Ethanol potentiates dopamine uptake and increases cell surface distribution of dopamine transporters expressed in SK-N-SH and HEK-293 cells.
Ethanol increases dopaminergic release in the reward and reinforcement areas of the brain. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT). In vitro electrophysiological and biochemical studies in Xenopus laevis oocytes have previously shown ethanol potentiates DAT function and increases transporter-binding sites. The potentiating effect of ethanol on the transporter is eliminated in Xenopus oocytes by the DAT mutation glycine 130 to threonine. However, ethanol's action on DAT functional regulation has yet to be examined in mammalian cell expression systems. To further understand the molecular mechanisms of ethanol's action on DAT, we determined the direct mechanistic action of short-term (< or =2 h) ethanol exposure on transporter function and cell surface distribution in non-neuronal human embryonic kidney cells-293 (HEK-293) and neuronal SK-N-SH neuroblastoma cells expressing the transporter. Wild-type or G130T mutant DAT were overexpressed in HEK-293 and SK-N-SH cells. Ethanol potentiated DAT mediated [(3)H]DA uptake in a dose (25, 50, 100 mM), but not time dependent manner in cells expressing wild-type DAT. Ethanol-induced potentiation of uptake was significantly reduced in cells expressing the G130T mutant. Analysis of DA uptake kinetic parameters indicates 100-mM ethanol exposure increased [(3)H]DA uptake velocity (V(max)), while affinity for DA (K(m)) remained unchanged. The effect of ethanol on wild-type DAT surface expression was measured by biotinylation cell surface labeling. DAT surface expression increased 40%-50% after 1-h, 100-mM ethanol exposure. These studies show ethanol potentiates DAT functional regulation in both neuronal and non-neuronal cells, suggesting a direct mechanistic action of ethanol on transporter trafficking in mammalian systems. Our findings demonstrate ethanol's action on DAT function and regulation is consistent across multiple model systems.
Riherd
D Nicole
DN
Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, TX 78712, USA.
Galindo
David G
DG
Krause
Lucretia R
LR
Mayfield
R Dayne
RD
D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D004357:Drug Synergism; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000196:embryology; D009447:Neuroblastoma
D050483:Dopamine Plasma Membrane Transport Proteins; D000431:Ethanol; D004298:Dopamine