NICOTINIC RECEPTOR SITES FOR ALCOHOL ACTIONS

酒精作用的烟碱受体位点

• 批准号: 2716390
• 负责人: STUART A FORMAN
• 金额: $ 14.99万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2716390
• 关键词: Xenopus oocyte; acetylcholine; active sites; electrophysiology; ethanol; gene mutation; hydropathy; ligands; membrane channels; molecular biology; molecular cloning; neurons; nicotinic receptors; physical model; polymerase chain reaction; protein structure; receptor binding; receptor expression; site directed mutagenesis; voltage /patch clamp; voltage gated channel

This is a request for an ADAMHA Scientist/Physician Career Development Award, which will enable the candidate to fully focus on research, developing new skills in molecular biology and electrophysiology. The long term objective of the proposed research is to define the molecular mechanisms whereby ethanol and long chain alcohols act at neurotransmitter gated ion channels in the brain. The working hypothesis, based on preliminary data, is that alcohols act at protein sites on ion channels from the nicotinic acetylcholine receptor superfamily. While there is both pharmacologic and physiologic data to implicate neuronal nicotinic receptors in ethanol-induced intoxication and addictive behavior, far more is known about the actions of alcohols at the homologous nicotinic receptors from electroplaque and neuromuscular junction. In these models, long-chain alcohols bind to a hydrophobic inhibitory site within the receptor pore formed by the M2 domains of receptor subunits. Ethanol also interacts with this site, but stabilizes the open channel state. The studies proposed here use site-directed mutagenesis and rapid kinetic electrophysiology techniques to test specific features of the hypothesis in cloned/expressed nicotinic receptors from both muscle and neural tissues. Specific aim 1 is to define the molecular determinants of alcohol actions on the muscle receptor in order to better formulate models for testing in neuronal receptors. The inhibitory site for alcohols in the receptor pore will be mapped by making mutations that alter the hydrophobicity of specific amino acid side-chains in the M2 domains. To determine why ethanol fails to inhibit channels and how channel stabilization is produced, ethanol's interactions with both the channel site and the receptor's agonist binding sites will be examined. Specific aim 2 extends these studies into the clinically relevant neuronal nicotinic receptors expressed by either clonal cell lines (PC-12) or by oocytes and cells expressing cloned neuronal subunits. Ethanol and long-chain alcohol actions will be determined in the known functional neuronal receptor subunit mixtures to determine if, as anticipated, the M2 domain structure is a major determinant of drug sensitivity. Specific mechanistic hypotheses based on findings from muscle receptor studies will be tested in neuronal receptors using chimeras or site-directed mutagenesis. This grant will contribute significantly to the candidate's career development by providing time, salary support and supplies necessary for education in neurobiology as well as training in research techniques and strategies to achieve the research goals.

这是对 ADAMHA 科学家/医生职业发展的请求 奖项，这将使候选人能够完全专注于研究， 发展分子生物学和电生理学的新技能。 长的 拟议研究的术语目标是定义分子 乙醇和长链醇作用于神经递质的机制 大脑中的门控离子通道。 工作假设基于 初步数据是，醇作用于离子通道上的蛋白质位点 来自烟碱乙酰胆碱受体超家族。 虽然两者都有 涉及神经元烟碱的药理学和生理学数据 乙醇引起的中毒和成瘾行为中的受体，更多 已知醇在同源烟碱上的作用 来自电斑和神经肌肉接头的受体。 在这些模型中， 长链醇与疏水性抑制位点结合 受体孔由受体亚基的M2结构域形成。 乙醇也 与该站点交互，但稳定开放通道状态。 这 这里提出的研究使用定点诱变和快速动力学 电生理学技术来测试假设的具体特征 从肌肉和神经组织克隆/表达烟碱受体。 具体目标 1 是定义酒精作用的分子决定因素 肌肉受体，以便更好地制定模型进行测试 神经元受体。 受体孔中醇的抑制位点 将通过改变疏水性的突变来绘制 M2 结构域中的特定氨基酸侧链。 确定原因 乙醇无法抑制通道以及通道稳定性如何 产生的乙醇与通道位点和 将检查受体的激动剂结合位点。 具体目标2延伸 这些研究涉及临床相关的神经元烟碱受体 由克隆细胞系 (PC-12) 或卵母细胞和细胞表达 表达克隆的神经元亚基。 乙醇和长链醇 作用将在已知的功能性神经元受体中确定 亚基混合物以确定 M2 结构域结构是否如预期 是药物敏感性的主要决定因素。 具体机理 基于肌肉受体研究结果的假设将在 使用嵌合体或定点诱变的神经元受体。 这笔补助金 将为候选人的职业发展做出重大贡献 提供教育所需的时间、工资支持和用品 神经生物学以及研究技术和策略的培训 达到研究目标。