Im W B, Pregenzer J F, Binder J A, Dillon G H, Alberts G L
CNS Diseases Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
J Biol Chem. 1995 Nov 3;270(44):26063-6. doi: 10.1074/jbc.270.44.26063.
Despite the presence of the multiple subunits (alpha, beta, gamma, and delta) and their isoforms for gamma-aminobutyric acid, type A (GABAA) receptors in mammalian brains, the alpha x beta 2 gamma 2 subtypes appear to be the prototype GABAA receptors sharing many properties with native neuronal receptors. In order to gain insight into their subunit stoichiometry and orientation, we prepared a tandem construct of the alpha 6 and beta 2 subunit cDNAs where the carboxyl-terminal of alpha 6 is linked to the amino-terminal of beta 2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cells with the tandem construct alone failed to induce GABA-dependent CI- currents, but its cotransfection with the cDNA for alpha 6 or gamma 2, but not beta 2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of gamma 2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [3H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the beta 2. No appreciable differences were found in their Kd (2.5 nM) and Bmax values (1.4 pmol/mg of protein). These data are consistent with the view that the polypeptides arising from the tandem construct were expressed with the high affinity GABA site, but unable to form GABA channels. The requirement of a specific monomeric subunit (alpha 6 or gamma 2) for the tandem construct to express Cl--currents supports a pentameric structure of GABAA receptors consisting of two alpha 6, two beta 2, and one gamma 2 for the alpha 6 beta 2 gamma 2 and three alpha 6 and two beta 2 for the alpha 6 beta 2 subtype.
尽管哺乳动物大脑中存在多种亚基（α、β、γ和δ）以及γ-氨基丁酸A型（GABAA）受体的亚型，但αxβ2γ2亚型似乎是与天然神经元受体具有许多共同特性的GABAA受体原型。为了深入了解它们的亚基化学计量和取向，我们制备了α6和β2亚基cDNA的串联构建体，其中α6的羧基末端通过编码10个谷氨酰胺残基的接头与β2的氨基末端相连。单独用串联构建体转染人胚肾293细胞未能诱导GABA依赖性氯离子电流，但将其与α6或γ2的cDNA共转染（而非与β2共转染）会导致出现对印防己毒素敏感的GABA电流，对于含γ2的受体，还会对苯二氮卓类激动剂U-92330产生反应。然而，在所有受体亚基组合中，包括单独的串联构建体或与β2组合，用[3H]蝇蕈醇结合检测到了高亲和力GABA位点。它们在Kd（2.5 nM）和Bmax值（1.4 pmol/mg蛋白质）方面没有发现明显差异。这些数据与以下观点一致，即串联构建体产生的多肽与高亲和力GABA位点一起表达，但无法形成GABA通道。串联构建体表达氯离子电流需要特定的单体亚基（α6或γ2），这支持了GABAA受体由两个α6、两个β2和一个γ2组成的五聚体结构（对于α6β2γ2亚型）以及由三个α6和两个β2组成的五聚体结构（对于α6β2亚型）。
