Ligand binding to the serotonin 5HT sub(3) receptor studied with a novel fluorescent ligand

The thermodynamics and kinetics of ligand binding to the purified serotonin 5HT sub(3) receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-ami no- (N-fluorescien-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiological investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissociation constant of 0.32 nM. Changes in the fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissociation constants of similar to 0.2 nM. Fluorescence measurements showed that selective 5HT sub(3) receptor ligands competed for GR-flu binding with a rank order of potency identical to that established with the radioligand [ super(3)H]-GR65630. The kinetics of GR-flu binding to the 5HT sub(3) receptor revealed a bimolecular association process with an on-rate constant of 1.17 x 10 super(6) s super(-1) M super(-1) and a biphasic dissociation reaction with off-rate constants of 275 x 10 super(-6) and 43 x 10 super(-6) s super(-1). The temperature dependence of the dissociation constant yielded an enthalpic term of -26 kJ mol super(-1) and an entropic term of 94 J K super(-1) mol super(-1) for the binding of GR-flu to the receptor, indicating that both quantities contribute equally to the reaction. An activation enthalpy Delta H super(#) sub(on) and entropy Delta S super(#) sub(on) of binding of 50 kJ mol super(-1) and 43 J mol super(-1) K super(-1) were obtained, indicating that the entropy facilitates the initial steps of GR-flu binding to the 5HT sub(3) receptor. The fluorescence anisotropy of receptor-bound GR-flu and the environmental sensitivity of the fluorescent probe suggest that the binding site has a wide entrance and that it is 0.8 pH unit more acidic than the bulk solution.