G Protein Modulation of Glycine Receptor Function and Ethanol Action

描述(申请人提供)：甘氨酸受体(GlyRs)在哺乳动物脑干和脊髓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，在天然神经元和异源表达系统中，低浓度的临床相关乙醇可以增强甘氨酸激活的氯电流。Aguayo等人的研究揭示了乙醇对GlyRs的一种新的作用机制，涉及到G蛋白激活的调制。我们(阿瓜约)最近证明了GlyRs是由G蛋白调节的？？通过含有A1的GlyRs的TM3-4细胞内环中的基本残基结构域。重要的是，我们的初步结果表明，突变株对G？二聚体的调节对乙醇的增强不敏感。值得注意的是，其他受体特性(明显的激动剂和拮抗剂亲和力和单通道电导)是正常的，全身麻醉剂对受体的调节没有改变，表明乙醇具有显著的选择性。总之，这些数据表明，细胞内大环中的碱性氨基酸调节GlyRs对生理相关浓度的乙醇的敏感性。然而，尚未得到回答的首要问题是：G的重要性是什么？乙醇暴露对整个动物行为的临床相关影响的表达对GlyR功能的调节？为了填补这一知识空白，我们建议将阿瓜约的开创性研究扩展到整个动物层面。我们的工作假设是乙醇通过游离G？影响GlyR功能。通过细胞内环中的特定区域，而G？GlyRs的调节对乙醇的行为效应至关重要。我们的主要目标是培育出对G？调节不敏感的突变A1-GlyR基因敲击小鼠，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.