Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique

摘要

GABAA receptor channels (GABARs) composed of varying combinations of alpha 1, beta 1, and gamma 2S subunits were transiently expressed in mammalian cell lines. The whole-cell patch-clamp recording technique was used to determine which combinations of GABAR subunits produced functional receptor channels and whether assembly of GABAR subunits into receptor channels followed a random or preferred sequence. To identify rapidly cells expressing GABARs, mammalian cell lines were cotransfected with combinations of GABAR subunit cDNAs and the Escherichia coli beta-galactosidase gene as a transfection marker. Positively transfected cells were identified by staining with the enzyme substrate fluorescein di-beta-galactopyranoside. Using this technique, we confirmed that functional alpha 1 beta 1 and alpha 1 beta 1 gamma 2S GABARs were assembled in transfected mouse L929 fibroblast cells, but surprisingly, functional alpha 1 gamma 2S and beta 1 gamma 2S GABARs were not expressed. It was determined that after transient transfection, levels of expressed receptors varied little among individual cells permitting comparison of absolute whole-cell GABA- evoked current values. Whole-cell currents recorded from cells coexpressing alpha 1 beta 1 gamma 2S subunits were three to four times larger than those recorded from cells coexpressing alpha 1 beta 1 subunits, and they were always enhanced by coapplied diazepam. The increase in whole-cell current was due in part to the larger single- channel current of the alpha 1 beta 1 gamma 2S GABARs. GABARs comprised of alpha 1 beta 1 gamma 2S subunits were formed preferentially over GABARs of alpha 1 beta 1 subunits alone, since only after substantially increasing the ratio of the beta 1 expression vector over the alpha 1 and gamma 2S subunit expression vectors were alpha 1 beta 1 GABARs formed in the presence of the gamma 2S subunit. These findings suggest that assembly of GABARs from constituent subunits did not proceed randomly to form all possible combinations, but that certain subunit combinations were preferred intermediates during the assembly process.