Ethanol-induced regulation of the human dopamine transporter
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The dopamine transporter (DAT) is a plasma membrane-bound protein, localized on peri-synaptic terminals of dopaminergic (DA) neurons. DAT is responsible for terminating DA signaling by rapid removal of the transmitter from the synaptic cleft region. DA signaling relies on a critical balance between release and removal of the neurotransmitter within synaptic clefts. Recycling of DAT between intracellular endosomal compartments and the plasma membrane regulates DAT function. This dynamic trafficking occurs in both a constitutive and regulated manner to increase or decrease the number of transporters on the cell surface available for transmitter reuptake. Therapeutic drugs and/or drugs of abuse, including psychostimulants and ethanol, cause maladaptive changes in DA signaling in mesolimbic areas of the brain, leading to addictive behaviors. DAT is the primary site of action for psychostimulants such as, cocaine, methylphenidate, and amphetamine. These drugs can alter the function and/or regulation of the transporter. Ethanol, one of the most widely abused drugs in society, is known to activate DA pathways in reward and reinforcement areas of the brain. However, the effect of ethanol on DAT function and regulation is less clear. The studies presented here explore the action of ethanol on DAT function in mammalian cell systems, and the subcellular trafficking mechanisms that regulate the transporter. To delineate mechanisms of ethanol action on DAT, several lines of HEK-293 cells stably expressing DAT or ethanol-insensitive DAT mutants were generated. Short-term ethanol exposure was found to potentiate DAT function, and ethanol sensitivity is mediated by specific amino acids in the first intracellular loop. This increase in function was accompanied by an enhancement of DAT expressed on the cell surface. The changes in DAT localization and the absence of consensus phosphorylation sites in the ethanol sensitive regions of the transporter, led to the hypothesis that ethanol modulates DAT uptake by altering the dynamic trafficking of the transporter. In the present studies, we found ethanol directly regulates DAT function by altering specific step of the endosomal recycling pathway. Further analysis of the ethanol-sensitive first intracellular loop revealed this region might also play a role in conformational changes required for substrate binding. The findings presented in these studies describe a novel molecular mechanism of ethanol action on DAT, and provide a framework to further understand the action of ethanol on synaptic dopamine regulation.