A single glycine residue at the entrance to the first membrane-spanning domain of the γ-aminobutyric acid type a receptor β2 subunit affects allosteric sensitivity to GABA and anesthetics
A single glycine residue at the entrance to the first membrane-spanning domain of the γ-aminobutyric acid type a receptor β2 subunit affects allosteric sensitivity to GABA and anesthetics
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DOI：
10.1124/mol.57.3.474
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发表时间：
2000-03-01
影响因子：
3.6
通讯作者：
Olsen, RW
中科院分区：
文献类型：
--
作者：
Carlson, BX;Engblom, AC;Olsen, RW
γ -氨基丁酸A（GABA(A)）受体β 2亚基的定点突变表明，位于第一个跨膜结构域入口的保守甘氨酸残基转化为同源的rho(1)残基苯丙氨酸，改变了四种不同静脉麻醉药物的调节作用：戊巴比妥、阿尔法xalone、依托咪酯和异丙酚。利用杆状病毒在frugiperda Spodoptera 9细胞中的表达系统，麻醉诱导的[H-3]muscimol和[H-3]氟硝西泮与含有β 2（G219F）点突变的受体结合的增强显示，所有四种静脉注射麻醉药的调节效果都显着降低。此外，含有α (1)（G223F）点突变的GABA(A)受体也显著降低了依托咪酯和异丙酚诱导的配体结合增强的最大效应。相反，rho(1)受体（F261G）的同源点突变改变了静脉麻醉不敏感受体，赋予麻醉调节[H-3]muscimol结合。与结合一致，全细胞膜片钳记录的戊巴比妥增强GABA电流的功能分析表明β 2（G219F）亚基突变消除了麻醉剂的增强作用。同样，异丙酚增强的GABA电流在α (1) β (2)(G219F) γ(2)受体中的增强程度低于α (1) β (2) γ(2)受体。尽管配体结合在野生型、α (1) β (2) γ(2)和突变受体中显示出相似的K-D值，但膜片钳记录显示，α (1) β (2)(G219F) γ(2)受体对GABA的反应明显比α (1) β (2) γ(2)或α (1)(G223F) β (2) γ(2)更有效。在缺乏GABA的情况下，α (1) β (2)(G219F) γ(2)受体对戊巴比妥和异丙酚的直接通道激活也更敏感。这些结果表明，β(2)亚基上的第一个跨膜甘氨酸残基可能对GABA和麻醉药通道门化的构象或变构相互作用很重要。
Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) receptor beta 2 subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [H-3]muscimol and [H-3]flunitrazepam binding in receptors containing the beta 2(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in rho(1) receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [H-3]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the beta 2(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in alpha(1)beta(2)(G219F)gamma(2) receptors than in alpha(1)beta(2)gamma(2) receptors. Although ligand binding displayed comparable K-D values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, patch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors had a significantly more potent response to GABA than did alpha(1)beta(2)gamma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.