P1otentiation of Glycine Receptors by Ethanol

乙醇对甘氨酸受体的 P1 增强

• 批准号: 7119212
• 负责人: LUIS GERARDO AGUAYO
• 金额: $ 17.14万
• 依托单位: UNIVERSITY OF CONCEPCION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7119212
• 关键词: G protein; alcoholism /alcohol abuse; biological signal transduction; brain electrical activity; brain stem; cell line; electrophysiology; ethanol; glycine receptors; laboratory mouse; molecular cloning; neurons; phosphorylation; receptor binding; receptor expression; receptor sensitivity; spinal cord; spinal ganglion; stoichiometry; tissue /cell culture; toxicant interaction; transfection; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): Glycine receptors (GlyRs), together with GABAA and nicotinic ACh receptors, form part of the ligand-activated ion channel superfamily and play an important role in the excitability of the mammalian brain stem and spinal cord. For instance, the activation of glycine receptors (GlyRs) in brain stem, spinal cord and brain provides a main inhibitory control for neuronal excitability. Studies from our and other laboratories have shown that clinically relevant concentrations of ethanol (1-1 O0 mM) enhanced the function of these receptors in hippocampal, cortical and spinal neurons. We have recently published that GlyRs are regulated by G protein activation. In addition, our study indicated that this novel modulation of GlyR was via the G-beta-gamma dimer. The mechanisms by which ethanol is able to affect GlyRs had remained largely unknown. An important line of research has dealt with the search of sites that permit the binding of ethanol within the GlyR. For instance, single residue mutations in S267 within the alpha1 GlyR was reported to change the sensitivity to 200 mM ethanol by more than 50%. Overall, these studies concluded that ethanol effects on GlyRs could be related to the amino acid sequence inTM2 andTM3: Based on our recent paper and preliminary results, we have proposed the hypothesis that the effects of low ethanol concentrations depend on the activation of G proteins, more than the presence of sites that directly bind the ethanol molecule, For example, GlyR sensitivity to ethanol was significantly modified by changing the stoichiometry of the heterotrimeric complex: More importantly, the potentiation of the GlyR was completely blocked by overexpression of Gl3y subunits (occlusion), as well as a G-beta-gamma scavenger peptide (ct-GRK). In order to characterize the importance- of G protein activation on ethanol sensitivity; we will study G protein modulators using heterologous expression of G protein subunits in HEK cells, electrophysiology, receptor mutagenesis and Western blot techniques. Key experiments will be performed in cultured spinal and dorsal root ganglion (DRG) neurons to confirm the major effects in a neuronal substrate. Such information will help us to understand the mechanisms by which ethanol affects these inhibitory receptors, which are important in functions such as convulsions, sensory integration, muscle tone and respiration. In summary, this proposal may provide support for a novel mechanism of ethanol effects in motor and sensory functions.

描述(申请人提供)：甘氨酸受体(GlyRs)与GABAA和尼古丁ACh受体一起，构成配体激活的离子通道超家族的一部分，在哺乳动物脑干和脊髓的兴奋性中发挥重要作用。例如，脑干、脊髓和脑中的甘氨酸受体(GlyRs)的激活为神经元的兴奋性提供了主要的抑制控制。我们和其他实验室的研究表明，临床上相关浓度的乙醇(1-10 mM)增强了海马神经元、皮质神经元和脊髓神经元中这些受体的功能。我们最近发表了GlyRs是由G蛋白激活来调节的。此外，我们的研究表明，这种对GlyR的新调节是通过G-β-Gamma二聚体实现的。 乙醇能够影响GlyRs的机制在很大程度上仍不清楚。一条重要的研究路线涉及搜索允许乙醇在GlyR内结合的位点。例如，据报道，Alpha1 GlyR中S267的单个残基突变会使人对200 mM乙醇的敏感度改变50%以上。总体而言，这些研究得出的结论是，乙醇对GlyR的影响可能与TM2和TM3中的氨基酸序列有关：根据我们最近的论文和初步结果，我们提出了这样的假设，即低浓度乙醇的影响取决于G蛋白的激活，而不是直接与乙醇分子结合的位点的存在，例如，通过改变异三聚体复合体的化学计量比，GlyR对乙醇的敏感性显著改善：更重要的是，GlyR的增强完全被Gl3y亚基(Occlusion)和G-β-γ清除肽(ct-GRK)的过度表达所阻断。 为了研究G蛋白激活在乙醇敏感性中的重要性，我们将利用G蛋白亚基在HEK细胞中的异源表达、电生理学、受体突变和Western印迹技术来研究G蛋白调节剂。将在培养的脊髓和背根神经节(DRG)神经元中进行关键实验，以确认神经元基质的主要作用。这些信息将帮助我们理解乙醇影响这些抑制性受体的机制，这些受体在抽搐、感觉整合、肌肉张力和呼吸等功能中非常重要。综上所述，这一建议可能为酒精对运动和感觉功能影响的新机制提供支持。