Location of the Polyamine Binding Site in the Vestibule of the Nicotinic Acetylcholine Receptor Ion Channel

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was incorporated into the receptor α-subunit. From proteolytic mapping studies, we conclude that the site of 125I-MR44 cross-linking is contained in the sequence αHis-186 to αLeu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist α-bungarotoxin had only minor influence on the photocross-linking of 125I-MR44. The site where the hydrophobic head group of 125I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with 125I-MR44. We conclude that the polyamine moiety of 125I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the α-subunit that is located in close proximity to the agonist binding site. The region of the α-subunit labeled by 125I-MR44 should therefore be accessible from the luminal side of the vestibule.