title: Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.
abstract: [(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Trinidad
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.forename: Michael R
authors.initials: MR
authors.lastname: Ziebell
authors.forename: Deirdre
authors.initials: D
authors.lastname: Sullivan
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D000664:Amphibian Venoms / Q000378:metabolism; D000818:Animals; D001389:Azirines / Q000378:metabolism* / Q000494:pharmacology; D001588:Benzoylcholine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002794:Choline / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007473:Ion Channels / Q000378:metabolism*; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000378:metabolism*; D018733:Nicotinic Antagonists / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium; D014466:Ultraviolet Rays; D014981:Xenopus
chemical_list: C472309:4-((3-trifluoromethyl)-3H-diazirin-3-yl)benzoylcholine; D000596:Amino Acids; D000664:Amphibian Venoms; D001389:Azirines; D002038:Bungarotoxins; D007457:Iodine Radioisotopes; D007473:Ion Channels; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D020033:Protein Isoforms; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D014316:Tritium; D001588:Benzoylcholine; C004792:perhydrohistrionicotoxin; D002794:Choline; D000109:Acetylcholine