Ligand binding to the serotonin 5HT3 receptor studied with a novel fluorescent ligand

The thermodn. and kinetics of ligand binding to the purified serotonin 5HT3 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-amino-(N-fluorescein-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiol. investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissocn. const. of 0.32 nM. Changes in the fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissocn. consts. of .apprx.0.2 nM. Fluorescence measurements showed that selective 5HT3 receptor ligands competed for GR-flu binding with a rank order of potency identical to that established with the radioligand [3H]-GR65630. The kinetics of GR-flu binding to the 5HT3 receptor revealed a bimol. assocn. process with an on-rate const. of 1.17*106 s-1 M-1 and a biphasic dissocn. reaction with off-rate consts. of 275*10-6 and 43*10-6 s-1. The temp. dependence of the dissocn. const. 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 to the reaction. An activation enthalpy DH#on and entropy DS#on of binding of 50 kJ mol-1 and 43 J mol-1 K-1 were obtained, indicating that the entropy facilitates the initial steps of GR-flu binding to the 5HT3 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 soln. [on SciFinder (R)