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Direct evidence that two cysteines in the dopamine transporter form a disulfide bond

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Abstract

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.

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Abbreviations

DA:

Dopamine

NE:

Norepinephrine

5-HT:

5-Hydroxytryptamine

DAT:

Dopamine transporter

NET:

NE transporter

SERT:

5-HT transporter

mDAT:

Mouse DAT

dmDAT:

Drosophila melanogaster DAT

TM:

Transmembrane domain

EL:

Extracellular loop

Reference

  1. Amara SG, Arriza JL (1993) Neurotransmitter transporters: three distinct gene families. Curr Opin Neurobiol 3:337–344

    Article  PubMed  CAS  Google Scholar 

  2. Rudnick G, Clark J (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters. Biochim et Biophys Acta 1144:249–263

    Article  CAS  Google Scholar 

  3. Goodnick PJ (1991) Pharmacokinetics of second generation antidepressants: bupropion. Psychopharmacol Bull 27:513–519

    PubMed  CAS  Google Scholar 

  4. Ritz MC, Lamb RJ, Goldberg SR, Kuhar MJ (1987) Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237:1219–1223

    Article  PubMed  CAS  Google Scholar 

  5. Giros B, Caron MG (1993) Molecular characterization of the dopamine transporter. Trends Pharmacol Sci 14:43–49

    Article  PubMed  CAS  Google Scholar 

  6. Gu H, Wall SC, Rudnick G (1994) Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. J Biol Chem 269:7124–7130

    PubMed  CAS  Google Scholar 

  7. Ascher JA, Cole JO, Colin JN, Feighner JP, Ferris RM, Fibiger HC, Golden RN, Martin P, Potter WZ, Richelson E, et al. (1995) Bupropion: a review of its mechanism of antidepressant activity. J Clin Psychiatry 56:395–401

    PubMed  CAS  Google Scholar 

  8. Seeman P, Madras BK (1998) Anti-hyperactivity medication: methylphenidate and amphetamine [In Process Citation]. Mol Psychiatry 3:386–396

    Article  PubMed  CAS  Google Scholar 

  9. Han DD, Gu HH (2006) Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs. BMC Pharmacol 6:6

    Article  PubMed  CAS  Google Scholar 

  10. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E (2005) Crystal structure of a bacterial homologue of Na+/Cl− dependent neurotransmitter transporters. Nature 437:215–223

    Article  PubMed  CAS  Google Scholar 

  11. Wang JB, Moriwaki A, Uhl GR (1995) Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression. J Neurochem 64:1416–1419

    Article  PubMed  CAS  Google Scholar 

  12. Chen JG, Liu-Chen S, Rudnick G (1997) External cysteine residues in the serotonin transporter. Biochemistry 36:1479–1486

    Article  PubMed  CAS  Google Scholar 

  13. Wu X, Gu HH (2003) Cocaine affinity decreased by mutations of aromatic residue phenylalanine 105 in the transmembrane domain 2 of dopamine transporter. Mol Pharmacol 63:653–658

    Article  PubMed  CAS  Google Scholar 

  14. Nelson RM, Long GL (1989) A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem 180:147–151

    Article  PubMed  CAS  Google Scholar 

  15. Chen R, Han DD, Gu HH (2005) A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate. J Neurochem 94:352–359

    Article  PubMed  CAS  Google Scholar 

  16. Seal RP, Leighton BH, Amara SG (1998) Transmembrane topology mapping using biotin-containing sulfhydryl reagents. Methods Enzymol 296:318–331

    Article  PubMed  CAS  Google Scholar 

  17. Chen R, Wu X, Wei H, Han DD, Gu HH (2006) Molecular cloning and functional characterization of the dopamine transporter from Eloria noyesi, a caterpillar pest of cocaine-rich coca plants. Gene 366:152–160

    Article  PubMed  CAS  Google Scholar 

  18. Hastrup H, Karlin A, Javitch JA (2001) Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment. Proc Natl Acad Sci USA 98:10055–10060

    Article  PubMed  CAS  Google Scholar 

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Acknowledgement

This work was supported by a grant from National Institute on Drug Abuse (DA014610).

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Authors and Affiliations

  1. Department of Pharmacology and Department of Psychiatry, The Ohio State University College of Medicine, 333 West 10th Avenue, Columbus, OH, 43210, USA

    Rong Chen, Hua Wei, Erik R. Hill, Lucy Chen, Liying Jiang, Dawn D. Han & Howard H. Gu

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  1. Rong Chen
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  2. Hua Wei
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  3. Erik R. Hill
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  4. Lucy Chen
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  5. Liying Jiang
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  6. Dawn D. Han
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  7. Howard H. Gu
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Corresponding author

Correspondence to Howard H. Gu.

Additional information

Rong Chen and Hua Wei contributed equally to this manuscript.

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Chen, R., Wei, H., Hill, E.R. et al. Direct evidence that two cysteines in the dopamine transporter form a disulfide bond. Mol Cell Biochem 298, 41–48 (2007). https://doi.org/10.1007/s11010-006-9348-7

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  • DOI: https://doi.org/10.1007/s11010-006-9348-7

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