studies

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Copyright © 1998 Society for Neuroscience  0270-6474/98/1872449-09$05.00/0

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