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的结合方式。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都发挥着作用。基础科学家使用竞争性拮抗剂特异性地阻断电流，标记受体结合部位，并探测结合部位的分子结构。医生在手术中使用肌型乙酰胆碱受体的竞争性拮抗剂作为肌肉松弛药。 最近，人们提出了乙酰胆碱受体配体结合部位的结构模型。这里的实验旨在测试这一模型，并为该模型添加动态和能量信息。这项建议主要围绕竞争性拮抗剂与突触离子通道结合的动力学的电生理学测量。这些结果推广了从平衡结合实验中获得的信息。它们允许计算可用于突触功能的真实动力学模型的速率常数，以深入了解它们的临床作用，并提供关于配体结合部位的分子结构的信息。 有四个具体目标。 1.测定人和小鼠肌乙酰胆碱受体在室温和生理温度下的竞争性拮抗动力学。 2.测定肌乙酰胆碱受体对乙酰胆碱结合口袋及其附近突变的拮抗作用动力学。 3.将动力学结果纳入神经肌肉接头的蒙特卡罗模拟。 4.用等温滴定量热法测定乙酰胆碱结合蛋白的配基结合热力学。乙酰胆碱结合蛋白是一种水溶性蛋白，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.