The nicotinic acetylcholine receptor translates the binding of acetylcholine into the flow of ions across the cell membrane and thus serves a major role in the communication between two nerve cells and between nerve and muscle cells. The receptor consists of five membrane- spanning subunits which surround a central pore that functions as an ion channel. Studies of the nicotinic acetylcholine receptor are of great relevance to the understanding of neuromuscular disease due to its involvement in myasthenia gravis and related syndromes. Several classes of pharmacological and toxicological agents interact with the acetylcholine receptor, some of which activate the ion channel (agonists) and others which prevent the activation (antagonists) or block the ion channel (noncompetitive blockers). The studies described in this proposal are designed to determine the structural details of the interaction of cholinergic effectors with nicotinic acetylcholine receptors using patch clamp recording, radioligand binding, NMR spectroscopy, and site-directed mutagenesis. Two sites of interaction will be studied: the agonist binding sites and the site(s) responsible for ion channel blockade. A number of systematically varying cholinergic agonists have been synthesized for these studies and will be used to correlate the structure of the compounds with the activation and blockade of the ion channel. Single channel recording will be used to study the kinetics of the activation of the ion channel, and radioligand binding will be used to study the thermodynamic properties associated with the binding interaction. Specific mutagenesis of the receptor molecule in conjunction with systematically varying channel blockers will help the mapping the binding sites within the ion channel and possibly to probe the structure of the channel. These studies should provide important information to aid in the production of neuromuscular blockers with specific properties such as high affinity binding or long channel openings. In addition to the structure-activity and mutagenesis experiments, these investigators will exploit the power of solid state NMR spectroscopy to study the conformation of an acetylcholine derivative while bound to the nicotinic acetylcholine receptor. By using a series of three isotopically labeled compounds (13C,15N), the bound conformation can be determined by the use of the rotational-echo double-resonance NMR experiment. They hope that the combination of spectroscopic techniques with the information gained from site-directed mutagenesis and structure-activity relationships will provide a more detailed picture of the binding sites on nicotinic acetylcholine receptors and provide a basis of the rational design of drugs for this system.
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