Structural basis for action by diverse antidepressants on biogenic amine transporters

Abstract

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin–noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs)1,2. Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function3. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter4, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT5,6,7 and act as non-competitive inhibitors of transport5. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket8,9,10,11,12,13,14,15,16. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

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Figure 1: LeuBAT design and pharmacology.
Figure 2: LeuBAT Δ13–sertraline complex adopts an outward-facing open conformation.
Figure 3: SSRIs, SNRIs, TCA and mazindol share similar binding features.
Figure 4: Implication for drug binding in human SERT and validation by mutational studies.

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Acknowledgements

We thank J. Michel for help in binding experiments, D. Claxton for comments and L. Vaskalis for assistance with illustrations. We also thank the beamline staff at the Advanced Light Source (beamlines 8.2.1 and 5.0.2) and Advanced Photon Source (Argonne National Laboratory, beamlines 24-ID-C and 24-ID-E). H.W. also thanks the presenters at 2012 CCP4/APS summer school for useful lectures and tutorials. This work was supported by the National Institutes of Health. E.G. is an Investigator with the Howard Hughes Medical Institute.

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Contributions

H.W. and E.G. designed the research; H.W., A.G. and R.R. performed protein expression and purification; H.W., A.G., K.H.W. and A.P. carried out ligand-binding and flux experiments; H.W. conducted crystallization and structure determination; H.W. and E.G. wrote the manuscript together with comments from all authors.

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Correspondence to Eric Gouaux.

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The authors declare no competing financial interests.

Additional information

Coordinates and structure factors for the LeuBAT Δ13-paroxetine, Δ13-sertraline, Δ13-duloxetine, Δ13-desvenlafaxine, Δ13-fluoxetine, Δ13-fluvoxamine, Δ13-clomipramine, Δ6-sertraline, Δ6-desvenlafaxine, Δ6-duloxetine, Δ6-mazindol and Δ5-mazindol crystal structures have been deposited in the Protein Data Bank with codes 4MM4, 4MM5, 4MM6, 4MM7, 4MM8, 4MM9, 4MMA, 4MMB, 4MMC, 4MMD, 4MME and 4MMF, respectively.

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Wang, H., Goehring, A., Wang, K. et al. Structural basis for action by diverse antidepressants on biogenic amine transporters. Nature 503, 141–145 (2013). https://doi.org/10.1038/nature12648

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