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Molecular Pharmacology

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Research ArticleArticle

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin Transporter

Subhodeep Sarker, René Weissensteiner, Ilka Steiner, Harald H. Sitte, Gerhard F. Ecker, Michael Freissmuth and Sonja Sucic
Molecular Pharmacology December 2010, 78 (6) 1026-1035; DOI: https://doi.org/10.1124/mol.110.067538
Subhodeep Sarker
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René Weissensteiner
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Ilka Steiner
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Harald H. Sitte
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Gerhard F. Ecker
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Michael Freissmuth
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Sonja Sucic
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Abstract

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the Austrian Science Fund FWF Cell Communication in Health and Disease [Grant SFB35]; and the Medical University of Vienna, Cell Communication in Health and Disease.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.067538.

  • ABBREVIATIONS:

    NSS
    neurotransmitter:sodium symporters
    WIN35,428
    -(−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane
    5-HT
    5-hydroxytryptamine (serotonin)
    MDMA
    s-(+)-3,4-methylenedioxy-methamphetamine
    MPP+
    1-methyl-4-phenylpyridinium ion
    PCA
    para-chloroamphetamine
    LeuTAa
    leucine transporter from Aquifex aeolicus
    HEK
    human embryonic kidney
    DAT
    dopamine transporter
    NET
    noradrenaline transporter
    SERT
    serotonin transporter
    TM
    transmembrane
    YFP
    yellow fluorescent protein
    TCA
    tricyclic antidepressant
    MOE
    Molecular Operating Environment
    PLIF
    protein ligand interaction fingerprint
    SSRI
    selective serotonin reuptake inhibitor.

  • Received July 14, 2010.
  • Accepted September 9, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (6)
Molecular Pharmacology
Vol. 78, Issue 6
1 Dec 2010
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Research ArticleArticle

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin Transporter

Subhodeep Sarker, René Weissensteiner, Ilka Steiner, Harald H. Sitte, Gerhard F. Ecker, Michael Freissmuth and Sonja Sucic
Molecular Pharmacology December 1, 2010, 78 (6) 1026-1035; DOI: https://doi.org/10.1124/mol.110.067538

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Research ArticleArticle

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin Transporter

Subhodeep Sarker, René Weissensteiner, Ilka Steiner, Harald H. Sitte, Gerhard F. Ecker, Michael Freissmuth and Sonja Sucic
Molecular Pharmacology December 1, 2010, 78 (6) 1026-1035; DOI: https://doi.org/10.1124/mol.110.067538
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