Cholesterol & protein-lipid effects on Ach Receptor

描述（由申请人提供）：结构和功能的变更 烟碱乙酰胆碱受体（AChR）与致病反应有关 对人体肌肉和大脑的影响这项研究计划的长期目标是 定义脂质-蛋白质相互作用在构象中的功能作用 ACbR的转换。本项目的目标是定义模式 疏水性变构相互作用与门控机制相联系 和弓胆固醇调节ACbR功能。核心假设 AChR通道动力学是由 受体上的特异性位点直接与 脂质界面这一假设是建立在强有力的 初步数据，这表明：（1）单个氨基酸替换， 的M3和M4跨膜区段的疏水变构位点。 AChR大大增强了对乙酰胆碱的宏观反应，并产生了一种抑制剂。 胆固醇对AChR的调节作用显著改变 胆固醇在AChRs的转换中起着新的作用 从沉默到功能性膜池。建议的理由 研究表明，膜脂质成分（和脂质蛋白）的变化 相互作用）代表了AChR调节的一个非常重要的机制 通道功能的胆碱能突触的肌肉和大脑，因此， 了解这些机制的分子基础将有助于确定 膜脂质组合物增强或抑制AChR的条件 功能将通过追求四个具体目标来检验中心假设： (1)研究五种疏水性变构物质的结构与功能关系 使用额外的侧链替换，（2）引入周期性的 色氨酸取代以完成M3跨膜沿着的所有位置 域以定义螺旋-螺旋接触，结构约束 位置和其他变构位点，（3）检查膜的影响 胆固醇水平对AChR通道动力学的影响和五种新的疏水性变构蛋白 M3突变和（4）在疏水变构位点引入非天然氨基酸 在M3域的位置，以确定;（a）电子密度的影响 （B）膜渗透深度 变构位置拟议的工作是创新的，因为它利用了 由申请人开发的新方法，以：1）实施胆固醇 卵母细胞质膜中的富集和消耗，以及2） 引入荧光非天然氨基酸以评估膜深度穿透 和环状脂质组合物。我们预计 这些方法将使我们能够确定新的机制， 脂质-蛋白质相互作用调节AChR的变构转换。的 从这些研究中获得的知识具有极其重要的意义，因为它将：1） 描述了完整突触中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， M4结构域; 3）描述了AChR的新结构-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.