title: Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor.
abstract: We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.
authors.affiliation: Laboratoire de Chimie Bioorganique UMR 7175 LC1 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, 74, Route du Rhin, BP24, 67401 Illkirch Cedex, France. krieger@bioorga.u-strasbg.fr
authors.forename: Florian
authors.initials: F
authors.lastname: Krieger
authors.forename: Alexandre
authors.initials: A
authors.lastname: Mourot
authors.forename: Romulo
authors.initials: R
authors.lastname: Araoz
authors.forename: Florence
authors.initials: F
authors.lastname: Kotzyba-Hibert
authors.forename: Jordi
authors.initials: J
authors.lastname: Molgó
authors.forename: Ernst
authors.initials: E
authors.lastname: Bamberg
authors.forename: Maurice
authors.initials: M
authors.lastname: Goeldner
mesh_terms: D000109:Acetylcholine / Q000138:chemical synthesis / Q000494:pharmacology; D000818:Animals; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D029941:Fish Proteins / Q000819:agonists* / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D018722:Nicotinic Agonists / Q000138:chemical synthesis / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013379:Substrate Specificity; D014101:Torpedo
chemical_list: D003374:Coumarins; D029941:Fish Proteins; D005456:Fluorescent Dyes; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D000109:Acetylcholine