Brain Region-Specific Trafficking of the Dopamine Transporter

Abstract

The dopamine (DA) transporter (DAT) controls dopaminergic neurotransmission by removing extracellular DA. Although DA reuptake is proposed to be regulated by DAT traffic to and from the cell surface, the membrane trafficking system involved in the endocytic cycling of DAT in the intact mammalian brain has not been characterized. Hence, we performed immunolabeling and quantitative analysis of the subcellular and regional distribution of DAT using the transgenic knock-in mouse expressing hemagglutinin (HA) epitope-tagged DAT (HA-DAT) and by using a combination of electron microscopy and a novel method for immunofluorescence labeling of HA-DAT in acute sagittal brain slices. Both approaches demonstrated that, in midbrain somatodendritic regions, HA-DAT was present in the plasma membrane, endoplasmic reticulum, and Golgi complex, with a small fraction in early and recycling endosomes and an even smaller fraction in late endosomes and lysosomes. In the striatum and in axonal tracts between the midbrain and striatum, HA-DAT was detected predominantly in the plasma membrane, and quantitative analysis revealed increased DAT density in striatal compared with midbrain plasma membranes. Endosomes were strikingly rare and lysosomes were absent in striatal axons, in which there was little intracellular HA-DAT. Acute administration of amphetamine in vivo (60 min) or to slices ex vivo (10 -60 min) did not result in detectable changes in DAT distribution. Altogether, these data provide evidence for regional differences in DAT plasma membrane targeting and retention and suggest a surprisingly low level of endocytic trafficking of DAT in the striatum along with limited DAT endocytic activity in somatodendritic areas.

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

“…We and others have shown that the closely related SLC6 transporters DAT and SERT, like NET, undergo considerable constitutive internalization (18,20,22,23), although a recent study has indicated that DAT only might undergo modest endocytic trafficking in vivo (63). Nonetheless, according to our previous findings and in contrast to NET, DAT and SERT are mainly sorted to late endosomes and lysosomes, and, if recycled, they are more likely to follow the Rab4 short loop recycling pathway (22,24).…”

Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

“…Gene silencing studies suggest that both constitutive and PKC-stimulated DAT internalization in nonneuronal cell lines is clathrindependent (37); however, whether chronic clathrin depletion artifactually skews these studies is uncertain. A recent study examining DAT trafficking in a knock-in mouse encoding a DAT extracellular epitope tag observed only modest DAT endocytosis and little/no clathrin colocalization under basal conditions (38). However, it is unclear whether antibody-bound DAT traffics similar to native DAT, as we investigate here.…”

Fast‐scan cyclic voltammetry demonstrates that L‐DOPA produces dose‐dependent, regionally selective bimodal effects on striatal dopamine kinetics in vivo

“…; Block et al . ), and thus, functional changes in DAT may be a major contributor to L‐DOPA‐associated kinetic changes in DA clearance. By contrast, other in vivo studies demonstrate that acute high doses of L‐DOPA significantly decrease striatal DAT binding in rodents, suggesting that L‐DOPA induces DAT internalization (Scheffel et al .…”