The University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712, United States.
University of Pittsburgh, Departments of Anesthesiology, Neurobiology, and Pharmacology & Chemical Biology, Pittsburgh, PA 15261, United States.
Neuropharmacology. 2017 Sep 1;123:201-209. doi: 10.1016/j.neuropharm.2017.06.013. Epub 2017 Jun 13.
Genes encoding the ρ1/2 subunits of GABA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABA ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans.
GABA 受体 ρ1/2 亚基的编码基因与人的酒精（乙醇）依赖有关，ρ1 也被证明可调节动物模型中乙醇的某些行为效应。乙醇抑制野生型（WT）ρ1 介导的 GABA 反应，但不抑制在非洲爪蟾卵母细胞中表达的 ρ1(T6'Y)突变受体，这表明在第二跨膜螺旋中存在乙醇的抑制性结合位点。在这项研究中，我们发现表达在卵母细胞中的 ρ1(T6'Y)受体对 GABA、内源性 GABA 调节剂（锌）和部分激动剂（β-丙氨酸和牛磺酸）的整体反应正常。我们使用 CRISPR/Cas9 生成 ρ1(T6'Y)敲入（KI）小鼠，以测试乙醇对该受体的抑制作用的行为重要性。与 WT 小鼠相比，ρ1 KI 和敲除（KO）小鼠均显示出更快从急性乙醇诱导的运动不协调中恢复。两种突变株（KI 和 KO）还显示出对乙醇引起的运动障碍的耐受性增加。与 WT 小鼠相比，KI 小鼠在其他行为作用方面没有差异，包括乙醇摄入和偏好、乙醇条件性味觉厌恶和乙醇诱导的翻正反射丧失的持续时间。WT 和 KI 小鼠在小脑的 ρ1 或 ρ2 mRNA 水平或乙醇清除率方面没有差异。我们的研究结果表明，GABA ρ1 受体中乙醇的抑制性结合位点调节急性功能性对中度乙醇中毒的耐受。我们注意到，对醉酒的低敏感性与人的酒精依赖发展风险有关。
