A substantial body of evidence suggests that the dopamine transporter (DAT) is the principal site for cocaine-induced reward and euphoria. Interactions between the DAT and its substrates and ligands may therefore be of clinical relevance. The pharmacological characteristics of DAT compounds were compared in wild type (WT) and mutant DATs. The DAT mutants chosen for study were those with reduced binding and uptake activities (aspartic acid 79 mutated to alanine, termed D79A), reduced binding but normal uptake (tyrosine 251 mutated to alanine, termed Y251A; tyrosine 273 mutated to alanine, termed, Y273A), and normal binding but reduced uptake (a double mutation: serines 356 and 359 mutated to alanine, termed S356,359A). The WT and mutant DATs were transfected into COS-7 cells, and their pharmacological activities were examined 3 days later. Different patterns of pharmacological activity emerged. GBR 12909, cocaine, and mazindol each showed reduced affinity for the Y251A and the Y273A mutants, but their affinity for the S356,359A mutant was similar to that of the WT DAT. d-Amphetamine, MPP+, and dopamine each showed reduced affinity for the S356,359A mutant. Benztropine and methylphenidate had a different effect. Relative to the WT DAT, they both showed reduced affinity for the S356,359A mutant when displacing radioactive carboxyfluorotropane (CFT) binding, but similar affinity when inhibiting radioactive dopamine uptake. These results indicate that methylphenidate and benztropine may interact with the DAT in a different fashion then other substrates and ligands.
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