The influence of an endogenous beta 3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA,) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant al and gamma 2 subunits. Our pharmacological and RT-PCR analyses of GABA, receptors and their mRNAs in WSS-1 cells confirm the presence of al and gamma 2 subunits and suggest the existence of an endogenous beta 3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma 2 subunit, WSS-1 cell receptors also contain beta 2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta 3 mRNA, We examined the contribution of the beta 3 subunit in the function of receptors formed by expression of al and gamma 2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with al and gamma 2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha 1 and gamma 2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha 1 and gamma 2S subunits alone do not form functional GABA, receptors and that receptors of WSS-1 cells contain al, beta 3 and gamma 2S subunits. (C) 2000 Elsevier Science Ltd. All rights reserved.