LIGAND BINDING SITES OF THE ACETYLCHOLINE RECEPTOR

描述：(摘自申请者摘要)该计划的长期目标 对这一应用的研究是为了了解其作用机制 烟碱型乙酰胆碱受体(AChR)门控的调节 乙酰胆碱结合的离子通道。这个项目的具体目标是 就是确定AChR上的神经递质结合位点。三 具体目标将付诸实施。第一，两个系列的同源 毒素，基于生物碱d-管胡萝卜碱和多肽α- 芋螺毒素，将被合成并表征为与天然的结合 感受器。第二，AChR中的一组扩展氨基酸 与毒素的相互作用将被定义。第三，具体互动 AChR氨基酸与毒素上官能团之间的能量 将被测量，以完成互补相互作用的地图 结合部位残基和毒素之间的关系。 实现这些具体目标的方法包括均衡。 毒素与乙酰胆碱受体的结合分析及新毒素的合成， 主要是多肽的合成和修饰。AchR亚单位嵌合体 将使用顺序较小的分段替换来构建。 这些嵌合体将被分析其毒素结合特性以 确定参与结合的氨基酸。光亲和性衍生物 毒素也将被合成，用于鉴定氨基酸 通过随后的蛋白水解图绘制结合位点。站点定向 与毒素相互作用的氨基酸的突变将是 建造的。然后是特定氨基酸-毒素的能量贡献 相互作用将通过双突变热力学循环来测量 分析。 骨骼肌乙酰胆碱受体(尤其是胞外区)是 自身免疫抗体是重症肌无力疾病的靶点。 这种蛋白的神经元同系物很可能与尼古丁有关。 上瘾，可能还有阿尔茨海默氏症。更好地理解 AChR胞外区的结构，特别是 神经递质部位对于全面了解是很重要的。 这些疾病，并可能导致洞察，以改善合理的药物 为这类受体设计的。

DESCRIPTION: (from applicant's abstract) The long term goal of the research in this application is to understand the mechanism of regulation of the gating of the nicotinic acetylcholine receptor (AChR) ion channel by acetylcholine binding. The specific goal of this project is to define the neurotransmitter binding sites on the AChR. Three specific aims will be carried out. First, two series of homologous toxins, based on the alkaloid d-tubocurarine and on the peptide alpha- conotoxins, will be synthesized and characterized for binding to native receptors. Second, an extended set of amino acids in the AChR that interact with the toxins will be defined. Third, specific interaction energies between AChR amino acids and functional groups on the toxins will be measured to then complete a map of complementary interactions between binding site residues and the toxins. The methods for carrying out these specific aims include equilibrium binding analysis of toxins to the AChR and synthesis of new toxins, principally peptide synthesis and modification. AChR subunit chimeras will be constructed with sequentially smaller segment replacement. These chimeras will be analyzed for their toxin binding properties to identify amino acids involved in binding. Photoaffinity derivatives of toxins will also be synthesized for identification of amino acids at the binding sites by subsequent proteolytic mapping. Site directed mutations of amino acids that interact with the toxins will be constructed. Then the energy contribution of specific amino acid-toxin interactions will be measured by double mutant thermodynamic cycle analysis. The skeletal muscle AChR (and particularly the extracellular domain) is the target of autoimmune antibodies in the disease Myasthenia gravis. The neuronal homologs of this protein are likely involved in nicotine addiction and possibly Alzheimer's disease. A better understanding of the structure of the extracellular domain of the AChR and especially the neurotransmitter sites will be important for a full understanding of these ailments and may lead to insights to improve rational drug design for this class of receptors.