The dopamine transporter, DAT, is a primary regulator of dopamine (DA) signaling at the synapse. Persistent stimulation with the substrate amphetamine (AMPH) promotes DAT internalization. AMPH rapidly elicits DA efflux, yet its effect on DAT trafficking at short times is unknown. We examined the rapid effect of AMPH on DAT trafficking in rat striatal synaptosomes using biotinylation to label surface DAT. Within 30 s of treatment with 3 μM AMPH, synaptosomal DAT surface expression increased to 163% of control and remained elevated through at least 1 min before returning to control levels at 2.5 min. The increase in surface DAT was cocaine-sensitive but was not produced by DA itself. A 1-min preincubation with AMPH did not alter [3H]DA uptake, but did result in a higher basal DA efflux and efflux elicited in the presence of AMPH as compared to vehicle pretreatment. Reversible biotinylation experiments demonstrated that the AMPH-stimulated rise in surface DAT is due to an increase in the delivery of DAT to the plasmalemmal membrane rather than a reduction of the endocytic process. These studies suggest that AMPH has a biphasic effect on DAT trafficking and acts rapidly to regulate DAT in the plasmalemmal membrane.
Bibliographical noteFunding Information:
Funded by NIH grant DA11697 and Pharmacological Sciences Training Grant GM07767. This work was supported in part by the Michigan Diabetes Research and Training Center Grant 5P60DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. We would like to thank Dr. Steven Lentz for his help in obtaining and processing the confocal images and Dr. Roxanne Vaughan for the MAB16 anti-DAT monoclonal antibody. We would also like to thank Dr. Ronald Holz, Dr. Jose Esteban, and Dr. Jeff Martens for their helpful discussions on the results in this manuscript.
- Dopamine transporter
- HEK293 cells
- Transporter regulation