WIN 35,428 and mazindol are mutually exclusive in binding to the cloned human dopamine transporter

It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.