PERMEABILITY CONTROL OF ACETYLCHOLINE RECEPTOR

乙酰胆碱受体的通透性控制

• 批准号: 3399584
• 负责人: JONATHAN D COHEN
• 金额: $ 28.94万
• 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 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3399584
• 关键词: Torpedo; Xenopus oocyte; affinity labeling; alternatives to animals in research; chemical binding; conformation; electrical measurement; fish electric organ; membrane channels; membrane lipids; membrane permeability; molecular cloning; molecular site; nicotinic receptors; point mutation; protein sequence; protein structure function; radiotracer; receptor binding; site directed mutagenesis; synapses

We wish to understand how the nicotinic acetylcholine receptor (AChR) functions as a ligand-gated ion channel and to understand how drugs and toxins interact with the AChR to alter its function. Torpedo electric tissue will be fractionated to. isolate postsynaptic membranes to be used in biochemical studies designed to: 1) identify functional domains (ligand binding sites, ion channel) in terms of the known amino acid sequences of AChR subunits and to identify differences in structure of those domains that distinguish between the binding of agonists and antagonists or between different conformational states of the AChR, and 2) provide a general description of the three dimensional structure of the AChR in terms of the regions of each subunit exposed at extracellular or cytoplasmic surfaces, at subunit interfaces, or in contact with lipid. Radiolabeled affinity labels and structural probes will be covalently incorporated into AChR, and labeled AChR subunits will be isolated and degraded so that sites of labeling will be determined by N-terminal sequence analysis of isolated labeled peptides. The structural studies will provide a definition of particular amino acids and regions contained within binding sites, at subunit interfaces, or at the protein- lipid interface, but they do not of their own assess the importance of the identified amino acids as determinants of ligand binding affinity or their involvement in the mechanism of channel gating. To address these issues a third research goal is to test models of AChR structure derived from the structural studies by analysis of functional properties of mutant AChRs expressed in Xenopus oocytes. The equilibrium binding affinity of agonists and antagonists will be assessed by radioligand binding assays, while AChR function will be assessed by electrophysiological techniques. Point mutations will be introduced to change amino acids predicted to be important for ligand binding affinity or for channel-gating, and additional mutations will be made of amino acids predicted to be important in the propagation of structural changes from the ACh binding site to the ion channel.

我们希望了解烟碱乙酰胆碱受体（AChR） 作为配体门控离子通道，并了解药物和 毒素与乙酰胆碱受体相互作用以改变其功能。 鱼雷电 组织将被分离成。分离突触后膜 在生物化学研究中，设计用于：1）鉴定功能结构域 （配体结合位点，离子通道）的已知氨基酸 AChR亚单位的序列，并确定结构的差异， 这些结构域区分激动剂和 拮抗剂之间或AChR的不同构象状态之间，和 2)提供了三维结构的一般描述， AChR的每个亚基暴露在细胞外的区域 或细胞质表面、亚基界面或与脂质接触。 放射性标记的亲和标记物和结构探针将共价结合。 掺入AChR中，并分离标记的AChR亚基， 降解，使得标记位点将由N-末端确定。 分离的标记肽的序列分析。 结构研究 将提供特定氨基酸和所含区域的定义 在结合位点内、在亚基界面处或在蛋白质-脂质处 接口，但他们自己不评估的重要性， 确定氨基酸作为配体结合亲和力的决定因素，或 它们参与了通道门控机制。 解决这些 问题的第三个研究目标是测试模型的AChR结构推导 从结构研究，通过分析功能特性， 在非洲爪蟾卵母细胞中表达的突变AChRs。 平衡结合 激动剂和拮抗剂的亲和力将通过放射性配体 结合试验，而AChR功能将通过 电生理技术。 点突变将被引入到 改变预计对配体结合亲和力重要的氨基酸 或用于通道门控，并且另外的突变将由氨基 预测在结构变化传播中起重要作用的酸 从乙酰胆碱结合位点到离子通道。