Competition kinetic analysis was performed to examine the interaction mechanism of the dopamine transporter with dopamine, (S)-amphetamine, and cocaine, which play a central role in drug abuse phenomena connected with the dopaminergic system. Efficient dopamine transporter inhibitor [3H]PE2I was used as a reporter ligand for this analysis as this compound initiates slow isomerization of the transporter–ligand complex and thus ensures reliable results of the filtration radioligand assay. It was shown that the three investigated compounds do not initiate slow isomerization of their complexes with the transporter, but their presence inhibits the isomerization step of the radioactive reporter ligand. Secondly, it was shown that (S)-amphetamine and dopamine do not interfere with the fast step of the inhibitor ligand binding, pointing to the formation of the ternary complex, including transporter protein, reporter ligand, and an unlabeled compound. This is possible if the two molecules bind to non-overlapping sites on the transporter. Binding of cocaine results in slightly improved binding of the reporter ligand, pointing to a positive allosteric interaction between these binding processes.
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