An isoflurane- and alcohol-insensitive mutant GABAA receptor α1 subunit with near-normal apparent affinity for GABA:: Characterization in heterologous systems and production of knockin mice
An isoflurane- and alcohol-insensitive mutant GABAA receptor α1 subunit with near-normal apparent affinity for GABA:: Characterization in heterologous systems and production of knockin mice
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DOI：
10.1124/jpet.106.104406
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发表时间：
2006-10-01
影响因子：
3.5
通讯作者：
Harrison, N. L.
中科院分区：
文献类型：
--
作者：
Borghese, C. M.;Werner, D. F.;Harrison, N. L.
挥发性麻醉剂和酒精可增强中枢神经系统中γ-氨基丁酸A型受体（GABA（A）Rs）介导的传递，这种作用可能是这些药物的某些行为作用的基础。用较大的组氨酸残基取代GABA（A）R α（1）亚基270位的关键丝氨酸残基，消除了异氟烷对受体的调节，但也影响了受体门控（增加GABA敏感性）。为了纠正这种突变体的GABA敏感性的变化，我们将第二个残基，277位的亮氨酸突变为丙氨酸。双突变体α（1）（S270 H，L277 A）β（2）γ（2S）GABA（A）R在非洲爪蟾卵母细胞和人胚肾（HEK）293细胞中表达，具有接近正常的GABA敏感性。然而，快速应用一个简短的GABA脉冲在HEK 293细胞中表达的受体显示，失活更快的双突变体比野生型受体。在所有异源系统中，异氟烷和乙醇的增强作用在双突变受体中大大降低。携带双突变的纯合子敲入小鼠是可行的，除了活动过度外，没有明显的异常。这种敲入小鼠系在确定挥发性麻醉剂和乙醇的哪些行为作用是由含有α（1）亚基的GABA（A）受体介导的方面应该是有用的。
Volatile anesthetics and alcohols enhance transmission mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) in the central nervous system, an effect that may underlie some of the behavioral actions of these agents. Substituting a critical serine residue within the GABA(A)R alpha(1) subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating ( increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant alpha(1)(S270H,L277A)beta(2)gamma(2S) GABA(A)R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA(A)Rs containing the alpha(1) subunit.