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 hi gh affinity to the nicotinic acetylcholine receptor in its closed channel c onformation. The binding stoichiometry was two molecules of MR44 per recept or monomer. Upon UV irradiation of the receptor-ligand complex, I-125-MR44 was incorporated into the receptor alpha -subunit. From proteolytic mapping studies, we conclude that the site of I-125-MR44 cross-linking is containe d in the sequence alpha His-186 to alpha Leu-199, which is part of the extr acellular domain of the receptor. This sequence partially overlaps in its C -terminal region with one of the three leaps that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha -bungaroto xin had only minor influence on the photocross-linking of I-125-MR44. The s ite where the hydrophobic head group of I-125-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 exp eriments, the luminal noncompetitive inhibitors ethidium and triphenylmethy lphosphonium were found to compete with I-125-MR44. We conclude that the po lyamine moiety of I-125-MR44 interacts with the high affinity noncompetitiv e inhibitor site deep in the channel of the nicotinic acetylcholine recepto r, 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 alpha -s ubunit that is located in close proximity to the agonist binding site. The region of the alpha -subunit labeled by I-125-MR44 should therefore be acce ssible from the luminal side of the vestibule.