title: Structural reorganization of the acetylcholine binding site of the torpedo nicotinic receptor as revealed by dynamic photoaffinity labeling.
abstract: We explored the structural changes that occur at the acetylcholine binding site of the Torpedo marmorata nicotinic receptor during activation by the tritiated photoactivatable agonist (diazocyclohexadienoylpropyl)trimethylammonium ([(3)H]DCTA). We quantified the incorporation of radioactivity into the receptor subunits as a function of the mixing time of [(3)H]DCTA with the receptor by using a rapid-mixing device adapted with a photochemical quenching system. A saturable increase of the specific photolabeling on the alpha and gamma subunits was observed with a half-time of about 2 minutes. We further analyzed this photoincorporation either after rapid mixing for 500 ms or after equilibration for 50 minutes. Under these conditions, [(3)H]DCTA explored transient state(s) and the stable desensitized state, respectively. Comparative analyses showed that at a probe concentration of 10 microM the relative variation of photoincorporation was more pronounced for the gamma subunit (three- to fourfold) than for the alpha subunit (about twofold). By contrast, the relative distribution of radioactivity among alpha-subunit labeled residues (alphaTyr190, alphaCys192, alphaCysC193, and alphaTyr198) did not change. Altogether, these results reveal that during the course of agonist-induced receptor desensitization, the site-lining peptide loops, which belong to adjacent alpha and gamma subunits, move closer to each other.
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D003979:Diazonium Compounds / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000378:metabolism* / Q000528:radiation effects; D014101:Torpedo / Q000378:metabolism*; D014316:Tritium; D014466:Ultraviolet Rays
chemical_list: D000596:Amino Acids; D003979:Diazonium Compounds; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D014316:Tritium; D000109:Acetylcholine