Analysis of Acetylcholine Receptor Subunit

乙酰胆碱受体亚基的分析

• 批准号: 7059803
• 负责人: RICHARD M HANN
• 金额: $ 14.44万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059803
• 关键词: Chondrichthyes; acetylcholine; alternatives to animals in research; binding proteins; cholinergic receptors; minority institution research support; molecular assembly /self assembly; point mutation; protein structure; receptor expression; site directed mutagenesis

The ionotropic acetylcholine receptor (AchR) from the electric organ of the marine ray Torpedo californica is a pentameric ligand-gated cation channel composed of 4 different but similar subunits with a subunit stoichiometry of alpha2-beta-gamma-delta. Each subunit presents two distinct faces, (+) and (-), to form a different interface with each of its neighboring subunits. The long-term research goals of this project are to identify the subunit residues that are essential for stabilization of these interfaces that form during receptor assembly and to identify within that set of stabilizing residues the subset of residues which determines the specificity of each subunit-subunit interaction. SPECIFIC AIM 1 is to identify alpha and gamma subunit residues that stabilize the alpha+/-gamma interface that forms in one of the earliest steps in AchR assembly. SPECIFIC AIM 2 is to identify alpha and beta subunit residues that stabilize the beta+/-alpha interface, which also forms early in AchR assembly. SPECIFIC AIM 3 is to identify residues in the delta+/-beta interface that influence delta subunit assembly with beta-alpha-gamma trimers, an intermediate step in AchR assembly. In these 3 aims, site-directed mutagenesis will be used to modify specific residues that will be identified from a homology model of the interfaces constructed from the published high-resolution structure of a snail acetylcholine-binding protein. The effect of each point mutation on cell-surface AchR expression and on the assembly of intracellular oligomers will be measured. SPECIFIC AIM 4 is to make a mutant gamma subunit (gamma') that can substitute the delta subunit in assembly of surface expressed AchR yet still present a "gamma-like" (-) face to its interface with the alpha subunit. This aim will confirm the specificity of the interactions and will produce a cell surface-expressed alpha2-beta-gamma-gamma' receptor with equivalent alpha-gamma ligand-binding domains. Such a molecule would greatly facilitate radioligand-binding studies on this receptor. The Torpedo1 AchR is an excellent model for the human muscle AchR, whose dysfunction occurs in numerous acquired, inherited and toxicological neuromuscular diseases. Understanding Torpedo AchR assembly at the molecular level will be a major step toward improving the diagnosis and treatment of such diseases.

来自海洋射线鱼雷的电动器官的离子型乙酰胆碱受体（ACHR）是 一个由4种不同但相似的亚基组成的五聚体门控阳离子通道 alpha2-beta-gamma-delta的化学计量法。每个亚基都会呈现两个不同的面（+）和（ - ），以与其每个相邻的界面形成不同的界面 亚基。该项目的长期研究目标是确定必不可少的亚基残基 为了稳定这些在受体组装过程中形成的界面，并在该组中识别 稳定残基的残基子集，该残基确定每个亚基 - 亚基的特异性 相互作用。具体目的1是识别稳定α+/-γ界面的α和伽马亚基残基 形式是ACHR组装中最早的步骤之一。特定目标2是识别α和β亚基残基 稳定β+/- alpha界面，该界面在ACHR组装中也很早就形成。特定目标3是确定 Delta +/-β界面中的残留物与Beta-Alpha-Gamma Trimers影响三角洲亚基组件，这是一个中间步骤 ACHR组装。在这三个目标中，将使用位置定向的诱变来修改特定残基 可以从已发表高分辨率构建的界面的同源模型中识别 蜗牛乙酰胆碱结合蛋白的结构。每个点突变对细胞表面ACHR的影响 将测量表达和细胞内低聚物的组装。特定目标4是 突变的伽马亚基（伽玛'）可以用表达ACHR的表面替代三角洲亚基替代Delta亚基 在与alpha子单位的接口上呈现“伽马状”（ - ）（ - ）。这个目标将确认 相互作用，将产生与等效α-γ配体结合的细胞表面表达表达的α2-beta-gamma-gamma'受体 域。这样的分子将极大地促进对该受体的放射性结合研究。鱼雷1 ACHR是人类肌肉ACHR的绝佳模型，其功能障碍发生在许多获得的中， 遗传和毒理学神经肌肉疾病。了解鱼雷ACHR组装 分子水平将是改善此类疾病诊断和治疗的主要步骤。