Ethanol alters endosomal recycling of human dopamine transporters

Dynamic membrane trafficking of the monoamine dopamine transporter (DAT) regulates dopaminergic signaling. Various intrinsic and pharmacological modulators can alter this trafficking. Previously we have shown ethanol potentiates in vitro DAT function and increases surface expression. However, the mechanism underlying these changes is unclear. In the present study, we found ethanol directly regulates DAT function by altering endosomal recycling of the transporter. We defined ethanol action on transporter regulation by [(3)H]DA uptake functional analysis combined with biochemical and immunological assays in stably expressing DAT HEK-293 cells. Short-term ethanol exposure potentiated DAT function in a concentration-, but not time-dependent manner. This potentiation was accompanied by a parallel increase in DAT surface expression. Ethanol had no effect on function or surface localization of the ethanol-insensitive mutant (G130T DAT), suggesting a trafficking-dependent mechanism in mediating the ethanol sensitivity of the transporter. The ethanol-induced increase in DAT surface expression occurred without altering the overall size of DAT endosomal recycling pools. We found ethanol increased the DAT membrane insertion rate while having no effect on internalization of the transporter. Ethanol had no effect on the surface expression or trafficking of the endogenously expressing transferrin receptor, suggesting ethanol does not have a nonspecific effect on endosomal recycling. These results define a novel trafficking mechanism by which ethanol regulates DAT function.