Cholesterol & protein-lipid effects on Ach Receptor

描述(由申请人提供)：结构和功能的变化 烟碱型乙酰胆碱受体(AChR)与致病反应有关 在人类的肌肉和大脑上。这项研究提案的长期目标是 确定脂质-蛋白质相互作用在构象中的功能作用 ACBR的过渡。本项目的目标是定义模式 其中疏水变构相互作用通过其与浇注机械相连 AChR和弓形胆固醇调节ACbR功能。中心假说 这项研究的重点是AChR通道动力学是由变构调节的 受体上与受体直接接触的特定部位 脂类界面。这一假设是在强势的基础上提出的 初步数据表明：(1)单一氨基酸在 猪瘟病毒M3和M4膜节段的疏水变构位点 乙酰胆碱受体显著增强对乙酰胆碱的宏观反应，并产生一种 胆固醇对乙酰胆碱受体调节作用的显著改变 功能；(2)胆固醇在AChRs的转变中起着新的作用 从无声的膜池到功能膜池。建议的理由是 研究表明，膜脂成分(和脂蛋白)的变化 相互作用)是调节AChR的一种非常重要的机制 肌肉和大脑胆碱能突触的通道功能，因此 了解这些机制的分子基础将有助于识别 膜脂成分增强或抑制AChR的条件 功能。核心假设将通过追求四个具体目标来检验： (1)研究五种疏水性变构化合物的构效关系 使用额外侧链替换的位置，(2)引入周期性 色氨酸取代完成M3跨膜上的所有位置 定义结构域以定义螺旋-螺旋接触、结构约束 位置和额外的变构位置，(3)考察膜的影响 胆固醇水平对乙酰胆碱受体通道动力学的影响及五种新的疏水类固醇 M3突变和(4)在疏水变构上引入非天然氨基酸 确定M3域中的位置；(A)电子密度的影响 对沟道浇注特性和(B)膜渗透深度的影响 变构位置。这项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：1)执行胆固醇 卵母细胞质膜中的浓缩和亏损；2) 引入荧光非天然氨基酸评价膜深度渗透 以及使用多光子共聚焦成像的环状脂质成分。我们期待着 这些方法将使我们能够确定新的机制，通过这些机制 脂-蛋白相互作用调节AChR的变构转变。这个 从这些研究中获得的知识具有至关重要的意义，因为它将：1) 描述完整突触中AChR调节的新机制，2)提供 关于M3的二级结构和空间组织的信息和 M4结构域，以及3)描述了AChR的新的结构-功能关系。

DESCRIPTION (provided by applicant): Changes in the structure and function of the Nicotinic Acetyicholine Receptor (AChR) are linked to pathogenic responses on human muscle and brain. The long-term goal of this research proposal is to define the functional role of lipid-protein interactions in the conformational transitions of the ACbR. The objective of this project is to define the modes by which hydrophobic allosteric interactions are linked to the gating machinery of the AChR and bow cholesterol modulates ACbR function. 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 lipid-interface. This hypothesis has been formulated on the basis of strong preliminary data, which suggest that: (1) single amino acid replacements at hydrophobic allosteric sites of the M3 and M4 iransmembrane segments of the AChR greatly enhance the macroscopic response to acetyicholine and produce a remarkable alteration of the modulatory role of cholesterol on the AChR function, and (2) cholesterol plays a novel role in the transition of AChRs from silent to functional membrane pools. The rationale for the proposed research is that changes in membrane lipid composition (and lipid-protein interactions) represent a very important mechanism for the regulation of AChR channel function in cholinergic synapses of muscle and brain, thus understanding the molecular basis of these mechanisms will help to identify conditions in which the membrane lipid composition enhance or inhibit AChR function. The central hypothesis will be tested by pursuing four specific aims: (1) To study the structure-function relationship of five hydrophobic allosteric positions using additional side chain replacements, (2) Introduce periodic tryptophan substitutions to complete all positions along the M3 transmembrane domains in order to define helix-helix contacts, structural constraint positions and additional allosteric sites, (3) Examine the effects of membrane cholesterol levels on AChR channel kinetics and five new hydrophobic ailosteric M3 mutations and (4) Introduce unnatural amino acids at hydrophobic allosteric positions in the M3 domain to determine; (a) the effect of electronic density on channel gating properties and (b) membrane penetration depth of the allosteric positions. The proposed work is innovative because it capitalizes on new approaches, developed by the applicants, to; 1) perform cholesterol enrichment and depletions in the plasmatic membrane of oocytes, and 2) introduce fluorescent unnatural amino acid to assess membrane depth penetration and annular lipid composition using multiphoton confocal imaging. We anticipate that these approaches will allow us to identify new mechanisms by which lipid-protein interactions modulate the allosteric transitions of AChR. The knowledge gained from these studies is of paramount significance as it will: 1) describe new mechanisms for AChR regulation in the intact synapse, 2) provide information on the secondary structure and spatial organization of the M3 and M4 domains, and 3) describe new structure-function relationships for the AChR.