Khan Z U, Gutiérrez A, De Blas A L
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110-2499.
J Neurochem. 1994 Oct;63(4):1466-76. doi: 10.1046/j.1471-4159.1994.63041466.x.
Three novel antisera to the gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor/benzodiazepine receptor (GABAAR/BZDR) complex have been made. Anti-gamma 2S and anti-gamma 2L are specific antibodies to synthetic peptides that recognize the gamma 2S (short) and gamma 2L (long) forms, respectively, of the gamma 2 subunit. An antibody (anti-gamma 2IL2) to staphylococcal protein A fusion protein of the large intracellular loop (gamma 2IL) located between the putative transmembrane segments M3 and M4 of gamma 2S recognizes both gamma 2S and gamma 2L subunits. The antibodies immunoprecipitated both the solubilized and affinity-purified GABAAR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity-purified receptor from bovine cortex show that anti-gamma 2S and anti-gamma 2L recognize peptides of 45,000 and 47,000 M(r), respectively. Immunoprecipitation experiments indicate that gamma 2S is more prevalent in hippocampus, whereas gamma 2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of gamma 2S and gamma 2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both gamma 2S and gamma 2L subunits and 41-48% of the gamma 2L subunit coexists with gamma 2S in the same receptor complex. In cerebellum, in 27% of the clonazepam-sensitive and 39% of the clonazepam-insensitive BZDRs the gamma 2S and gamma 2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain alpha 6. In addition, almost all (93%) the clonazepam-insensitive BZDRs that contain gamma 2L also contain a gamma 2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain alpha 6, gamma 2S, and gamma 2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain alpha 6 subunit(s) have neither gamma 2S nor gamma 2L subunits. There are also species differences with respect to the relative abundance of gamma 2S and gamma 2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABAAR/BZDR-mediated effects of ethanol intoxication involving cerebellar granule cells.
已制备出三种针对γ-氨基丁酸A（GABAA）受体/苯二氮䓬受体（GABAAR/BZDR）复合物γ2亚基的新型抗血清。抗γ2S和抗γ2L是针对合成肽的特异性抗体，分别识别γ2亚基的γ2S（短）和γ2L（长）形式。一种针对位于γ2S假定跨膜片段M3和M4之间的大细胞内环（γ2IL）的葡萄球菌蛋白A融合蛋白的抗体（抗γ2IL2）可识别γ2S和γ2L亚基。这些抗体可免疫沉淀大鼠和牛脑中可溶解的以及亲和纯化的GABAAR/BZDR。用大鼠脑皮质膜以及牛皮质亲和纯化受体进行的免疫印迹表明，抗γ2S和抗γ2L分别识别分子量为45,000和47,000的肽段。免疫沉淀实验表明，γ2S在海马体中更为普遍，而γ2L在小脑中更为丰富。在大脑皮质中发现每种形式的中间值。结果表明，在大鼠脑中，γ2S和γ2L在同一受体复合物中有相当数量的共定位。在大脑皮质中，15%的BZDR同时含有γ2S和γ2L亚基，41 - 48%的γ2L亚基与γ2S在同一受体复合物中共存。在小脑中，27%对氯硝西泮敏感和39%对氯硝西泮不敏感的BZDR中，γ2S和γ2L在同一受体复合物中共存。后者可能定位于颗粒细胞中，并且还含有α6。此外，几乎所有（93%）含有γ2L的对氯硝西泮不敏感的BZDR在同一受体复合物中也含有γ2S亚基。对结果最可能的解释是，有大量颗粒细胞受体在同一受体复合物中同时含有α6、γ2S和γ2L。然而，31%含有α6亚基的小脑受体既不含有γ2S也不含有γ2L亚基。关于γ2S和γ2L的相对丰度也存在物种差异。这些结果可能与理解一些涉及小脑颗粒细胞的乙醇中毒的GABAAR/BZDR介导效应的分子机制相关。
