Abstract
Previous studies have shown that ethanol enhanced [(3)H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity.
Highlights
The family of Naϩ and ClϪ-dependent transporters, which includes the dopamine (DA)1 and norepinephrine (NE) transporters (DAT and NET, respectively), functions to clear released neurotransmitters from the synaptic cleft [1]
These results suggest that ethanol enhancement of transporter function may involve redistribution of DAT at the cell surface
NET, which belongs to the Naϩ and ClϪ-dependent family of neurotransmitter transporters, shares a high degree of homology (64%) with DAT [14], but in vivo electrochemical studies have shown that ethanol inhibits rather than enhances NET function [13]
Summary
The family of Naϩ and ClϪ-dependent transporters, which includes the dopamine (DA) and norepinephrine (NE) transporters (DAT and NET, respectively), functions to clear released neurotransmitters from the synaptic cleft [1]. DAT regulates the spatial and temporal aspects of dopaminergic synaptic transmission and is an integral part of the mesostriatal DA system This system plays a central role in mediating the rewarding and reinforcing effects of various drugs of abuse, including ethanol [2,3,4]. Experiments carried out in human embryonic kidney (HEK-293) cells demonstrated that acute exposure to cocaine enhances DAT activity in a time-dependent manner by increasing the number of functional transporters at the cell surface [8]. Acute ethanol (10 –100 mM) enhances DAT-mediated [3H]DA uptake and transporter-associated currents in a time- and concentrationdependent manner [12] This potentiation of transporter function was accompanied by an increase in the number of functional cell surface transporters, suggesting that ethanol affects transporter function by altering the steady state trafficking of DAT to the cell surface. Site-directed mutagenesis experiments were carried out, and mutant transporters were functionally analyzed to pinpoint individual amino acid residues that may be crucial for ethanol enhancement of DAT function
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