Connor J X, Boileau A J, Czajkowski C
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 1998 Oct 30;273(44):28906-11. doi: 10.1074/jbc.273.44.28906.
gamma-Aminobutyric acid, type A (GABAA) receptors, the major inhibitory neurotransmitter receptors in the central nervous system, are heteropentameric proteins assembled from distinct subunit classes with multiple subtypes, alpha(1-6), beta(1-4), gamma(1-3), delta(1), and epsilon(1). To examine the process of receptor assembly and targeting, we tagged the carboxyl terminus of the GABAA receptor alpha1 subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, alpha1-EGFP, alone or in combination with cRNA of GABAA receptor beta2, gamma2, or beta2+gamma2 subunits. Within 72 h after injection, EGFP fluorescence was visible in all fusion protein-injected cells. The fluorescence was associated with the plasmalemma only when the beta2 subunit was co-injected with alpha1-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the alpha1 subunit is extracellular. Two-electrode voltage clamp of alpha1-EGFPbeta2- and alpha1-EGFPbeta2 gamma2-injected oocytes demonstrates that these cells express functional receptors, with EC50 values for GABA and diazepam similar to wild-type receptors. Thus, a COOH-terminal tag of the alpha1 subunit appears to be functionally silent, providing a useful marker for studies of GABAA receptor expression, assembly, transport, targeting, and clustering. Moreover, the beta2 subunit is required for receptor assembly and surface expression.
γ-氨基丁酸A型（GABAA）受体是中枢神经系统中主要的抑制性神经递质受体，是由不同亚基类别组装而成的异五聚体蛋白，具有多种亚型，包括α(1 - 6)、β(1 - 4)、γ(1 - 3)、δ(1)和ε(1)。为了研究受体组装和靶向过程，我们用红移增强型绿色荧光蛋白（EGFP）标记了GABAA受体α1亚基的羧基末端。将这种融合蛋白α1-EGFP的cRNA单独或与GABAA受体β2、γ2或β2 + γ2亚基的cRNA一起注射到非洲爪蟾卵母细胞中。注射后72小时内，在所有注射融合蛋白的细胞中都能看到EGFP荧光。只有当β2亚基与α1-EGFP共同注射时，荧光才与质膜相关。对未通透细胞上的EGFP进行德克萨斯红偶联免疫标记表明，EGFP定位于细胞外。因此，α1亚基的COOH末端位于细胞外。对注射了α1-EGFPβ2和α1-EGFPβ2γ2的卵母细胞进行双电极电压钳实验表明，这些细胞表达功能性受体，GABA和地西泮的EC50值与野生型受体相似。因此，α1亚基的COOH末端标签在功能上似乎是沉默的，为研究GABAA受体的表达、组装、运输、靶向和聚集提供了一个有用的标记。此外，受体组装和表面表达需要β2亚基。
