SUBUNIT STOICHIOMETRY OF OLIGOMERIC MEMBRANE-PROTEINS - GABA(A) RECEPTORS ISOLATED BY SELECTIVE IMMUNOPRECIPITATION FROM THE CELL-SURFACE

GABA(A) receptors are hetero-oligomeric proteins of unknown subunit st oichiometry. In this study alpha 1 beta 3 GABA(A) receptor channels we re functionally expressed in Xenopus oocytes. Direct immunoprecipitati on from the oocyte surface was used to exclusively isolate mature GABA (A) receptors. The subunit ratio was determined by quantitation of the amount of [S-35]methionine incorporated into individual receptor subu nits. Antibody released from the antigen or antibody not reacted was p revented 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. Th is indicates that the subunit stoichiometry generated is independent o f the pools of newly synthesized subunit monomers and supports the vie w that the receptor assembly is a regulated process. The ratio of alph a 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 fa vors a receptor with the composition 3 alpha 2 beta. The method develo ped here is applicable to the determination of the subunit stoichiomet ry of other recombinant oligomeric membrane proteins. Copyright (C) 19 96 Elsevier Science Ltd.