Wingrove P B, Wafford K A, Bain C, Whiting P J
Merck Sharp and Dohme Research Laboratories, Harlow, Essex, United Kingdom.
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4569-73. doi: 10.1073/pnas.91.10.4569.
Type A gamma-aminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligand-gated ion channels probably formed from the coassembly of different subunits (alpha 1-6, beta 1-3, gamma 1-3, delta) in the arrangement alpha beta gamma or alpha beta delta. The activation of these receptors by GABA can be modulated by a range of compounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the beta subunit of the GABAA receptor. The action of loreclezole depends on the type of beta subunit present in the receptor complex; receptors containing beta 2 or beta 3 subunits have > 300-fold higher affinity for loreclezole than receptors containing a beta 1 subunit. We have used this property to identify the amino acid residue in the beta subunit that determines the subunit selectivity of loreclezole. Chimeric beta 1/beta 2 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human alpha 1 and gamma 2s subunits. The chimera beta 1/beta 2Lys237-Gly334 conferred sensitivity to 1 microM loreclezole. Within this region there are four amino acids that are conserved in beta 2 and beta 3 but differ in beta 1. By mutating single amino acids of the beta 1 subunit to the beta 2/beta 3 equivalent, only the beta 1 mutation of Ser-290-->Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the beta 2 and beta 3 subunits to the beta 1 equivalent (Asn-->Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors containing the mutated beta subunits. Thus, a single amino acid, beta 2 Asn-289 (beta 3 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.
哺乳动物神经系统中的A型γ-氨基丁酸（GABAA）受体是一类配体门控离子通道家族，可能由不同亚基（α1-6、β1-3、γ1-3、δ）以αβγ或αβδ的排列方式共同组装而成。GABA对这些受体的激活可被一系列作用于不同变构位点的化合物所调节。其中一种化合物是广谱抗惊厥药氯雷唑，我们最近发现它通过GABAA受体β亚基上的一个特定调节位点起作用。氯雷唑的作用取决于受体复合物中存在的β亚基类型；含有β2或β3亚基的受体对氯雷唑的亲和力比对含有β1亚基的受体高300倍以上。我们利用这一特性确定了β亚基中决定氯雷唑亚基选择性的氨基酸残基。构建了嵌合的β1/β2人GABAA受体亚基，并与人类α1和γ2s亚基在非洲爪蟾卵母细胞中共表达。嵌合体β1/β2Lys237-Gly334赋予了对1微摩尔氯雷唑的敏感性。在该区域内，有四个氨基酸在β2和β3中保守，但在β1中不同。通过将β1亚基的单个氨基酸突变为β2/β3中的对应氨基酸，只有β1亚基的Ser-290→Asn突变赋予了氯雷唑的增强作用。同样，将β2和β3亚基中的同源残基突变为β1中的对应氨基酸（Asn→Ser）导致对氯雷唑的敏感性丧失。含有突变β亚基的受体对GABA的亲和力和氟硝西泮的增强作用没有改变。因此，位于假定的通道内衬结构域TM2羧基末端的单个氨基酸β2 Asn-289（β3 Asn-290）赋予了对氯雷唑调节作用的敏感性。
