The binding of the fluorescein-labeled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino- (N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilized ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT3R), was characterized by frequency-domain time-resolved fluorescence spectroscopy (TRFS). Detailed understanding of how ligands interact with the homopentameric receptor was obtained. While a 1:1 stoichiometry was obsd. for the GR-flu - receptor complex, the agonist quipazine bound cooperatively to the receptor, suggesting multiple binding sites for this ligand. The GR-flu-binding site of the receptor was proven to provide an acidic environment as shown by detg. the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation expts. indicated a hindered but still high mobility for the receptor-bound GR-flu. Hence, the binding site is expected to present a wide opening to the ligand. Finally, the authors succeeded in measuring the binding of GR-flu to 5HT3 receptors in live cells. These results show that the purified and the native receptor behave identically and demonstrate that time-resolved fluorescence measurements are suited to selectively investigate biomol. interactions in live cells. [on SciFinder (R)]