EXTENSIVE HETEROGENEITY OF RECOMBINANT GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS EXPRESSED IN ALPHA(4)BETA(3)GAMMA(2)-TRANSFECTED HUMAN EMBRYONIC KIDNEY 293-CELLS

Human embryonic kidney 293 cells transiently transfected with alpha(4) -, beta(3)- and gamma(2)-subunits of gamma-aminobutyric acid(A) (GABA( A)) receptors from the rat exhibited specific high affinity binding si tes for [H-3]muscimol, [H-3]Ro 15-4513 and [S-35]t-butylbicyclophospho rothionate (TBPS). B-max values obtained, however, were dramatically d ifferent for these compounds. In addition, GABA was able to inhibit on ly 20% of specific [S-35]TBPS binding to membranes from alpha(4) beta( 3) gamma(2)-transfected cells. In order to investigate possible recept or heterogeneity, receptors were extracted from alpha(4) beta(3) gamma (2)-transfected cells and were fractionated by chromatography on an an ti-gamma(2)-, followed by an anti-alpha(4)- and an anti-beta(3)-immuno affinity column. western blot analysis of the column eluates indicated the separate existence of GABA(A) receptors consisting of alpha(4) be ta(3) gamma(2)-, alpha(4) beta(3)- or beta(3)-subunits in alpha(4) bet a(3) gamma(2)-transfected cells. This, and the finding that only alpha (4) beta(3) gamma(2)- but not alpha(4) beta(3)- or beta(3)-receptors p ossess high affinity binding sites for all three radiolabeled ligands investigated, combined with the observation that [S-35]TBPS binding to receptors consisting of beta(3)-subunits cannot be inhibited by GABA, can explain most of the binding data obtained. The present results su ggest an inefficient assembly of gamma(2)- with alpha(4)- and/or beta( 3)-subunits under the conditions used, and indicate that recombinant r eceptors expressed in HEK cells are not necessarily homogeneous. Copyr ight (C) 1996 Elsevier Science Ltd.