Kinetics of Ligand Binding to Synaptic Ion Channels

描述（由申请人提供）： 该项目的总体目标是了解配体与突触离子通道结合的方式。 配体门控离子通道，如乙酰胆碱受体，允许神经系统中细胞之间的快速突触通讯。与这些通道结合的配体在基础和临床医学中发挥作用。基础科学家使用竞争性拮抗剂来特异性地阻断电流，标记受体结合位点并探测结合位点的分子结构。医生在手术中使用肌肉型乙酰胆碱受体的竞争性拮抗剂作为肌肉松弛剂。 最近，已经提出了乙酰胆碱受体的配体结合位点的结构模型。这里的实验是为了测试这个模型，并添加动态和充满活力的信息的模型。该建议的重点是围绕竞争性拮抗剂结合突触离子通道的动力学的电生理测量。这些结果扩展了从平衡结合实验中获得的信息。它们允许计算速率常数，可用于突触功能的现实动力学模型，以提供对其临床作用的深入了解，并提供有关配体结合位点的分子结构的信息。 有四个具体目标。 1.测量在室温和生理温度下对人类和小鼠肌肉乙酰胆碱受体的竞争性拮抗作用的动力学。 2.测量拮抗剂对肌肉乙酰胆碱受体的作用动力学，所述肌肉乙酰胆碱受体在推定的乙酰胆碱结合口袋中或附近含有突变。 3.将动力学结果纳入神经肌肉接头的Monte Carlo模拟。 4.使用等温滴定量热法测量配体与乙酰胆碱结合蛋白结合的热力学-乙酰胆碱结合蛋白是一种水溶性蛋白质，其结构被用作乙酰胆碱受体的模板。

DESCRIPTION (provided by applicant): The overall goal of the project is to understand the way in which ligands bind to synaptic ion channels. Ligand-gated ion channels, such as the acetylcholine receptor, permit rapid synaptic communication between cells in the nervous system. Ligands that bind to these channels play roles in both basic and clinical medical science. Basic scientists use competitive antagonists to block currents specifically, to label the receptor binding site and to probe the molecular structure of the binding site. Physicians use competitive antagonists to the muscle-type acetylcholine receptor as muscle relaxants during surgery. Recently, a structural model for the ligand binding site of the acetylcholine receptor has been proposed. The experiments here are designed to test this model and to add dynamic and energetic information to the model. This proposal is focused around electrophysiological measurements of the kinetics of competitive antagonist binding to synaptic ion channels. These results extend information obtained from equilibrium binding experiments. They allow calculation of rate constants that can be used in realistic kinetic models of synaptic function to provide insight into their clinical action and also provide information about the molecular structure of ligand binding sites. There are four specific aims. 1. Measure the kinetics of competitive antagonism on human and mouse muscle acetylcholine receptors at room and physiological temperatures. 2. Measure the kinetics of antagonist actions on muscle acetylcholine receptors containing mutations in and near the putative acetylcholine binding pocket. 3. Incorporate the kinetic results into a Monte Carlo simulation of the neuromuscular junction. 4. Use isothermal titration calorimetry to measure the thermodynamics of ligand binding to acetylcholine binding protein - a water soluble protein whose structure is being used as a template for the acetylcholine receptor.