Abstract

The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - i.e., the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives) and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

Data availability

We uploaded original data onto Dyrad.The generated DOI is: doi.org/10.5061/dryad.fqz612jvz.

The following data sets were generated

Article and author information

Author details

  1. Shreyas Bhat

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  2. Ali El-Kasaby

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  3. Ameya Kasture

    Department of Neurobiology, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  4. Danila Boytsov

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  5. Julian B Reichelt

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  6. Thomas Hummel

    Department of Neurobiology, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8108-9307
  7. Sonja Sucic

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  8. Christian Pifl

    Center for Brain Research, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  9. Michael Freissmuth

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  10. Walter Sandtner

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria
    For correspondence
    walter.sandtner@meduniwien.ac.at
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3637-260X

Funding

Austrian Science Fund (P31813)

  • Walter Sandtner

Austrian Science Fund (P31255-B27)

  • Sonja Sucic

Vienna Science and Technology Fund (LSC17-026)

  • Michael Freissmuth

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Version history

  1. Received: August 11, 2022
  2. Preprint posted: August 13, 2022 (view preprint)
  3. Accepted: January 17, 2023
  4. Accepted Manuscript published: January 17, 2023 (version 1)
  5. Version of Record published: January 27, 2023 (version 2)

Copyright

© 2023, Bhat et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Shreyas Bhat
  2. Ali El-Kasaby
  3. Ameya Kasture
  4. Danila Boytsov
  5. Julian B Reichelt
  6. Thomas Hummel
  7. Sonja Sucic
  8. Christian Pifl
  9. Michael Freissmuth
  10. Walter Sandtner
(2023)
A mechanism of uncompetitive inhibition of the serotonin transporter
eLife 12:e82641.
https://doi.org/10.7554/eLife.82641

Share this article

https://doi.org/10.7554/eLife.82641

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