Substrate-induced trafficking of the dopamine transporter in heterologously expressing cells and in rat striatal synaptosomal preparations

Dopamine transporter (DAT) trafficking was assessed by functional measurements of dopamine uptake and by biotinylation of surface proteins followed by gel electrophoresis and Western blotting. In human embryonic kidney (HEK)-293 cells expressing human DAT ( HEK-hDAT), pretreatment with dopamine (0.1-100 muM) followed by washout caused reductions in subsequent dopamine uptake (reflected in V-max) with effective dopamine concentrations in the 10 to 100 muM range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 muM dopamine corresponded with decreases measured in surface DAT by the noncleavable biotin method, which were caused, at least in part, by enhanced endocytosis as monitored with cleavable biotin. Pretreatment of rat striatal synaptosomes with dopamine ( 10 and 100 muM) also caused reductions in DAT uptake activity (V-max), and again the underlying mechanism seemed to be a diminished presence of DAT at the surface of synaptosomes as measured by the noncleavable biotin method. The copresence of cocaine during pretreatment with dopamine prevented the down-regulation of surface DAT. The present results show that DAT surface residency can be regulated by substrate acting on it, not only in cells heterologously expressing DAT but also in situ in rat brain tissue.