LIGAND BINDING SITES OF THE ACETYLCHOLINE RECEPTOR

本研究的长期目标是了解神经递质结合对配体门控离子通道的调节。本申请的特定目标是理解电荷-电荷相互作用在通过乙酰胆碱的结合调节烟碱乙酰胆碱受体中的重要性。具体地说，d-筒箭毒碱的烟碱乙酰胆碱受体的结构活性关系将通过分析的d-筒箭毒碱类似物的天然和突变的乙酰胆碱受体的结合检查。将检查静电吸引在结合中的作用，以确定电荷-电荷吸引是否控制乙酰胆碱结合的快速速率并有助于激动剂阳离子的稳定。与乙酰胆碱受体构象转变相关的通道运动将通过通道窄孔附近残基的诱变以及通过使用荧光寿命光谱法直接测量静电势来测量。这些实验将定义受体结构的功能相关组分，这些组分有助于配体结合、离子通道结构和功能。这将提高我们对突触传递的理解，突触传递是学习、记忆和思维等复杂现象的基础。骨骼肌烟碱乙酰胆碱受体（特别是细胞外结构域）是重症肌无力疾病中自身免疫抗体的靶标。这种蛋白质的神经元同源物与尼古丁成瘾有关，可能与阿尔茨海默病有关。更好地了解这种蛋白质的结构，尤其是乙酰胆碱结合位点，对于全面了解这些疾病和开发治疗方法至关重要。结合位点狭窄的基本理解将提高这种蛋白质的合理药物设计，而这里提出的方法可能构成一个新的范例，为受体靶点的合理药物设计。

The long-term aim of this research is to understand the regulation of ligand-gated ion channels by the binding of neurotransmitters. The particular goal of this application is to understand the importance of charge-charge interactions in the regulation of the nicotinic acetylcholine receptor by the binding of acetylcholine. Specifically, the structure activity relationship of d-tubocurarine to the nicotinic acetylcholine receptor will be examined by analyzing the binding of d-tubocurarine analogs to native and mutated acetylcholine receptors. The role of electrostatic attraction in binding will be examined to determine whether charge- charge attraction governs the rapid rate of acetylcholine binding and contributes to the stabilization of the agonist cation. The channel movements associated with the conformational transition of the acetylcholine receptor will be measured by mutagenesis of residues near the narrow pore of the channel, and by direct measurements of electrostatic potential using fluorescence lifetime spectroscopic methods. The experiments will define functionally relevant components of the receptor structure that contribute to ligand binding, to ion channel structure, and to function. This will improve our understanding of synaptic transmission, a process that underlies the complex phenomena of learning, memory and thought. The skeletal muscle nicotinic acetylcholine receptor (and particularly the extracellular domain) is the target of autoimmune antibodies in the disease Myasthenia Gravis. The neuronal homologues of this protein are involved in nicotine addiction and possibly in Alzheimer's disease. A better understanding of the structure of this protein and especially the acetylcholine binding sites will be important for a full understanding of these ailments and for developing treatments. A fundamental understanding of the binding site stricture will improve rational drug design for this protein while the methodology proposed here may constitute a new paradigm for rational drug design for receptor targets in general.