Biophysical Characterization of the Cocaine Binding Pocket in the Serotonin Transporter Using a Fluorescent Cocaine Analogue as a Molecular Reporter

To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3β-(4-methylphenyl)tropane-2β-carboxylic acidN-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); KI = 14 nm) that contained the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specific binding of RTI-233 to the rat serotonin transporter, purified from Sf-9 insect cells, was demonstrated by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (2β-carbomethoxy-3β-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measurement of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound RTI-233 displayed an emission maximum (λmax) of 532 nm, corresponding to a 4-nm blue shift as compared with the λmax of RTI-233 in aqueous solution and corresponding to the λmax of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (KSV = 1.7m−1), although not to the same extent as free RTI-233 (KSV = 7.2m−1). Conversely, the hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.