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 co ntribution of endogenous proteins in the assembly of mature channels. We ex amined whether an endogenous subunit was required for the functional expres sion of gamma-aminobutyric acid type A (GABA,) receptors in WSS-1 cells, HE K293 cells stably expressing recombinant al and gamma 2 subunits. Our pharm acological 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 exist ence of an endogenous beta 3 subunit. Whole-cell GABA-evoked currents recor ded from untransfected WSS-1 cells were blocked by bicuculline methiodide a nd enhanced by anesthetics and anticonvulsants including the subunit-select ive compounds diazepam and loreclezole. These data suggest that, in additio n to the gamma 2 subunit, WSS-1 cell receptors also contain beta 2/3 subuni ts. 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. Untransfect ed HEK293 cells were unresponsive to GABA. Cells transfected with al and ga mma 2S cDNAs displayed small diazepam and loreclezole responsive GABA-activ ated 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 receptor s of WSS-1 cells contain al, beta 3 and gamma 2S subunits. (C) 2000 Elsevie r Science Ltd. All rights reserved.