The tethered agonist approach to mapping ion channel proteins - Toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor

Background: The integral membrane proteins of neurons and other excitable cells are generally resistant to high resolution structural tools. Structure-function studies, especially those enhanced by the nonsense suppression methodology for unnatural amino acid incorporation, constitute one of the most powerful probes of ion channels and related structures. The nonsense suppression methodology can also be used to incorporate functional side chains designed to deliver novel structural probes to membrane proteins. In this vein, we sought to generalize a potentially powerful tool - the tethered agonist approach - for mapping the agonist binding site of ligand-gated ion channels. Results: Using the in vivo nonsense suppression method for unnatural amino acid incorporation, a series of tethered quaternary ammonium derivatives of tyrosine have been incorporated into the nicotinic acetylcholine receptor. At three sites a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position α149, there is a clear preference for a three-carbon tether, while at position α93 tethers of 2-5 carbons are comparably effective. At position γ55/δ57 all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the receptor binding site can be developed by analogy to the acetylcholine esterase crystal structure. Conclusion: Through the use of nonsense suppression techniques, the tethered agonist approach has been made into a general tool for probing receptor structures. When applied to the nicotinic receptor, the method places new restrictions on developing models for the agonist binding site.