title: Allosteric transitions of the acetylcholine receptor probed at the amino acid level with a photolabile cholinergic ligand.
abstract: Structural changes occurring upon desensitization of the Torpedo marmorata acetylcholine receptor were monitored with tritiated p-(N,N-dimethyl)aminobenzenediazonium fluoroborate, a reversible competitive antagonist in the dark, which may serve as a photoaffinity probe of the area of the receptor molecule with which cholinergic ligands interact. Addition of meproadifen, an allosteric effector that stabilizes the high-affinity desensitized state of the receptor upon binding to a site topographically distinct from the cholinergic ligand-binding domains, caused a major increase in labeling of the alpha subunit, a smaller increase in the delta subunit, and decreased labeling in the gamma subunit, thus revealing changes in the alpha and non-alpha subunits' contribution to cholinergic ligand binding. Also, in agreement with the tighter binding of cholinergic ligands to the desensitized receptor, differential labeling of three peptide loops of the alpha subunit was detected: while Tyr-190, Cys-192, and Cys-193 were labeled in a roughly identical manner in both resting and desensitized conformations, the labeling of Tyr-93 and Trp-149 increased up to 6-fold in the desensitized state. Tyr-93 and Trp-149 belong to separate regions containing strictly conserved "canonical" amino acids, common to all nicotinic, gamma-aminobutyrate, and glycine receptor subunits. These regions are thus likely to play a critical role in the regulation of ligand-gated ion channels.
authors.affiliation: Institut Pasteur, Laboratoire de Neurobiologie Moléculaire associé au Centre National de la Recherche Scientifique (Unité Recherche Associée D 1284, Département des Biotechnologies, Paris, France.
authors.forename: J L
authors.initials: JL
authors.lastname: Galzi
authors.forename: F
authors.initials: F
authors.lastname: Revah
authors.forename: F
authors.initials: F
authors.lastname: Bouet
authors.forename: A
authors.initials: A
authors.lastname: Ménez
authors.forename: M
authors.initials: M
authors.lastname: Goeldner
authors.forename: C
authors.initials: C
authors.lastname: Hirth
authors.forename: J P
authors.initials: JP
authors.lastname: Changeux
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D003979:Diazonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011335:Proadifen / Q000494:pharmacology / Q000031:analogs & derivatives; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D003979:Diazonium Compounds; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; C028214:4-dimethylaminobenzenediazonium fluoroborate; C019001:meproadifen; D011335:Proadifen; D014403:Tubocurarine