Wong G, Sei Y, Skolnick P
Laboratory of Neuroscience, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1992 Dec;42(6):996-1003.
Expression plasmids were constructed with cDNAs encoding the rat gamma-aminobutyric acid-A (GABAA) receptor alpha 1, beta 2, and gamma 2 subunits and were cotransfected into cultured human embryonic kidney 293 cells. A single cell line (WS-1) was established after G-418 treatment and clonal selection. This cell line contained saturable, high affinity binding sites for the benzodiazepines [3H] Ro 15-4513 and [3H]flunitrazepam that were modulated by GABA. Competition experiments with benzodiazepine receptor ligands suggest a profile characteristic of native "type I" benzodiazepine receptors, because strong correlations were observed between the Ki values of these ligands in WS-1 cells and in both cerebellar homogenates (r = 0.97, p < 0.0001) and 293 cells transiently transfected with the corresponding cDNAs (r = 0.96, p < 0.001). Fluorescence intensity in WS-1 cells loaded with the Cl(-)-specific probe 6-methoxy-N-(3-sulfopropyl)-quinolinium was reliably increased by GABA. This effect was blocked by bicuculline and augmented by midazolam, consistent with the presence of GABA-gated, benzodiazepine receptor-modulated, Cl- channels. Northern blot analysis revealed the presence of mRNAs encoding alpha 1 and gamma 2 receptor subunits. Southern blot analysis confirmed genomic integration of transfected alpha 1 and gamma 2 cDNAs. The beta 2 subunit was not detected in either Northern or Southern blot analysis, indicating that a functional type I GABAA/benzodiazepine receptor complex can be constituted without a beta subunit.
构建了表达质粒，其含有编码大鼠γ-氨基丁酸-A（GABAA）受体α1、β2和γ2亚基的cDNA，并将其共转染到培养的人胚肾293细胞中。经G-418处理和克隆筛选后建立了单一细胞系（WS-1）。该细胞系含有可饱和的、对苯二氮䓬类[3H]Ro 15-4513和[3H]氟硝西泮具有高亲和力的结合位点，这些位点受γ-氨基丁酸（GABA）调节。用苯二氮䓬受体配体进行的竞争实验表明其具有天然“Ⅰ型”苯二氮䓬受体的特征，因为在WS-1细胞中以及在小脑匀浆（r = 0.97，p < 0.0001）和用相应cDNA瞬时转染的293细胞中（r = 0.96，p < 0.001），观察到这些配体的Ki值之间存在强相关性。用Cl(-)特异性探针6-甲氧基-N-(3-磺丙基)-喹啉鎓装载的WS-1细胞中的荧光强度可靠地被GABA增加。该效应被荷包牡丹碱阻断，并被咪达唑仑增强，这与存在GABA门控、苯二氮䓬受体调节的Cl-通道一致。Northern印迹分析揭示了编码α1和γ2受体亚基的mRNA的存在。Southern印迹分析证实了转染的α1和γ2 cDNA的基因组整合。在Northern或Southern印迹分析中均未检测到β2亚基，表明功能性Ⅰ型GABAA/苯二氮䓬受体复合物可以在没有β亚基的情况下构成。
