PERMEABILITY CONTROL BY ACETYLCHOLINE RECEPTOR

乙酰胆碱受体的渗透性控制

• 批准号: 3399592
• 负责人: JONATHAN D COHEN
• 金额: $ 16.07万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3399592
• 关键词: Torpedo; acetylcholine; anticholinergic agent; binding proteins; cell membrane; cholinergic agents; gel electrophoresis; ion transport; laboratory mouse; ligands; lipid structure; membrane channels; membrane lipids; membrane permeability; monoclonal antibody; neurochemistry; nicotinic receptors; protein sequence; proteins; radiotracer; saltwater environment; synapses; synaptic vesicles

We wish to understand how the nicotinic acetylcholine receptor functions as a ligand-gated ion channel and also to define the interactions within the post-synaptic memberane that are necessary to maintain acetylcholine receptors in high density clusters restricted to regions of the muscle plasma membrane directly underlying the nerve terminal. Torpedo electric tissue will be fractionated to isolate post-synaptic membranes to be used in studies of receptor structure and function. Functional domains within the receptor to be identified include the site of binding of agonists and competitive antagonists as well as the separate binding site for amine non-competitive antagonists. Binding sites will be labeled covalently by appropriate radiolabeled affinity reagents, and the receptor will be degraded to identify the labeled peptides in terms of the known amino acid sequences of the receptor subunits. To determine how the ligand binding sites are oriented within the three-dimensional structure of the receptor, biochemical and immunological labeling procedures will be used to identify receptor domains exposed at the extracellular surface, buried within the lipid bilayer, or exposed on the cytoplasmic surface. These techniques will also be used to identify regions of each receptor subunit in contact with each other or with non-receptor proteins of the post-synamptic membrane. Receptors in the isolated Torpedo post-synaptic membrane have very restricted motility unless the non-receptor peripheral proteins are removed. We will characterize by biochemical and immunological means the structure of the principal non-receptor protein (43kDa protein) and determine whether interactions between this protein and the receptor restrict motility of the receptor.

我们希望了解烟碱乙酰胆碱受体如何 充当配体门控离子通道并定义 突触后膜内的相互作用是 维持高密度乙酰胆碱受体所必需的 簇仅限于肌肉质膜区域 直接位于神经末梢下方。 鱼雷电组织 将被分级分离以分离突触后膜 用于受体结构和功能的研究。 功能性 待鉴定的受体内的结构域包括以下位点 激动剂和竞争性拮抗剂的结合以及 胺非竞争性拮抗剂的单独结合位点。 结合位点将通过适当的共价标记 放射性标记的亲和试剂，受体会被降解 根据已知氨基酸识别标记肽 受体亚基的序列。 确定配体如何 结合位点在三维结构内定向 受体、生化和免疫学标记 程序将用于识别暴露于的受体域 埋在脂质双层内的细胞外表面，或 暴露于细胞质表面。 这些技术也将 用于识别每个受体亚基接触的区域 彼此或与突触后非受体蛋白 膜。 孤立鱼雷突触后的受体 膜的运动性非常有限，除非非受体 外周蛋白被去除。 我们将表征为 生化和免疫学手段的结构 主要非受体蛋白（43kDa 蛋白）并确定 该蛋白质与受体之间是否存在相互作用 限制受体的运动。