Carlson B X, Engblom A C, Kristiansen U, Schousboe A, Olsen R W
Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen, Denmark.
Mol Pharmacol. 2000 Mar;57(3):474-84. doi: 10.1124/mol.57.3.474.
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 [(3)H]muscimol and [(3)H]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 [(3)H]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.
γ-氨基丁酸A型（GABA(A)）受体β(2)亚基的定点诱变表明，位于第一个跨膜结构域入口处的保守甘氨酸残基转换为同源的rho(1)残基苯丙氨酸，会改变四种不同静脉麻醉药的调节作用：戊巴比妥、α-香豆素、依托咪酯和丙泊酚。利用杆状病毒表达系统在草地贪夜蛾9细胞中进行实验，在含有β(2)(G219F)点突变的受体中，麻醉药诱导的[(3)H]蝇蕈醇和[(3)H]氟硝西泮结合增强在所有四种测试的静脉麻醉药调节下，显示出显著降低的效力。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丙泊酚诱导的配体结合增强的最大效应。相反，rho(1)受体中的同源点突变（F261G）改变了对静脉麻醉药不敏感的受体，使其对[(3)H]蝇蕈醇结合具有麻醉药调节作用。与结合情况一致，用全细胞膜片钳记录戊巴比妥增强的GABA电流的功能分析表明，β(2)(G219F)亚基突变消除了麻醉药的增强作用。同样，在α(1)β(2)(G219F)γ(2)受体中，丙泊酚增强的GABA电流的增强程度低于α(1)β(2)γ(2)受体。尽管配体结合在野生型、α(1)β(2)γ(2)和突变型受体之间显示出对蝇蕈醇具有可比的K(D)值，但膜片钳记录表明，α(1)β(2)(G219F)γ(2)受体对GABA的反应比α(1)β(2)γ(2)或α(1)(G223F)β(2)γ(2)受体显著更强。α(1)β(2)(G219F)γ(2)受体对戊巴比妥和丙泊酚在无GABA时直接激活通道也更敏感。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和麻醉药介导通道门控的构象或变构相互作用很重要。
