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

Sathya Challasivakanaka,Juan Zhen,Margaret E Smith,Maarten E.A Reith,James D Foster,Roxanne A Vaughan

doi:10.1074/jbc.m117.787002

Sathya Challasivakanaka, Juan Zhen + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m117.787002

Copy DOI

Abstract

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.

Highlights

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)
Phosphorylation of Thr-53 was significantly increased by AMPH (152 Ϯ 9% of basal, p Ͻ 0.05), similar to levels induced by okadaic acid (OA) (177 Ϯ 19% of basal, p Ͻ 0.01)
Characterization of additional substrates in rDAT-LLCPK1 cells showed that Thr-53 phosphorylation was increased by AMPH (142 Ϯ 8% of basal, p Ͻ 0.05) and METH (159 Ϯ 24% of basal, p Ͻ 0.01), with a possible trend toward enhanced phosphorylation induced by DA (125 Ϯ 6% of basal) (Fig. 1B)

Summary

To whom correspondence should be addressed

Temporal control of DA signaling [1]. DAT is a primary target for psychostimulant drugs that elevate extracellular DA including inhibitors such as cocaine that block reuptake, and substrates such as amphetamine (AMPH) and methamphetamine (METH) that stimulate efflux of intracellular transmitter [2]. Uptake and efflux activities are tightly regulated by signaling pathways including protein kinase C (PKC) and extracellular signal–regulated kinase (ERK), providing for acute modulation of reuptake in response to physiological demands [3,4,5,6] Dysregulation of these processes has been hypothesized to contribute to DA imbalances in disorders such as Parkinson’s disease, bipolar disorder, attention deficit hyperactivity disorder, and dopamine transporter deficiency syndrome [7], with recent studies supporting DAT regulatory alterations in some of these conditions (8 –10). T53A DAT showed reduced cocaine analog binding affinity, which was not rescued by Zn2ϩ These findings support a role for Thr-53 phosphorylation in regulation of DAT kinetic properties that may impact transporter responsiveness to amphetamines and cocaine

Results

Discussion

Experimental procedures