Regulation of the dopamine transporter: a role for ethanol and protein interactions

The dopamine (DA) transporter (DAT) serves to clear released DA from the synaptic cleft and is an important part of the mesolimbic DA system, which mediates the rewarding and reinforcing effects of various drugs of abuse. Several studies suggest that the function of DAT is regulated by protein-protein interactions and signaling systems that alter cellular trafficking of DAT. Ethanol potentiates DAT function in Xenopus oocytes expressing DAT in a manner consistent with altered cellular trafficking. In contrast to ethanol’s effects on DAT, the function of the related norepinephrine transporter (NET) is inhibited by ethanol. To delineate mechanisms of ethanol action on DAT, chimeras were generated between DAT and NET. The results of these as well as site directed mutagenesis experiments revealed ethanol sensitive sites in the first intracellular loop of DAT. The absence of consensus phosphorylation sites in this loop led to the hypothesis that ethanol modulates the interaction between DAT and a putative regulatory protein important for ethanol-induced trafficking of DAT and that this interaction occurs at the first intracellular loop. To identify proteins and signaling pathways that might regulate DAT function, an interaction proteomics based approach was used to isolate and identify proteins associated with DAT. These studies revealed that DAT is part of a large multiprotein complex consisting of 21 proteins that can be classified as ion channels, trafficking proteins, extracellular matrix associated and cytoskeletal proteins. Finally, the effects of ethanol on DAT trafficking were ascertained by examining ethanol-induced changes in DAT function in several cell types. Studies on MDCK cells stably expressing GFP-DAT suggest that ethanol potentiates DAT function in this cell type. SH-SY5Y cells stably expressing DAT were also examined for ethanol effects on DAT function. Ethanol produced a 25% enhancement in DAT function in these cells, which was not statistically significant. The effects of ethanol on DAT trafficking in neuronal cells were observed by using a sindbis viral construct encoding GFP-DAT. The experiments outlined above have led to the identification of a novel role for DAT in ethanol-induced neuroadaptation and in the identification of several novel proteins that could modulate DAT function.