A novel pH-sensitive reporter reveals the cocaine-regulated trafficking of dopamine transporters in neuronal processes

The dopamine transporter (DAT) mediated DA reuptake is a major molecular mechanism for termination of dopaminergic signaling in the brain. Psychoactive substances such as cocaine act by inhibition of plasma membrane DAT function as well as by altering its expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. We have now engineered a novel pH-sensitive reporter for DAT by conjugating pHluorin to the second exofacial loop of human DAT. We show that DAT-pHluorin can be used to study DAT localization and its dynamic trafficking at neuronal processes. Using DAT-pHluorin we show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while varicosities contain abundant intracellular DAT within acidic intracellular structures. Using this novel reporter, we show, for the first time, that cocaine exposure leads to a brief DAT internalization followed by membrane reinsertion that lasts for days. We further show that the cocaine-induced DAT trafficking is sensitive to the activities of Synaptojanin1 phosphatase. Thus, our study using the newly engineered DAT optical reporter reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.