LIGAND-BINDING TO THE SEROTONIN 5HT(3) RECEPTOR STUDIED WITH A NOVEL FLUORESCENT LIGAND

The thermodynamics and kinetics of ligand binding to the purified sero tonin 5HT(3) receptor and the local environment of the bound ligand we re studied by fluorescence spectroscopy using a novel fluorescein-labe led ligand GR-flu rescien-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. E lectrophysiological investigations demonstrated GR-flu to be an antago nist, and radioligand competition assays delivered a dissociation cons tant of 0.32 nM. Changes in the fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissociation constants o f similar to 0.2 nM. Fluorescence measurements showed that selective 5 HT(3) receptor ligands competed for GR-flu binding with a rank order o f potency identical to that established with the radioligand [H-3]-GR6 5630. The kinetics of GR-flu binding to the 5HT(3) receptor revealed a bimolecular association process with an on-rate constant of 1.17 x 10 (6) s(-1) M-1 and a biphasic dissociation reaction with off-rate const ants of 275 x 10(-6) and 43 x 10(-6) s(-1). The temperature dependence of the dissociation constant yielded an enthalpic term of -26 kJ mol( -1) and an entropic term of 94 J K-1 mol(-1) for the binding of GR-flu to the receptor, indicating that both quantities contribute equally t o the reaction. An activation enthalpy Delta H-on(#), and entropy Delt a S-on(#), of binding of 50 kT mol(-1) and 43 J mol(-1) K-1 were obtai ned, indicating that the entropy facilitates the initial steps of GR-f lu binding to the 5HT(3) receptor. The fluorescence anisotropy of rece ptor-bound GR-flu and the environmental sensitivity of the fluorescent probe suggest that the binding site has a wide entrance and that it i s 0.8 pH unit more acidic than the bulk solution.