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

Abstract.

Human dopamine transporters, stably expressed by human embryonic kidney-293 cells, were reacted with 3β-(3p-chlorophenyl)tropan-2β-carboxylic acid p-isothiocyanatophenylethyl ester (RTI-76) under varying conditions. Exposure to RTI-76 (1 µM) at 0°C, followed by extensive wash-out, reduced subsequent binding of the cocaine analog [³H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428), which was caused by an increase in K _d in the absence of a B _max change. Exposure to RTI-76 (50 nM–1 µM) at 37°C, however, caused concentration-dependent reductions in binding B _max; increases in K _d were observed only at high levels of RTI-76 (0.5–1 µM). The reductions in B _max are consonant with acylation of transporters, the increases in K _d 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 B _max at 37°C. Reductions in binding B _max generated by varying concentrations of RTI-76 up to 300 nM at 37°C correlated with reductions in [³H]dopamine uptake V _max on a one-to-one basis, with K _m 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.