Mapping the antagonist binding site of the serotonin type 3 receptor by fluorescence resonance energy transfer

We have measured fluorescence resonance energy transfer (FRET) between a fl uorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the mice lle surrounding the detergent-solubilized receptor. The experimentally obse rved FRET efficiency was evaluated on the basis of the characteristic dimen sions of the receptor-micelle complex and the average number of acceptor mo lecules in such micelles. The binding site was determined to be 5.4 +/- 0.9 nm above the center of the detergent micelle. The experiments were perform ed below the critical micellar concentration of the detergent (C12E9) used to solubilize the receptor, under which conditions it was demonstrated that the ligand binding activity was fully preserved. This reduces considerably the fluorescence background arising from probes not associated with the re ceptor, allowing a precise determination of the transfer efficiency.