Psychostimulants Affect Dopamine Transporter Lateral Mobility and Membrane Microdomain Distribution

Neurotransmitter reuptake by transporters is a major mechanism for terminating synaptic transmission. The human dopamine transporter (hDAT) is one of the main targets for psychostimulants, and is critical to DA homeostasis. Lipid rafts are specialized membrane microdomains that serve as organizing centers to regulate different cellular processes such as neurotransmission and trafficking. To begin to understand how psychostimulants, methamphetamine (METH) and amphetamine (AMPH), affect hDAT microdomain association, we utilized fluorescence recovery after photobleaching (FRAP) and density-gradient centrifugation. Our FRAP studies revealed significant changes in the rate (D) and extent (Mf) of the fluorescence recovery into the bleached region of the plasma membrane in cells expressing YFP-hDAT in the presence of METH but not AMPH. Substitution of five N-terminal Ser with Ala, (cannot be phosphorylated) or Asp (pseudo-phosphorylated), and removal of the 22 N-terminal amino acids restored the diffusion rate of the transporter to control levels. Using density-gradient centrifugation, we found that YFP-hDAT is distributed into both, classically defined, membrane raft and non-raft fractions. Incubating with METH and AMPH shifted YFP-hDAT from non-raft to raft fractions. We have previously shown that METH uniquely modulates the biophysical properties of DAT. Our present findings suggest that METH and AMPH cause hDAT to partition into lipid raft membrane microdomains, and the decrease in the hDAT diffusion rate evoked by METH- vs. AMPH-occupied DAT could suggest that the N-terminal domain of the transporter is associated with a distinct group of proteins when exposed to these psychostimulants, and may thus describe an underlying mechanism behind the addictive biological differences between these psychostimulants.