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.

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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.

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.

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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.

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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.

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Correspondence to Eric Gouaux.

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The authors declare no competing financial interests.

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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

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