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 rrceptor, 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 solution structures of molecules which interact with the acetylcholine receptor and the details of that interaction using patch clamp recording, radioligand binding, and NMR spectroscopy. Two major groups of studies are proposed, one concerned with agonists and the other with polypeptide toxins that function as very high affinity antagonists. 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 of the ion channel. NMR spectroscopy will be used to study the structure of the compounds, 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. These compounds also produce a characteristic channel blockade under some conditions, so that we will be able to study the relationship of structure not only to agonist activity but also to noncompetitive inhibition of the channel. These studies should provide important information that will allow us to produce neuromuscular blockers with specific properties such as high affinity binding or long channel openings. The second group of studies is designed to investigate the structure of polypeptide neurotoxins from snake venom. These proteins (e.g., alpha-bungarotoxin, neuronal bungarotoxin) bind with extremely high affinity to the acetylcholine receptor and block the binding of acetylcholine. Our solution structure for neuronal bungarotoxin will be refined further, and the interaction of neuronal bungarotoxin and alpha- bungarotoxin with the acetylcholine receptor and portions of muscle and neuronal nicotinic acetylcholine receptors will be studied using one and two dimensional NMR spectroscopy. These studies should provide us with a detailed picture of the interaction of these toxins with subtypes of nicotinic acetylcholine receptors and potentially a greater understanding of the differences between these subtypes.
Showing the most recent 10 out of 16 publications
