Ethanol-sensitive sites on the human dopamine transporter.
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.
Maiya
Rajani
R
Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, 2500 Speedway, Austin, TX 78712, USA.
Buck
Kari J
KJ
Harris
R Adron
RA
Mayfield
R Dayne
RD
D000818:Animals; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D051381:Rats; D013329:Structure-Activity Relationship; D027981:Symporters / Q000187:drug effects; D014982:Xenopus laevis
D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; C493214:SLC6A3 protein, human; C493226:Slc6a2 protein, rat; C493216:Slc6a3 protein, rat; D027981:Symporters; D000431:Ethanol; D004298:Dopamine