LIGAND BINDING SITES OF THE ACETYLCHOLINE RECEPTOR

描述：（来自申请人的摘要） 在这种应用中的研究是为了了解 烟碱乙酰胆碱受体（AChR）门控的调节 离子通道通过乙酰胆碱结合。 本项目的具体目标 是确定乙酰胆碱受体上的神经递质结合位点。三 将实现具体目标。 一、两个系列的同源 毒素，基于生物碱d-筒箭毒碱和肽α- 芋螺毒素，将被合成和表征结合天然的 受体。 其次，AChR中的一组扩展的氨基酸， 与毒素的相互作用将被定义。 三、具体互动 AChR氨基酸和毒素上官能团之间的能量 将被测量，然后完成互补相互作用的地图 结合位点残基和毒素之间的联系 实现这些具体目标的方法包括均衡 毒素与AChR的结合分析和新毒素的合成， 主要是肽合成和修饰。 AChR亚单位嵌合体 将被构造成具有顺序较小的段替换。 将分析这些嵌合体的毒素结合特性， 鉴定参与结合的氨基酸。 的光亲和衍生物 毒素也将被合成，用于鉴定氨基酸， 通过随后的蛋白水解作图确定结合位点。 定点 与毒素相互作用的氨基酸的突变将 构建了 特定氨基酸-毒素的能量贡献 将通过双突变热力学循环测量相互作用 分析. 骨骼肌AChR（特别是细胞外结构域）是 自身免疫抗体在重症肌无力中的作用靶点。 这种蛋白质的神经元同源物可能与尼古丁有关 可能还有老年痴呆症 更好地了解 AChR胞外区的结构， 神经递质位点对于全面理解 这些疾病，并可能导致见解，以改善合理的药物 设计这类受体。

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.