Structure and Function of Nicotinic Receptors

烟碱型乙酰胆碱受体(AChR)结构和功能的改变与人类肌肉和大脑的致病反应有关。这项研究计划的长期目标是确定脂-蛋白相互作用在AChR构象转变中的功能作用。该项目的目标是确定脂蛋白相互作用与AChR的门控机制相联系的模式。这项研究的中心假设是AChR通道动力学受AChR上特定位点的变构调节 与膜脂直接接触的受体。这一假说是基于强大的初步数据提出的，这些数据表明，AChR的M4跨膜片段上离散脂质暴露位置的单一氨基酸替换极大地增强了对乙酰胆碱的宏观反应。这项研究的理论基础是，脂-蛋白相互作用可能代表着肌肉和大脑胆碱能突触中AChR通道功能调节和理解的一个非常重要的机制 这些机制的分子基础将有助于识别AChR功能受损导致疾病的条件。中心假设将得到检验，应用的目标将通过追求三个具体目标来实现：(1)定义肌型AChR的螺旋-螺旋接触、结构约束位置和额外的变构位置，使用沿M4跨膜结构域的周期性色氨酸替代；(2)定义11个脂质暴露位置对不同类型AChR的功能差异的作用 鱼雷和肌肉型AChR。(3)用荧光光漂白恢复法(FRAP)估计鱼雷AChRs和肌型AChRs在非洲爪哇卵母细胞质膜中的平移扩散。这项拟议的工作是创新的，因为它利用了申请者开发的新方法来估计活体内卵母细胞中AChR的翻译扩散常数。我们期望，这些方法将定义新的机制，通过这些机制，脂质-蛋白质相互作用将调节 AChR的变构转变。结果将是重要的，因为预计新的知识将揭示AChR在完整突触中调节的新机制。这项研究将具有额外的意义，因为它还将提供关于M4跨膜片段的二级结构和空间组织以及AChR的结构与功能关系的信息。 该项目的另一个生物学意义是，它将提供对AChR中脂质暴露区域的作用的新理解，以及这种脂质-蛋白质相互作用如何有助于AChR物种之间的功能差异。分析AChR的脂-蛋白相互作用不仅对了解配体门控离子通道的功能很重要，而且可能有助于深入了解某些治疗药物的作用机制，如酒精和全身麻醉药。

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.