Potentiation of Glycine Receptors by Ethanol

描述（由申请人提供）：脑干、脊髓和脑中甘氨酸受体（GlyR）的激活为神经元兴奋性提供了主要抑制控制。毒素和药物如乙醇对这些受体的修饰导致从镇静和肌肉松弛到抽搐、呼吸停止和死亡的广泛影响。然而，乙醇影响这些受体的机制在很大程度上是未知的。最近的研究表明，GlyR的功能被GTP结合蛋白活性的变化所改变。具体而言，使用一系列GlyR突变体，发现了负责G β γ结合和受体调节的碱性氨基酸基序的存在。更重要的是，初步结果显示，GlyR的主要胞内环中的碱性氨基酸决定了受体对临床相关浓度的乙醇（10-100 mM）的敏感性。这些氨基酸还调节GlyR与G β γ的相互作用。与这些发现一致，发现GlyR对乙醇的敏感性通过改变异源三聚体复合物的化学计量而显著改变。因此，假设乙醇敏感性的重要决定因素在细胞内环内，并且GlyR对乙醇的敏感性取决于G蛋白活化的状态。值得注意的是，显示这些位点不参与丙泊酚、异氟烷和长链醇对GlyR的调节，表明乙醇的新的和选择性的新位点。此外，这些位点的突变不影响GlyR的生理特性，表明乙醇抗性表型与通道门控的变化无关。因此，本研究的主要目的是通过使用受体诱变、GTP结合G蛋白的动力学分析、G蛋白亚基的异源表达、膜片钳和Western印迹技术来表征负责G蛋白活化和GlyR对乙醇敏感性的细胞内位点。此外，将使用在神经元背景（即DRG神经元）中表达的WT和突变GlyR进行关键实验。从这项研究中获得的信息将有助于了解乙醇影响甘氨酸受体的机制，甘氨酸受体在惊厥、感觉统合、肌张力和呼吸等功能中非常重要。因此，这些结果可能有助于开发新的酒精中毒治疗方法。乙醇的过度消费是美国和世界范围内的一个主要健康相关问题。然而，乙醇产生的大脑功能变化的主要决定因素在很大程度上仍然是未知的。因此，重要的是要确定乙醇作用的机制和位点，以了解乙醇如何影响中枢神经系统，并提出有用的治疗方法，以减轻与过度消费相关的神经健康问题。

DESCRIPTION (provided by applicant): Activation of glycine receptors (GlyRs) in brain stem, spinal cord and brain provides a main inhibitory control for neuronal excitability. Modification of these receptors by toxins and drugs such as ethanol leads to a wide range of effects varying from sedation and muscle relaxation to convulsions, respiratory arrest and death. However, the mechanism by which ethanol affects these receptors is largely unknown. Recent studies have indicated that GlyR function is modified by changes in GTP-binding protein activity. Specifically, using a series of GlyR mutants, the presence of basic amino acid motifs that are responsible for G beta gamma binding and receptor modulation were discovered. More importantly, preliminary results show that basic amino acids in the main intracellular loop of the GlyR determine the sensitivity of the receptor to clinically relevant concentrations of ethanol (10-100 mM). These amino acids also regulate the interaction of the GlyR with G beta gamma. In agreement with these findings, it was found that GlyR sensitivity to ethanol was significantly modified by changing the stoichiometry of the heterotrimeric complex. Therefore, it was hypothesized that important determinants for ethanol sensitivity are within the intracellular loop and that the sensitivity of GlyR to ethanol depends on the state of G protein activation. Remarkably, it was shown that these sites did not participate in the modulation of GlyRs by propofol, isoflurane and long chain alcohols indicating a novel and selective new site for ethanol. Additionally, mutations in these sites did not affect the physiological properties of the GlyR indicating that the ethanol resistant phenotype was not related to changes on channel gating. Therefore, the primary objective of this study is to characterize the intracellular sites responsible for G protein activation and GlyR sensitivity to ethanol by using receptor mutagenesis, kinetic analysis of GTP binding to G proteins, heterologous expression of G protein subunits, patch-clamp and Western blot techniques. In addition, key experiments will be performed using WT and mutant GlyRs expressed in a neuronal background (i.e. DRG neurons). The information obtained from this study will help to understand the mechanism by which ethanol affects glycine receptors, which are important in functions such as convulsions, sensory integration, muscle tone and respiration. Therefore, these results may contribute to the development of new treatments for alcoholism. PUBLIC HEALTH RELEVANCE Excessive consumption of ethanol is a main health-related problem in the US and world wide. However, the main determinants for the changes in the brain functions produced by ethanol are still largely unknown. Therefore, it is important to identify the mechanism and sites of ethanol action in order to understand how ethanol affects the central nervous system and to propose useful therapies to alleviate neurological health problems associated with its excessive consumption.