Nishikawa K, Jenkins A, Paraskevakis I, Harrison N L
C.V. Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology, Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021, USA.
Neuropharmacology. 2002 Mar;42(3):337-45. doi: 10.1016/s0028-3908(01)00189-7.
Previous studies have suggested that two specific amino acid residues in transmembrane segments 2 and 3 of the GABA(A) receptor alpha 2 subunit, Ser270 and Ala291, are critical for the enhancement of GABA(A) receptor function by inhaled anesthetics. The aim of this study was to determine the effects of amino acid substitutions in alpha 1 beta 2 gamma 2s GABA(A) receptors at alpha 1(S270) and at the homologous beta 2(N265) on receptor gating and anesthetic potentiation of GABA-induced responses. The wild-type and mutant receptors were transiently expressed in HEK 293 cells and GABA-induced currents were recorded using whole-cell voltage clamp. Potentiation of responses to a submaximal concentration of GABA by the anesthetics halothane and isoflurane was also examined. Some of the point mutations caused shifts in the GABA dose-response curve, indicating that the mutations changed the apparent affinity of the receptor for GABA. In receptors mutated at alpha 1(S270), the GABA EC(50) is inversely correlated with the volume of the residue of 270. On the contrary, there was no clear relationship between the physical properties of the amino acid residue at 265 in the beta 2 subunit and either the GABA EC(50) or anesthetic modulation, although mutations at N265 altered both parameters in a quantitative manner. These data are consistent with the results of previous work using other subunit combinations, in confirming that alpha 1(S270) may be involved in channel gating, and also may be important in anesthetic binding; the role of beta 2(N265) is less clear.
先前的研究表明，GABA(A)受体α2亚基跨膜片段2和3中的两个特定氨基酸残基Ser270和Ala291，对于吸入麻醉药增强GABA(A)受体功能至关重要。本研究的目的是确定α1β2γ2s GABA(A)受体中α1(S270)和同源β2(N265)处的氨基酸替换对受体门控以及GABA诱导反应的麻醉增强作用的影响。野生型和突变型受体在HEK 293细胞中瞬时表达，使用全细胞膜片钳记录GABA诱导的电流。还检测了麻醉药氟烷和异氟烷对亚最大浓度GABA反应的增强作用。一些点突变导致GABA剂量反应曲线发生偏移，表明这些突变改变了受体对GABA的表观亲和力。在α1(S270)处发生突变的受体中，GABA EC(50)与270位残基的体积呈负相关。相反，β2亚基中265位氨基酸残基的物理性质与GABA EC(50)或麻醉调节之间没有明确的关系，尽管N265处的突变以定量方式改变了这两个参数。这些数据与先前使用其他亚基组合的研究结果一致，证实α1(S270)可能参与通道门控，并且在麻醉药结合中也可能很重要；β2(N265)的作用尚不清楚。
