Pregenzer J F, Im W B, Carter D B, Thomsen D R
Upjohn Company, CNS Diseases Research, Kalamazoo, Michigan 49001.
Mol Pharmacol. 1993 May;43(5):801-6.
The alpha 1 beta 2 and alpha 1 beta 2 gamma 2 subtypes, common subtypes of gamma-aminobutyric acid (GABA)A receptors in the brain, are known to share many ligands, but only the latter interacts with benzodiazepines. In this study, we attempted to examine whether the presence of the gamma 2 subunit in the cloned receptors alters the binding properties of GABA and t-butylbicyclophosphorothionate (TBPS) (a highly sensitive probe for conformational changes in the chloride ionophore of GABAA receptors) and their interactions. Using a high-level expression system of SF-9 cells infected with baculovirus, we produced a group of cloned GABAA receptors with variations in the ratio (0 to 3) of the virion carrying the cDNA for the gamma 2 subunit to those carrying the cDNAs for the alpha 1 and beta 2 subunits. The number of benzodiazepine binding sites increased as the level of the gamma 2 virion was raised and reached that of GABA high affinity sites at a gamma 2 to alpha 1 beta 2 ratio of 0.5 or more. It appears that the conversion of the alpha 1 beta 2 to the alpha 1 beta 2 gamma 2 subtype is favorable and complete in the presence of a sufficient level of the gamma 2 subunit, assuming the number of the GABA sites to be equal to the total number of the cloned GABAA receptors. In all preparations, the dissociation constants for flunitrazepam, muscimol, and TBPS were fairly constant, and the maximal number of binding sites for TBPS appeared to be equal to that for muscimol, with no dependence on the gamma 2 virion levels. The effect of GABA on TBPS binding, however, were markedly altered by the gamma 2 subunit. With the alpha 1 beta 2 subtype GABA at concentrations occupying its high affinity sites markedly stimulated but at higher concentrations (micromolar ranges) inhibited TBPS binding, whereas with the alpha 1 beta 2 gamma 2 subtype GABA inhibited TBPS binding without the early stimulatory phase. We also confirmed the selective interaction of Zn2+ (50 microM) with the alpha 1 beta 2 subtype, as probed with TBPS binding, and observed a progressive disappearance of Zn2+ sensitivity as the gamma 2 virion level increased.(ABSTRACT TRUNCATED AT 400 WORDS)
α1β2和α1β2γ2亚型是大脑中γ-氨基丁酸（GABA）A受体的常见亚型，已知它们共享许多配体，但只有后者与苯二氮䓬相互作用。在本研究中，我们试图研究克隆受体中γ2亚基的存在是否会改变GABA和叔丁基双环磷硫代酸盐（TBPS，一种对GABAA受体氯离子通道构象变化高度敏感的探针）的结合特性及其相互作用。利用感染杆状病毒的SF-9细胞的高水平表达系统，我们制备了一组克隆的GABAA受体，携带γ2亚基cDNA的病毒粒子与携带α1和β2亚基cDNA的病毒粒子的比例（0至3）有所不同。随着γ2病毒粒子水平的提高，苯二氮䓬结合位点的数量增加，当γ2与α1β2的比例达到0.5或更高时，达到GABA高亲和力位点的数量。假设GABA位点的数量等于克隆的GABAA受体的总数，那么在存在足够水平的γ2亚基时，α1β2向α1β2γ2亚型的转变似乎是有利且完全的。在所有制剂中，氟硝西泮、蝇蕈醇和TBPS的解离常数相当恒定，TBPS结合位点的最大数量似乎与蝇蕈醇的相等，且不依赖于γ2病毒粒子水平。然而，γ2亚基显著改变了GABA对TBPS结合的影响。对于α1β2亚型，GABA在占据其高亲和力位点的浓度下显著刺激但在更高浓度（微摩尔范围）下抑制TBPS结合，而对于α1β2γ2亚型，GABA抑制TBPS结合而没有早期刺激阶段。我们还证实了Zn2 +（50μM）与α1β2亚型的选择性相互作用，通过TBPS结合进行探测，并观察到随着γ2病毒粒子水平的增加，Zn2 +敏感性逐渐消失。（摘要截断于400字）
