In vitro and in vivo ligand binding to the 5HT(3) serotonin receptor characterised by time-resolved fluorescence spectroscopy

The binding of the fluorescein-labelled antagonist GR-flu ([1,2,3,9-tetrahy dro-3-[(5-metyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluoresceinthiocarba moyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilised ligan d-gated ion channel, the type-3 serotonin [5-hydroxytryptamine, 5HT) recept or (5HT(3)R), was characterised by frequency-domain time-resolved fluoresce nce spectroscopy (TRFS). Detailed understanding of how ligands interact wit h the homopentameric receptor was obtained While a 1:1 stoichiometry was ob served for the GR-flu - receptor complex, the agonist quipazine bound coope ratively to the receptor, suggesting multiple binding sites for this ligand . The GR-flu-binding site of the receptor was proven to provide an acidic e nvironment as shown by determining the fraction of bound GR-flu in the prot onated state. Fluorescence anisotropy relaxation experiments indicated a hi ndered but still high mobility for the receptor-bound GR;flu Hence, the bin ding site is expected to present a wide opening to the ligand. Finally, we succeeded in measuring the binding of GR-flu to 5HT(3) receptors in live ce lls; These results:show that the purified and the native receptor behave id entically and demonstrate: that time-resolved fluorescence measurements are -suited to selectively investigate biomolecular interactions in live cells.