title: Tryptophan 86 of the alpha subunit in the Torpedo nicotinic acetylcholine receptor is important for channel activation by the bisquaternary ligand suberyldicholine.
abstract: Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Ankur
authors.initials: A
authors.lastname: Kapur
authors.forename: Martin
authors.initials: M
authors.lastname: Davies
authors.forename: William F
authors.initials: WF
authors.lastname: Dryden
authors.forename: Susan M J
authors.initials: SM
authors.lastname: Dunn
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D002794:Choline / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D018678:Cholinergic Agents / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D018733:Nicotinic Antagonists / Q000378:metabolism / Q000494:pharmacology; D009865:Oocytes; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D014101:Torpedo / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014403:Tubocurarine / Q000378:metabolism / Q000494:pharmacology; D014981:Xenopus
chemical_list: D018678:Cholinergic Agents; D018733:Nicotinic Antagonists; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C009335:subecholine; D014364:Tryptophan; D002794:Choline; D000109:Acetylcholine; D014403:Tubocurarine