Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding

present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl-- dependent neurotransmitter transporters, in conformational changes of the dopamine ( DA) transporter. Asparagine substitution ( D345N) resulted in near normal transporter expression on the cell surface but caused extremely low V-max and K-m values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine- like inhibitor 2 beta- carbomethoxy-3beta ( 4- fluorophenyl) tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate- binding site to the extracellular side, leading to a loss of the outward- facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane- impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na+- dependent conformational change, which may contribute to its Na+ -dependent uptake inhibitory activity.