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 c ells are generally resistant to high resolution structural tools. Structure -function studies, especially those enhanced by the nonsense suppression me thodology for unnatural amino acid incorporation, constitute one of the mos t powerful probes of ion channels and related structures. The nonsense supp ression methodology can also be used to incorporate functional side chains designed to deliver novel structural probes to membrane proteins. In this v ein, we sought to generalize a potentially powerful tool - the tethered ago nist approach - for mapping the agonist binding site of ligand-gated ion ch annels. 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 o f activation as a function of chain length is different. At position alpha 149, there is a clear preference for a three-carbon tether, while at positi on alpha 93 tethers of 2-5 carbons are comparably effective. At position ga mma 55/delta 57 all tethers except the shortest one call activate the recep tor. Based on these and other data, a model for the receptor binding site c an be developed by analogy to the acetylcholine esterase crystal structure. Conclusion: Through thr: use of nonsense suppression techniques, the tether ed agonist approach has been made into a general tool for probing receptor structures. When applied to the nicotinic receptor. the method places neu r estrictions on developing models For the agonist binding site. (C) 2001 Els evier Science Ltd. All rights reserved.