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 sites for [H-3]muscimol, [H-3]Ro 15-4513 and [S-35]t-butylbicyclophosphorothionate (TBPS). B-max values obtained, however, were dramatically different for these compounds. In addition, GABA was able to inhibit only 20% of specific [S-35]TBPS binding to membranes from alpha(4) beta(3) gamma(2)-transfected cells. In order to investigate possible receptor heterogeneity, receptors were extracted from alpha(4) beta(3) gamma(2)-transfected cells and were fractionated by chromatography on an anti-gamma(2)-, followed by an anti-alpha(4)- and an anti-beta(3)-immunoaffinity column. western blot analysis of the column eluates indicated the separate existence of GABA(A) receptors consisting of alpha(4) beta(3) gamma(2)-, alpha(4) beta(3)- or beta(3)-subunits in alpha(4) beta(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 possess 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 suggest an inefficient assembly of gamma(2)- with alpha(4)- and/or beta(3)-subunits under the conditions used, and indicate that recombinant receptors expressed in HEK cells are not necessarily homogeneous. Copyright (C) 1996 Elsevier Science Ltd.