Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

X-ray structure of dopamine transporter elucidates antidepressant mechanism

Abstract

Antidepressants targeting Na+/Cl-coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Architecture of Drosophila DATcryst.
Figure 2: Antidepressant-binding site.
Figure 3: Ion-binding sites.
Figure 4: Cholesterol site.
Figure 5: Extracellular and cytoplasmic gates and the C-terminal latch.
Figure 6: Mechanisms of antidepressants and cholesterol.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

Data deposits

The coordinates for the structure have been deposited in the Protein Data Bank under the accession code 4M48.

References

  1. Jessell, T. M. & Kandel, E. R. Synaptic transmission: a bidirectional and self-modifiable form of cell-cell communication. Cell 72 (Suppl). 1–30 (1993)

    Article  Google Scholar 

  2. Masson, J., Sagne, C., Hamon, M. & El Mestikawy, S. Neurotransmitter transporters in the central nervous system. Pharmacol. Rev. 51, 439–464 (1999)

    CAS  PubMed  Google Scholar 

  3. Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011)

    Article  CAS  Google Scholar 

  4. Rudnick, G. Ion-coupled neurotransmitter transport: thermodynamic vs. kinetic determinations of stoichiometry. Methods Enzymol. 296, 233–247 (1998)

    Article  CAS  Google Scholar 

  5. Radian, R., Bendahan, A. & Kanner, B. I. Purification and identification of the functional sodium- and chloride-coupled γ-aminobutyric acid transport glycoprotein from rat brain. J. Biol. Chem. 261, 15437–15441 (1986)

    CAS  PubMed  Google Scholar 

  6. Waldman, I. D. et al. Association and linkage of the dopamine transporter gene and attention-deficit hyperactivity disorder in children: heterogeneity owing to diagnostic subtype and severity. Am. J. Hum. Genet. 63, 1767–1776 (1998)

    Article  CAS  Google Scholar 

  7. Shannon, J. R. et al. Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N. Engl. J. Med. 342, 541–549 (2000)

    Article  CAS  Google Scholar 

  8. Meldrum, B. S. Neurotransmission in epilepsy. Epilepsia 36 (suppl. 1). 30–35 (1995)

    Article  Google Scholar 

  9. Kurian, M. A. et al. Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia. J. Clin. Invest. 119, 1595–1603 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kuhn, R. The treatment of depressive states with G 22355 (imipramine hydrochloride). Am. J. Psychiatry 115, 459–464 (1958)

    Article  CAS  Google Scholar 

  11. Axelrod, J., Whitby, L. G. & Hertting, G. Effect of psychotropic drugs on the uptake of H3-norepinephrine by tissues. Science 133, 383–384 (1961)

    Article  ADS  CAS  Google Scholar 

  12. Berton, O. & Nestler, E. J. New approaches to antidepressant drug discovery: beyond monoamines. Nature Rev. Neurosci. 7, 137–151 (2006)

    Article  CAS  Google Scholar 

  13. Pletscher, A. The discovery of antidepressants: a winding path. Experientia 47, 4–8 (1991)

    Article  CAS  Google Scholar 

  14. Anderson, I. M. Selective serotonin reuptake inhibitors versus tricyclic antidepressants: a meta-analysis of efficacy and tolerability. J. Affect. Disord. 58, 19–36 (2000)

    Article  CAS  Google Scholar 

  15. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters. Nature 437, 215–223 (2005)

    Article  ADS  CAS  Google Scholar 

  16. Singh, S. K., Piscitelli, C. L., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008)

    Article  ADS  CAS  Google Scholar 

  17. Krishnamurthy, H. & Gouaux, E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481, 469–474 (2012)

    Article  ADS  CAS  Google Scholar 

  18. Beuming, T. et al. The binding sites for cocaine and dopamine in the dopamine transporter overlap. Nature Neurosci. 11, 780–789 (2008)

    Article  CAS  Google Scholar 

  19. Sørensen, L. et al. Interaction of antidepressants with the serotonin and norepinephrine transporters: mutational studies of the S1 substrate binding pocket. J. Biol. Chem. 287, 43694–43707 (2012)

    Article  Google Scholar 

  20. Pörzgen, P., Park, S. K., Hirsh, J., Sonders, M. S. & Amara, S. G. The antidepressant-sensitive dopamine transporter in Drosophila melanogaster: a primordial carrier for catecholamines. Mol. Pharmacol. 59, 83–95 (2001)

    Article  Google Scholar 

  21. Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Sci. USA 105, 877–882 (2008)

    Article  ADS  CAS  Google Scholar 

  22. Tatsumi, M., Groshan, K., Blakely, R. D. & Richelson, E. Pharmacological profile of antidepressants and related compounds at human monoamine transporters. Eur. J. Pharmacol. 340, 249–258 (1997)

    Article  CAS  Google Scholar 

  23. Torres, G. E. et al. Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter. J. Biol. Chem. 278, 2731–2739 (2003)

    Article  CAS  Google Scholar 

  24. Sitte, H. H., Farhan, H. & Javitch, J. A. Sodium-dependent neurotransmitter transporters: oligomerization as a determinant of transporter function and trafficking. Mol. Interv. 4, 38–47 (2004)

    Article  CAS  Google Scholar 

  25. Li, L. B. et al. The role of N-glycosylation in function and surface trafficking of the human dopamine transporter. J. Biol. Chem. 279, 21012–21020 (2004)

    Article  CAS  Google Scholar 

  26. Chen, R. et al. Direct evidence that two cysteines in the dopamine transporter form a disulfide bond. Mol. Cell. Biochem. 298, 41–48 (2007)

    Article  CAS  Google Scholar 

  27. Norregaard, L., Frederiksen, D., Nielsen, E. O. & Gether, U. Delineation of an endogenous zinc-binding site in the human dopamine transporter. EMBO J. 17, 4266–4273 (1998)

    Article  CAS  Google Scholar 

  28. Buck, K. J. & Amara, S. G. Structural domains of catecholamine transporter chimeras involved in selective inhibition by antidepressants and psychomotor stimulants. Mol. Pharmacol. 48, 1030–1037 (1995)

    CAS  PubMed  Google Scholar 

  29. Chen, J. G., Sachpatzidis, A. & Rudnick, G. The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding. J. Biol. Chem. 272, 28321–28327 (1997)

    Article  CAS  Google Scholar 

  30. Henry, L. K. et al. Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants. J. Biol. Chem. 281, 2012–2023 (2006)

    Article  CAS  Google Scholar 

  31. Bismuth, Y., Kavanaugh, M. P. & Kanner, B. I. Tyrosine 140 of the γ-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition. J. Biol. Chem. 272, 16096–16102 (1997)

    Article  CAS  Google Scholar 

  32. Kitayama, S. et al. Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc. Natl Acad. Sci. USA 89, 7782–7785 (1992)

    Article  ADS  CAS  Google Scholar 

  33. Andersen, J. et al. Location of the antidepressant binding site in the serotonin transporter: importance of Ser-438 in recognition of citalopram and tricyclic antidepressants. J. Biol. Chem. 284, 10276–10284 (2009)

    Article  CAS  Google Scholar 

  34. Talvenheimo, J., Fishkes, H., Nelson, P. J. & Rudnick, G. The serotonin transporter-imipramine “receptor”. J. Biol. Chem. 258, 6115–6119 (1983)

    CAS  PubMed  Google Scholar 

  35. Singh, S. K., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007)

    Article  ADS  CAS  Google Scholar 

  36. Zhou, Z. et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317, 1390–1393 (2007)

    Article  ADS  CAS  Google Scholar 

  37. Zhou, Z. et al. Antidepressant specificity of serotonin transporter suggested by three LeuT–SSRI structures. Nature Struct. Mol. Biol. 16, 652–657 (2009)

    Article  CAS  Google Scholar 

  38. Harding, M. M. Metal–ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr. D 58, 872–874 (2002)

    Article  Google Scholar 

  39. Forrest, L. R., Tavoulari, S., Zhang, Y. W., Rudnick, G. & Honig, B. Identification of a chloride ion binding site in Na+/Cl-dependent transporters. Proc. Natl Acad. Sci. USA 104, 12761–12766 (2007)

    Article  ADS  CAS  Google Scholar 

  40. Zomot, E. et al. Mechanism of chloride interaction with neurotransmitter:sodium symporters. Nature 449, 726–730 (2007)

    Article  ADS  CAS  Google Scholar 

  41. Kantcheva, A. K. et al. Chloride binding site of neurotransmitter sodium symporters. Proc. Natl Acad. Sci. USA 110, 8489–8494 (2013)

    Article  ADS  CAS  Google Scholar 

  42. Tavoulari, S., Forrest, L. R. & Rudnick, G. Fluoxetine (Prozac) binding to serotonin transporter is modulated by chloride and conformational changes. J. Neurosci. 29, 9635–9643 (2009)

    Article  CAS  Google Scholar 

  43. Scanlon, S. M., Williams, D. C. & Schloss, P. Membrane cholesterol modulates serotonin transporter activity. Biochemistry 40, 10507–10513 (2001)

    Article  CAS  Google Scholar 

  44. North, P. & Fleischer, S. Alteration of synaptic membrane cholesterol/phospholipid ratio using a lipid transfer protein. Effect on γ-aminobutyric acid uptake. J. Biol. Chem. 258, 1242–1253 (1983)

    CAS  PubMed  Google Scholar 

  45. Hong, W. C. & Amara, S. G. Membrane cholesterol modulates the outward facing conformation of the dopamine transporter and alters cocaine binding. J. Biol. Chem. 285, 32616–32626 (2010)

    Article  CAS  Google Scholar 

  46. Bennett, E. R., Su, H. & Kanner, B. I. Mutation of arginine 44 of GAT-1, a (Na+ + Cl-coupled γ-aminobutyric acid transporter from rat brain, impairs net flux but not exchange. J. Biol. Chem. 275, 34106–34113 (2000)

    Article  CAS  Google Scholar 

  47. Cao, Y., Li, M., Mager, S. & Lester, H. A. Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters. J. Neurosci. 18, 7739–7749 (1998)

    Article  CAS  Google Scholar 

  48. Loland, C. J., Norregaard, L., Litman, T. & Gether, U. Generation of an activating Zn2+ switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proc. Natl Acad. Sci. USA 99, 1683–1688 (2002)

    Article  ADS  CAS  Google Scholar 

  49. Holton, K. L., Loder, M. K. & Melikian, H. E. Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization. Nature Neurosci. 8, 881–888 (2005)

    Article  CAS  Google Scholar 

  50. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

    Article  CAS  Google Scholar 

  51. Dukkipati, A., Park, H. H., Waghray, D., Fischer, S. & Garcia, K. C. BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies. Protein Expr. Purif. 62, 160–170 (2008)

    Article  CAS  Google Scholar 

  52. Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002)

    Article  ADS  CAS  Google Scholar 

  53. Baconguis, I. & Gouaux, E. Structural plasticity and dynamic selectivity of acid-sensing ion channel-spider toxin complexes. Nature 489, 400–405 (2012)

    Article  ADS  CAS  Google Scholar 

  54. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  55. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  56. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  57. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    Article  CAS  Google Scholar 

  58. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D 64, 61–69 (2008)

    Article  CAS  Google Scholar 

  59. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  60. Quick, M. & Javitch, J. A. Monitoring the function of membrane transport proteins in detergent-solubilized form. Proc. Natl Acad. Sci. USA 104, 3603–3608 (2007)

    Article  ADS  CAS  Google Scholar 

  61. Giros, B. et al. Cloning, pharmacological characterization, and chromosome assignment of the human dopamine transporter. Mol. Pharmacol. 42, 383–390 (1992)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Cawley for generating monoclonal antibodies and S. Amara for providing the wild-type Drosophila DAT construct. We would like to thank H. Wang and D. Claxton for comments and suggestions along with other Gouaux laboratory members for discussions during manuscript preparation. We thank L. Vaskalis for assistance with figures and H. Owen for help with manuscript preparation. We thank the staff of the Northeastern Collaborative Access Team (NECAT) at the Advanced Photon Source (APS) for assistance with data collection. This work was supported by a postdoctoral fellowship from the American Heart Association (A.P.), a National Institute of Mental Health research award (K.H.W.) and by the National Institutes of Health (E.G.) E.G. is an investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

A.P., K.H.W. and E.G. designed the project. A.P. and K.H.W. performed protein purification, crystallography and biochemical assays. A.P., K.H.W. and E.G. wrote the manuscript.

Corresponding author

Correspondence to Eric Gouaux.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3 and Supplementary Figures 1-8. (PDF 9370 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Penmatsa, A., Wang, K. & Gouaux, E. X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature 503, 85–90 (2013). https://doi.org/10.1038/nature12533

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12533

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing