RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, asa tool for irreversibly inactivating dopamine transporter function in vitro

Human dopamine transporters, stably expressed by human embryonic kidney-293 cells, were reacted with 3 beta-(3p-chlorophenyl)tropan-2 beta-carboxylic acid p-isothiocyanatophenylethyl ester (RTI-76) under varying conditions. E xposure to RTI-76 (1 mu M) at 0 degrees C, followed by extensive wash-out, reduced subsequent binding of the cocaine analog [H-3]2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane (WIN 35,428), which was caused by an increas e in K-d in the absence of a B-max change. Exposure to RTI-76 (50 nM-1 mu M ) at 37 degrees C, however, caused concentration-dependent reductions in bi nding B-max; increases in K-d were observed only at high levels of RTI-76 ( 0.5-1 mu M). The reductions in B-max are consonant with acylation of transp orters, the increases in K-d with incomplete wash-out observed also for the amine precursor of RTI-76 which lacks the isothiocyanate group for irrever sible binding and which did not lower B-max at 37 degrees C. Reductions in binding B-max generated by varying concentrations of RTI-76 up to 300 nM at 37 degrees C correlated with reductions in [H-3]dopamine uptake V-max on a one-to-one basis, with K-m values showing a tendency towards a small reduc tion as a function of transporter inactivation, but the potency of WIN 35,4 28 in inhibiting uptake not showing a change. The results are discussed in the context of possible oligomeric assemblies of dopamine transporters carr ying multiple recognition sites for cocaine analogs and dopamine.