Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding.

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.