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 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
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.