Structure and Function of Nicotinic Receptors

烟碱受体的结构和功能

• 批准号: 6766367
• 负责人: JOSE Antonio LASALDE-DOMINICCI
• 金额: $ 18.7万
• 依托单位: UNIVERSITY OF PUERTO RICO RIO PIEDRAS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766367
• 关键词: Torpedo; Xenopus; Xenopus oocyte; acetylcholine; allosteric site; diffusion; electrophysiology; gene expression; genetic regulation; intermolecular interaction; membrane activity; membrane channels; membrane lipids; membrane structure; molecular assembly /self assembly; nicotinic receptors; protein structure function; temperature; voltage /patch clamp

Changes in the structure and function of the Nicotinic Acetylcholine Receptor (AChR) are linked to pathogenic responses on human muscle and brain. The long-range goal of this research proposal is to define the functional role of lipid-protein interactions in the conformational transitions of the AChR. The objective of this project is to define the modes by which lipid protein interactions are linked to the gating machinery of the AChR. The central hypothesis of this research is that AChR channel kinetics is modulated allosterically by specific sites on the receptor that are in direct contact with the membrane lipids. This hypothesis has been formulated on the basis of strong preliminary data, which suggest that single amino acid replacements at discrete lipid-exposed positions on the M4 transmembrane segments of the AChR greatly enhanced the macroscopic response to acetylcholine. The rationale for the proposed research is that lipid-protein interactions could represent a very important mechanism for the regulation of AChR channel function in cholinergic synapses of the muscle and brain and understanding the molecular basis of these mechanisms would help to identify conditions in which the impairment of AChR function can lead to disease. The central hypothesis will be tested and the objective of the application will be accomplished by pursuing three specific aims: (1) to define helix-helix contacts, structural constraint positions and additional allosteric sites of the muscle-type AChR, using periodic tryptophan substitutions along the M4 transmembrane domains, (2) Define the role of 11 lipid exposed positions on the functional differences between the Torpedo and muscle-type AChR. (3) Perform Fluorescence Photobleaching Recovery (FRAP) to estimate translational diffusion measurements of AChRs of the Torpedo and muscle type AChRs in the plasmatic membrane of Xenopus oocytes. The proposed work is innovative because it capitalizes on new approaches developed by the applicants to estimate AChR translational diffusion constant in oocytes in vivo. It is our expectation that these approaches will define new mechanisms by which lipid-protein interactions that modulate the allosteric transitions of AChR. The outcomes will be significant because it is expected that the new knowledge will disclose new mechanisms for AChR regulation in the intact synapse. This research will be of additional significance, because it will also provide information on the secondary structure and the spatial organization of the M4 transmembrane segment, as well as to the structure-function relationships of the AChR. Another biological significance of this project is that it will provide new understanding of the role of lipid-exposed domains within the AChR and how such lipid-protein interactions can contribute to functional differences between AChR species. Analysis of the lipid-protein interactions of the AChR are not only important for understanding the functioning of ligand-gated ion channels, but may also provide insight into the mechanisms of action of certain therapeutic drugs, such as alcohols and general anesthetics.

烟碱乙酰胆碱受体（ACHR）的结构和功能的变化与人类肌肉和大脑的致病反应有关。该研究建议的远程目标是定义脂质 - 蛋白质相互作用在ACHR构象转变中的功能作用。该项目的目的是定义脂质蛋白相互作用与ACHR的门控机械相关的模式。这项研究的核心假设是，ACHR通道动力学是由特定位点的变构调节 与膜脂质直接接触的受体。该假设是基于强的初步数据提出的，该数据表明，在ACHR的M4跨膜段上，单个氨基酸替换在离散的脂质暴露位置上大大增强了对乙酰胆碱的宏观反应。拟议的研究的基本原理是，脂质 - 蛋白质相互作用可能代表了调节肌肉和大脑胆碱能突触中ACHR通道功能的非常重要的机制，并理解 这些机制的分子基础将有助于确定ACHR功能损害会导致疾病的疾病。将测试中心假设，并通过追求三个具体目的来实现应用程序的目标：（1）使用沿M4跨膜型域的定期替代方案来定义肌肉型ACHR的螺旋 - 螺旋触点，结构约束位置和其他肌肉型ACHR的其他变构位点，（2） 鱼雷和肌肉型Achr。 （3）执行荧光光漂白恢复（FRAP），以估计鱼雷和肌肉类型AChR的AChR的翻译扩散测量结果。拟议的工作具有创新性，因为它利用了申请人开发的新方法，以估计体内卵母细胞中ACHR翻译扩散常数。我们期望这些方法将定义调节脂质 - 蛋白质相互作用的新机制 ACHR的变构转变。结果将是重要的，因为预计新知识将披露完整突触中ACHR调节的新机制。这项研究将具有更大的意义，因为它还将提供有关M4跨膜段的二级结构和空间组织以及ACHR的结构 - 功能关系的信息。 该项目的另一个生物学意义是，它将对ACHR中脂质暴露结构域的作用以及这种脂质蛋白相互作用如何有助于ACHR物种之间的功能差异提供新的了解。对ACHR的脂质 - 蛋白相互作用的分析不仅对于了解配体门控离子通道的功能很重要，而且还可以洞悉某些治疗药物的作用机理，例如酒精和全身麻醉药。