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

The binding of the fluorescein-labelled 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-solubilised ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT(3)R), was characterised 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 observed 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 determining the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation experiments 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, we succeeded in measuring the binding of GR-flu to 5HT(3) 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 biomolecular interactions in live cells.