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

Christian B. Billesbølle, Jonas S. Mortensen, Azmat Sohail, Solveig G. Schmidt, Lei Shi, Harald H. Sitte, Ulrik Gether and Claus J. Loland ()
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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

Nature Communications, 2016, vol. 7, issue 1, 1-12

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms12755

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