PERMEABILITY CONTROL OF ACETYLCHOLINE RECEPTOR

我们希望了解烟碱型乙酰胆碱受体(AChR)是如何 作为配基门控离子通道的功能，并理解药物和 毒素与AChR相互作用，改变其功能。鱼雷电动 组织将被分割成。分离要使用的突触后膜 在生物化学研究中，旨在：1)识别功能域 (配体结合部位，离子通道)与已知氨基酸的关系 AChR亚基的序列并鉴定其结构的差异 那些区分激动剂和激动剂结合的区域 拮抗剂或AChR的不同构象状态之间，以及 2)提供对的三维结构的一般描述 细胞外暴露的每个亚基区域的乙酰胆碱受体 或细胞质表面，在亚单位界面，或与脂质接触。 放射性标记的亲和标记和结构探针将共价 整合到AChR中，并标记AChR亚单位将被分离和 降解，以便标记的位置将由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包含的区域的定义 在结合部位、亚单位界面或蛋白质-脂类之间 接口，但它们自己并不评估 确定氨基酸是配体结合亲和力的决定因素或 它们参与了通道门控的机制。要解决这些问题 问题的第三个研究目标是测试AChR结构衍生的模型 从结构研究出发，通过分析其功能特性 突变型AChRs在非洲爪哇卵母细胞中的表达。平衡约束 激动剂和拮抗剂的亲和力将通过放射性配基进行评估 结合分析，而AChR功能将通过 电生理技术。点突变将被引入到 氨基酸变化预计对配体结合亲和力很重要 或用于通道门控，其他突变将由氨基组成 预测在结构变化的传播中很重要的酸 从ACh结合部位到离子通道。

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.