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.
