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Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter

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  • Christian B. Billesbølle

    (Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen)

  • Jonas S. Mortensen

    (Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen)

  • Azmat Sohail

    (Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna)

  • Solveig G. Schmidt

    (Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen)

  • Lei Shi

    (Computational Chemistry and Molecular Biophysics Unit, National Institute on Drug Abuse, NIH)

  • Harald H. Sitte

    (Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna)

  • Ulrik Gether

    (Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen)

  • Claus J. Loland

    (Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen)

Abstract

Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle.

Suggested Citation

  • Christian B. Billesbølle & Jonas S. Mortensen & Azmat Sohail & Solveig G. Schmidt & Lei Shi & Harald H. Sitte & Ulrik Gether & Claus J. Loland, 2016. "Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter," Nature Communications, Nature, vol. 7(1), pages 1-12, November.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms12755
    DOI: 10.1038/ncomms12755
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    Cited by:

    1. Andreas Nygaard & Linda G. Zachariassen & Kathrine S. Larsen & Anders S. Kristensen & Claus J. Loland, 2024. "Fluorescent non-canonical amino acid provides insight into the human serotonin transporter," Nature Communications, Nature, vol. 15(1), pages 1-14, December.

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