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

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