G Protein Modulation of Glycine Receptor Function and Ethanol Action

描述（由申请人提供）：甘氨酸受体（GlyR）在哺乳动物脑干和脊髓中的神经元兴奋性中起关键作用。它们的激活降低了与感觉信息、运动控制和呼吸相关的神经元的兴奋性，这些功能在乙醇（EtOH）中毒期间显著改变。我们和其他实验室的研究表明，甘氨酸激活的Cl电流可以通过天然神经元和异源表达系统中的低临床相关EtOH浓度来增强。Aguayo和其他人的研究揭示了EtOH对GlyR作用的新机制，涉及G蛋白活化的调节。我们（Aguayo）最近证明，GlyRs是由G蛋白？？通过含α 1的GlyR的TM 3 -4细胞内环内的碱性残基结构域，重要的是，我们的初步结果表明，突变体抗G？二聚体调节对EtOH增强不敏感。值得注意的是，其他受体特性（表观激动剂和拮抗剂亲和力和单通道电导）正常，全身麻醉剂对受体的调节没有改变，表明对EtOH具有显著的选择性。总之，这些数据表明，在大的细胞内环中的碱性氨基酸调节GlyR的敏感性生理相关浓度的乙醇。然而，尚未回答的主要问题是：G的重要性是什么？在表达EtOH暴露对整个动物行为的临床相关影响方面，GlyR功能的调节？为了填补我们知识的空白，我们建议将Aguayo的开创性研究扩展到整个动物水平。我们的工作假设是，乙醇影响GlyR功能的自由G？通过细胞内环中的特定区域，而G？GlyR的调节对于EtOH的行为效应是关键的。我们的主要目标是产生基因敲入小鼠，这些小鼠携带突变的α 1 GlyR，这些突变的α 1 GlyR对G β的调节不敏感，并测试这些小鼠对乙醇的细胞和行为反应。

DESCRIPTION (provided by applicant): Glycine receptors (GlyRs) play a critical role in neuronal excitability in the mammalian brain stem and spinal cord. Their activation reduces the excitability of neurons associated with sensory information, motor control and respiration, functions that are significantly altered during ethanol (EtOH) intoxication. Studies from our and other laboratories demonstrated that the glycine-activated Cl current can be potentiated by low, clinically relevant EtOH concentrations in native neurons and heterologous expression systems. Studies by Aguayo and others have revealed a novel mechanism of EtOH action on GlyRs that involves modulation by G protein activation. We (Aguayo) recently demonstrated that GlyRs are modulated by G protein ?? subunits via basic residue domains within the TM3-4 intracellular loop of a1-containing GlyRs. Importantly, our preliminary results showed that mutants resistant to G??? dimer modulation were insensitive to potentiation by EtOH. Noteworthy, other receptor properties (apparent agonist and antagonist affinities and single channel conductance) were normal and receptor modulation by general anesthetics was not altered, indicating significant selectivity for EtOH. Altogether, these data demonstrate that basic amino acids in the large intracellular loop regulate the sensitivity of GlyRs to physiologically relevant concentrations of EtOH. However, the prime question that has not yet been answered is: What is the importance of G??? modulation of GlyR function in terms of expression of the clinically relevant effects of EtOH exposure on whole animal behavior? To fill this gap in our knowledge, we propose to extend the pioneering studies of Aguayo to the whole animal level. Our working hypothesis is that EtOH affects GlyR function by free G??? through specific regions in the intracellular loop, and that G??? modulation of GlyRs is critical for the behavioral effects of EtOH. Our primary objectives are to generate gene knockin mice that harbor mutant a1 GlyRs that are insensitive to modulation by G???, and test these mice for cellular and behavioral responses to EtOH.