Ligand Selectivity among the Dopamine and Serotonin Transporters Specified by the Forward Binding Reaction.

The membrane transporters for the monoamines serotonin (SERT) and dopamine (DAT) are prominent targets of various psychoactive substances, including competitive inhibitors, such as tricyclic antidepressants, methylphenidate, and cocaine. Upon rapid application of a substrate, SERT and DAT display an inwardly directed current comprised of a peak and a steady-state component. Binding of a competitive inhibitor to the transporter leads to reduction of the peak current amplitude because occupancy of the transporter by an inhibitor prevents the induction of the peak current by the substrate. We show that the inhibitory effect on the peak current can be used to study the association rate constant (k(on)), dissociation rate constant (k(off)), and equilibrium dissociation constant (K-D) of chemically distinct SERT and DAT inhibitors, with high temporal precision and without the need of high-affinity radioligands as surrogates. We exemplify our approach by measuring the kinetics of cocaine, methylphenidate, and desipramine binding to SERT and DAT. Our analysis revealed that the selectivity of methylphenidate and desipramine for DAT and SERT, respectively, can be accounted for by their rate of association and not by the residence time in their respective binding sites.