Department of Anesthesiology and Intensive Care Medicine, Karolinska University Hospital, Stockholm, Sweden.
Br J Anaesth. 2010 Apr;104(4):472-81. doi: 10.1093/bja/aeq023. Epub 2010 Feb 23.
Propofol is an i.v. anaesthetic commonly used during general anaesthesia and intensive care. It is known that the second transmembrane segment of the beta subunit in the GABA(A) receptor is an important target for the effects of propofol; however, this has not been investigated in human receptors. The aim of this study was to investigate the effect of propofol on human beta2 and beta3 GABA(A) subunits with point mutations corresponding to the N265M mutation in the rat beta2 and beta3 subunits.
Asparagine-to-methionine replacement at amino acid position 289 and 290 (N289M and N290M) in the beta2 and beta3 GABA(A) receptor subunits, respectively, was accomplished by site-directed mutagenesis. Thereafter, subunits for three human wild-type (alpha1beta2gamma2, alpha2beta2gamma2, and alpha2beta3gamma2) and two mutant GABA(A) receptor channels [alpha1beta2(N289M)gamma2 and alpha2beta3(N290M)gamma2] were introduced into Xenopus oocytes and studied with two-electrode voltage clamp.
The mutant receptors left-shifted the GABA concentration-response curve. In comparison with the wild-type receptors, both the positive modulatory and the agonistic effects of propofol were strongly reduced in potency and amplitude at both mutated GABA(A) channels.
We demonstrate that N289M or N290M mutation in human GABA(A) beta2 and beta3 subunits increases sensitivity to GABA, which is in contrast to the corresponding rat N265M mutation. Furthermore, the N289M and N289M mutations reduce both the potentiation of GABA-induced currents and the direct effect of propofol on channels incorporating either of the mutated subunits, which confirms earlier findings concerning the corresponding mutation in rat receptors and knock-in mice.
异丙酚是一种常用于全身麻醉和重症监护的静脉麻醉剂。已知 GABA(A) 受体的β亚基的第二个跨膜段是异丙酚作用的重要靶点；然而，这在人类受体中尚未得到研究。本研究的目的是研究异丙酚对人类β2 和 β3 GABA(A) 亚基的影响，这些亚基对应于大鼠β2 和 β3 亚基中的 N265M 突变。
通过定点诱变，分别在β2 和β3 GABA(A) 受体亚基的氨基酸位置 289 和 290 处将天冬酰胺替换为蛋氨酸（N289M 和 N290M）。此后，将三个野生型（α1β2γ2、α2β2γ2 和α2β3γ2）和两个突变 GABA(A) 受体通道（α1β2(N289M)γ2 和α2β3(N290M)γ2）的亚单位引入非洲爪蟾卵母细胞，并通过双电极电压钳进行研究。
突变受体使 GABA 浓度-反应曲线向左移位。与野生型受体相比，在两个突变的 GABA(A) 通道中，异丙酚的正变构和激动作用的效力和幅度都明显降低。
我们证明，人类 GABA(A)β2 和β3 亚基中的 N289M 或 N290M 突变增加了对 GABA 的敏感性，这与相应的大鼠 N265M 突变相反。此外，N289M 和 N289M 突变降低了 GABA 诱导电流的增强作用以及异丙酚对包含突变亚基的通道的直接作用，这证实了先前关于大鼠受体和敲入小鼠的相应突变的发现。
