Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: Evidence that the homopentameric 5-hydroxytryptamine type 3(As) receptor binds only one ligand

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily acces sible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3,9-tetrahydro-3-[(5-met hyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)-4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7-nitrobenz-2- oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large p art of the visible electromagnetic spectrum. It is shown that the photophys ical and chemical properties have a direct influence on the measurement qua lity (duration of measurement, signal-to-noise ratio) and the ligand-recept or interactions (dissociation constants), respectively. This makes it neces sary to choose a suitable label or a combination of labels for receptor stu dies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding exp eriments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experim ental results showed that only one antagonist binds to the receptor, in agr eement with measurements previously published [Tairi et al. (1998) Biochemi stry 37, 15850-15864].