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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.

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


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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