Jechlinger M, Pelz R, Tretter V, Klausberger T, Sieghart W
Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria.
J Neurosci. 1998 Apr 1;18(7):2449-57. doi: 10.1523/JNEUROSCI.18-07-02449.1998.
In cerebellum, GABAA receptors containing alpha6 subunits are expressed exclusively in granule cells. The number of alpha6 receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on alpha6 subunit-specific antibodies indicated that 45% of GABAA receptors in cerebellar extracts contained alpha6 subunits. Western blot analysis demonstrated that alpha1, beta1, beta2, beta3, gamma2, and delta subunits co-purified with alpha6 subunits, suggesting the existence of multiple alpha6 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 alpha6 receptors remaining in the extract, the proportion of alpha6 receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that alpha6 receptors in cerebellum are composed predominantly of alpha6betaxgamma2 (32%), alpha1alpha6betaxgamma2 (37%), alpha6betaxdelta (14%), or alpha1alpha6betaxdelta (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of alpha6 receptors contained homogeneous beta1, beta2, or beta3 subunits, respectively, whereas two different beta subunits were present in 18% of all alpha6 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.
在小脑中，含有α6亚基的GABAA受体仅在颗粒细胞中表达。目前尚不清楚这些细胞中形成的α6受体亚型的数量及其亚基组成。利用α6亚基特异性抗体进行免疫亲和层析表明，小脑提取物中45%的GABAA受体含有α6亚基。蛋白质免疫印迹分析表明，α1、β1、β2、β3、γ2和δ亚基与α6亚基共同纯化，提示存在多种α6受体亚型。这些亚型是通过一种新方法鉴定的，该方法基于在平行或后续实验中逐一免疫层析去除含有共同纯化亚基的受体。通过对提取物中剩余的α6受体进行定量和蛋白质免疫印迹分析，可以计算出含有被去除亚基的α6受体的比例，并确定剩余受体的亚基组成。结果表明，小脑中的α6受体主要由α6βxγ2（32%）、α1α6βxγ2（37%）、α6βxδ（14%）或α1α6βxδ（15%）亚基组成。其他实验表明，分别有10%、51%或21%的α6受体含有均一的β1、β2或β3亚基，而18%的所有α6受体中存在两种不同的β亚基。所提出的方法可用于解析脑中GABAA受体亚型的总数、亚基组成和丰度，也可应用于其他异源寡聚体受体的研究。
