Membrane trafficking regulates the activity of the human dopamine transporter

The trafficking of synaptic proteins is unquestionably a major determinant of the properties of synaptic transmission. Here, we present a detailed ana lysis of the downregulation and intracellular trafficking of the cocaine- a nd amphetamine-sensitive dopamine transporter (DAT), a presynaptic plasma m embrane protein responsible for the regulation of extracellular DA concentr ations. Using PC12 cells stably transfected with human DAT cDNA, we observe that phorbol ester activation of protein kinase C (PKC) results in decreas ed transporter capacity and a parallel decrease in the amount of DAT on the cell surface that is attributable to intracellular transporter sequestrati on. After internalization, DAT diverges to the recycling, as opposed to the degradative, arm of the endocytic pathway. This study demonstrates, for th e first time, DAT endocytosis, establishes the pathways through which DAT t raffics both at steady state and in response to PKC activation, and suggest s that DAT recycling is likely to occur.