Ethanol-sensitive sites on the human dopamine transporter

Rajani Maiya, Kari Buck, R. Adron Harris, R. Dayne Mayfield

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Previous studies have shown that ethanol enhanced [ 3H] 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.

Original languageEnglish (US)
Pages (from-to)30724-30729
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number34
DOIs
StatePublished - Aug 23 2002

Fingerprint

Dopamine Plasma Membrane Transport Proteins
Ethanol
Norepinephrine Plasma Membrane Transport Proteins
Xenopus
Oocytes
Amino Acids
Complementary RNA
Mutagenesis
Mutation
Site-Directed Mutagenesis
HeLa Cells
Dopamine
Modulation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ethanol-sensitive sites on the human dopamine transporter. / Maiya, Rajani; Buck, Kari; Adron Harris, R.; Dayne Mayfield, R.

In: Journal of Biological Chemistry, Vol. 277, No. 34, 23.08.2002, p. 30724-30729.

Research output: Contribution to journalArticle

Maiya, R, Buck, K, Adron Harris, R & Dayne Mayfield, R 2002, 'Ethanol-sensitive sites on the human dopamine transporter', Journal of Biological Chemistry, vol. 277, no. 34, pp. 30724-30729. https://doi.org/10.1074/jbc.M204914200
Maiya, Rajani ; Buck, Kari ; Adron Harris, R. ; Dayne Mayfield, R. / Ethanol-sensitive sites on the human dopamine transporter. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 34. pp. 30724-30729.
@article{dffe841393be4e23b678a1237a92a557,
title = "Ethanol-sensitive sites on the human dopamine transporter",
abstract = "Previous studies have shown that ethanol enhanced [ 3H] 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.",
author = "Rajani Maiya and Kari Buck and {Adron Harris}, R. and {Dayne Mayfield}, R.",
year = "2002",
month = "8",
day = "23",
doi = "10.1074/jbc.M204914200",
language = "English (US)",
volume = "277",
pages = "30724--30729",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "34",

}

TY - JOUR

T1 - Ethanol-sensitive sites on the human dopamine transporter

AU - Maiya, Rajani

AU - Buck, Kari

AU - Adron Harris, R.

AU - Dayne Mayfield, R.

PY - 2002/8/23

Y1 - 2002/8/23

N2 - Previous studies have shown that ethanol enhanced [ 3H] 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.

AB - Previous studies have shown that ethanol enhanced [ 3H] 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.

UR - http://www.scopus.com/inward/record.url?scp=0037163127&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037163127&partnerID=8YFLogxK

U2 - 10.1074/jbc.M204914200

DO - 10.1074/jbc.M204914200

M3 - Article

C2 - 12070173

AN - SCOPUS:0037163127

VL - 277

SP - 30724

EP - 30729

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -