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Molecular Pharmacology

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Research ArticleArticle

Substrate and Inhibitor–Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

Pella C. Söderhielm, Jacob Andersen, Lachlan Munro, Anne T. Nielsen and Anders S. Kristensen
Molecular Pharmacology October 2015, 88 (4) 676-688; DOI: https://doi.org/10.1124/mol.115.099911
Pella C. Söderhielm
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Jacob Andersen
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Lachlan Munro
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Anne T. Nielsen
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Anders S. Kristensen
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Abstract

The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations.

Footnotes

    • Received May 13, 2015.
    • Accepted July 13, 2015.
  • This work was supported by the Carlsberg Foundation, Lundbeck Foundation, Novo Nordisk Foundation, Hørslev Foundation, and Aase and Ejner Danielsen Foundation.

  • dx.doi.org/10.1124/mol.115.099911.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

  • Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 88 (4)
Molecular Pharmacology
Vol. 88, Issue 4
1 Oct 2015
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Research ArticleArticle

Structural Correlates of Inhibitor Function at hSERT

Pella C. Söderhielm, Jacob Andersen, Lachlan Munro, Anne T. Nielsen and Anders S. Kristensen
Molecular Pharmacology October 1, 2015, 88 (4) 676-688; DOI: https://doi.org/10.1124/mol.115.099911

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Research ArticleArticle

Structural Correlates of Inhibitor Function at hSERT

Pella C. Söderhielm, Jacob Andersen, Lachlan Munro, Anne T. Nielsen and Anders S. Kristensen
Molecular Pharmacology October 1, 2015, 88 (4) 676-688; DOI: https://doi.org/10.1124/mol.115.099911
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