Angelotti T P, Uhler M D, Macdonald R L
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
J Neurosci. 1993 Apr;13(4):1418-28. doi: 10.1523/JNEUROSCI.13-04-01418.1993.
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.
由α1、β1和γ2S亚基的不同组合构成的GABAA受体通道（GABARs）在哺乳动物细胞系中瞬时表达。采用全细胞膜片钳记录技术来确定GABAR亚基的哪些组合产生功能性受体通道，以及GABAR亚基组装成受体通道是遵循随机顺序还是优先顺序。为了快速鉴定表达GABARs的细胞，将GABAR亚基cDNA组合与大肠杆菌β-半乳糖苷酶基因作为转染标记共同转染哺乳动物细胞系。通过用酶底物荧光素二-β-吡喃半乳糖苷染色来鉴定阳性转染细胞。使用该技术，我们证实功能性α1β1和α1β1γ2S GABARs在转染的小鼠L929成纤维细胞中组装，但令人惊讶的是，功能性α1γ2S和β1γ2S GABARs未表达。经测定，瞬时转染后，各个细胞中表达的受体水平变化很小，从而允许比较绝对全细胞GABA诱发电流值。共表达α1β1γ2S亚基的细胞记录到的全细胞电流比共表达α1β1亚基的细胞记录到的全细胞电流大3至4倍，并且它们总是被共同应用的地西泮增强。全细胞电流的增加部分归因于α1β1γ2S GABARs的较大单通道电流。由α1β1γ2S亚基组成的GABARs比单独的α1β1亚基的GABARs优先形成，因为只有在将β1表达载体与α1和γ2S亚基表达载体的比例大幅增加后，α1β1 GABARs才会在γ2S亚基存在的情况下形成。这些发现表明，由组成亚基组装GABARs并非随机进行以形成所有可能的组合，而是某些亚基组合在组装过程中是优先中间体。
