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. Exposure 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 increase 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 binding 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 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 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 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
RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, as a tool for irreversibly inactivating dopamine transporter function in vitro
Wang, LJC., Berfield, JL., Kuhar, MJ., Carroll, F., & Reith, MEA. (2000). RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, as a tool for irreversibly inactivating dopamine transporter function in vitro. Naunyn-Schmiedeberg's Archives of Pharmacology, 362(3), 238-247.