Reduced effect of propofol at human α1β2(N289M)γ2 and α2β3(N290M)γ2 mutant GABAA receptors
Reduced effect of propofol at human α1β2(N289M)γ2 and α2β3(N290M)γ2 mutant GABAA receptors
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DOI：
10.1093/bja/aeq023
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发表时间：
2010-04-01
影响因子：
9.8
通讯作者：
Dabrowski, M. A.
中科院分区：
文献类型：
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作者：
Fagerlund, M. Jonsson;Sjodin, J.;Dabrowski, M. A.
异丙酚是一种静脉注射药物。全身麻醉和重症监护期间常用的麻醉剂。已知GABA(A)受体β亚基的第二跨膜片段是异丙酚作用的重要靶点；然而，尚未在人类受体中对此进行研究。本研究的目的是研究异丙酚对人β2和β3GABA(A)亚基的影响，这些点突变对应于大鼠β2和β3亚基中的N265M突变。分别完成了β2和β3GABA(A)受体亚基中氨基酸位置289和290(N289M和N290M)的天冬酰胺到蛋氨酸的替换通过定点诱变。此后，将三种人类野生型（α1β2γ2、α2β2γ2和α2β3γ2）和两种突变型GABA（A）受体通道[α1β2（N289M）γ2和α2β3（N290M）γ2]的亚基引入非洲爪蟾卵母细胞中，并用两电极电压钳进行研究。突变型受体GABA 浓度-反应曲线左移。与野生型受体相比，异丙酚的正调节和激动作用在两个突变的 GABA(A) 通道上的效力和幅度均大幅降低。我们证明，人 GABA(A) β 2 和 β 3 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 诱导电流的增强以及异丙酚对包含任一突变亚基的通道的直接影响，这证实了有关大鼠受体和敲入小鼠中相应突变的早期发现。
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 beta 2 and beta 3 GABA(A) subunits with point mutations corresponding to the N265M mutation in the rat beta 2 and beta 3 subunits.Asparagine-to-methionine replacement at amino acid position 289 and 290 (N289M and N290M) in the beta 2 and beta 3 GABA(A) receptor subunits, respectively, was accomplished by site-directed mutagenesis. Thereafter, subunits for three human wild-type (alpha 1 beta 2 gamma 2, alpha 2 beta 2 gamma 2, and alpha 2 beta 3 gamma 2) and two mutant GABA(A) receptor channels [alpha 1 beta 2(N289M)gamma 2 and alpha 2 beta 3(N290M)gamma 2] 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) beta 2 and beta 3 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.