Kellenberger S, Eckenstein S, Baur R, Malherbe P, Buhr A, Sigel E
Department of Pharmacology, University of Bern, Switzerland.
Neuropharmacology. 1996;35(9-10):1403-11. doi: 10.1016/s0028-3908(96)00034-2.
GABAA receptors are hetero-oligomeric proteins of unknown subunit stoichiometry. In this study alpha 1 beta 3 GABAA receptor channels were functionally expressed in Xenopus oocytes. Direct immunoprecipitation from the oocyte surface was used to exclusively isolate mature GABAA receptors. The subunit ratio was determined by quantitation of the amount of [35S]methionine incorporated into individual receptor subunits. Antibody released from the antigen or antibody not reacted was prevented from reassociation with labeled antigen by addition of excess unlabeled antigen. Variation of the alpha 1 beta 3 ratio of injected cRNAs only slightly affected the subunit ratio in mature receptors. This indicates that the subunit stoichiometry generated is independent of the pools of newly synthesized subunit monomers and supports the view that the receptor assembly is a regulated process. The ratio of alpha 1/beta 3 subunits was found to be 1.1 +/- 0.1 (SEM, n = 6). Our data are in best agreement with a tetrameric receptor with the composition 2 alpha 2 beta. For a pentameric receptor the ratio found slightly favors a receptor with the composition 3 alpha 2 beta. The method developed here is applicable to the determination of the subunit stoichiometry of other recombinant oligomeric membrane proteins.
GABAA受体是亚基化学计量未知的异源寡聚蛋白。在本研究中，α1β3 GABAA受体通道在非洲爪蟾卵母细胞中实现了功能表达。利用从卵母细胞表面进行的直接免疫沉淀来专门分离成熟的GABAA受体。通过对掺入各个受体亚基中的[35S]甲硫氨酸量进行定量来确定亚基比例。通过添加过量未标记抗原，防止从抗原释放的抗体或未反应的抗体与标记抗原重新结合。注射的cRNAs的α1β3比例变化仅对成熟受体中的亚基比例有轻微影响。这表明所产生的亚基化学计量独立于新合成的亚基单体库，并支持受体组装是一个受调控过程的观点。发现α1/β3亚基的比例为1.1±0.1（SEM，n = 6）。我们的数据与组成是2α2β的四聚体受体最为一致。对于五聚体受体，所发现的比例略倾向于组成是3α2β的受体。这里开发的方法适用于确定其他重组寡聚膜蛋白的亚基化学计量。
