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Nat Commun. 2016 Sep 28;7:12755. doi: 10.1038/ncomms12755.

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

Author information

1
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark.
2
Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna, 1090 Vienna, Austria.
3
Computational Chemistry and Molecular Biophysics Unit, National Institute on Drug Abuse, NIH, Baltimore, Maryland 20852, USA.

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.

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