Dopamine Transporter Is Rarely Endocytosed in Striatum

The dopamine transporter (DAT) limits the spatial and temporal spread of dopaminergic signaling by taking up extracellular dopamine. This function is thought to be modulated by endogenous signaling pathways. For example, phosphorylation of DAT by protein kinase C (PKC) reduces dopamine uptake, whereas phosphorylation by extracellular signal-regulated kinase increases uptake. Although phosphorylation might alter uptake by changing the Vmax of the transporter, PKC-mediated phosphorylation has been proposed to reduce dopamine uptake primarily by promoting DAT endocytosis. It has further been proposed that sorting of endocytosed DAT to recycling endosomes underlies short-term reductions in uptake, whereas targeting the transporter for degradation in lysosomes underlies longer term modulation (Vaughan and Foster, 2013, Trends Pharmacol Sci 34:489). To investigate these hypotheses and examine which endocytic pathways predominate under physiological conditions, Block et al. used knock-in mice expressing hemagglutinin-tagged DAT and examined DAT localization in acute brain slices and by electron microscopy. In the midbrain, most DAT ( 75%) was associated with intracellular membranes, particularly within somata, where it was found primarily on nuclear, endoplasmic reticulum, and Golgi membranes. DAT was occasionally colocalized with markers of early, recycling, or intermediate/sorting endosomes in vesicular structures in the midbrain, but it never colocalized with markers of late endosomes or lysosomes. Plasma membrane expression of DAT was higher in dopaminergic axons in the striatum than in the midbrain. Furthermore, DAT density was greater in axonal varicosities (putative presynaptic sites) than along intervening shafts, and most axonal DAT ( 85%) was associated with the plasma membrane. Surprisingly, markers of early and recycling endosomes were rarely observed in dopaminergic axons, and DAT never colocalized with these markers. Moreover, markers of intermediate/sorting endosomes and lysosomes were never detected in dopaminergic axons. Finally, although amphetamine can induce DAT endocytosis in cultured neurons, amphetamine administration in vivo or in slices did not significantly change the subcellular distribution of DAT. These results indicate that constitutive endocytosis rarely occurs in the axons of dopaminergic neurons, and when it does occur, internalized proteins are likely recycled to the plasma membrane rather being degraded in lysosomes. Thus, modulation of dopamine uptake must be regulated primarily by changes in DAT Vmax or lateral diffusion of DAT within the membrane.