title: Structural differences in the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor revealed by time-resolved fluorescence spectroscopy.
abstract: The nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata carries two nonequivalent agonist binding sites at the alphadelta and alphagamma subunit interfaces. These sites have been characterized by time-resolved fluorescence with the partial nicotinic agonist dansyl-C(6)-choline (Dnscho). When bound to the detergent-solubilized receptor, the fluorescence lifetime distribution of Dnscho displays a characteristic signature, with four separable components at 0.2, 1.8, 7.2, and 18.3 ns, respectively. Competition experiments with the antagonist d-tubocurarine (dTC), known to bind preferentially to the alphagamma site, result in substantial changes of this signature, associated with a strong decrease in average fluorescence lifetime. Comparisons with two other competitive antagonists, alpha-conotoxin M1 and alpha-bungarotoxin, demonstrate that Dnscho binds with a similar affinity to the two sites but that the microenvironment of the probe is different for each site. Using a two-site binding model together with published equilibrium constants to describe the competitive binding of dTC and Dnscho, we reach a satisfactory description of the changes in fluorescence lifetimes and propose characteristic fluorescence parameters of the probe bound to each type of site. This analysis indicates that Dnscho at the alphadelta site is principally associated with a 8.7 ns lifetime, while it has a 20.2 ns major lifetime at the alphagamma site. Therefore, the observed fluorescence heterogeneity arises in large part from the structural differences of the two binding sites. As a result, this signal can be used to identify the binding preferences of competitive ligands of unknown pharmacology.
authors.affiliation: Laboratoire pour l'Utilisation du Rayonnement Electromagnétique, Centre Universitaire Paris-Sud, B.P. 34, 91898 Orsay Cedex, France.
authors.forename: K L
authors.initials: KL
authors.lastname: Martinez
authors.forename: P J
authors.initials: PJ
authors.lastname: Corringer
authors.forename: S J
authors.initials: SJ
authors.lastname: Edelstein
authors.forename: J P
authors.initials: JP
authors.lastname: Changeux
authors.forename: F
authors.initials: F
authors.lastname: Mérola
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000494:pharmacology; D020916:Conotoxins / Q000494:pharmacology; D003619:Dansyl Compounds / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D018722:Nicotinic Agonists / Q000378:metabolism*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014101:Torpedo / Q000378:metabolism*; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: D002038:Bungarotoxins; D020916:Conotoxins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; C027933:(1-(5-dimethylaminonaphthalene)sulfonamido)-n-hexanoic acid-beta-N-trimethylammonium ethyl ester; C036155:conotoxin MI; D014403:Tubocurarine