Transition metal ion FRET uncovers K+regulation of a neurotransmitter/sodium symporter.
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.
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark.
Christian B
CB
Billesbølle
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark.
Jonas S
JS
Mortensen
Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna, 1090 Vienna, Austria.
Azmat
A
Sohail
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark.
Solveig G
SG
Schmidt
Computational Chemistry and Molecular Biophysics Unit, National Institute on Drug Abuse, NIH, Baltimore, Maryland 20852, USA.
Lei
L
Shi
Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna, 1090 Vienna, Austria.
Harald H
http://orcid.org/0000-0002-1339-7444
HH
Sitte
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark.
Ulrik
http://orcid.org/0000-0002-0020-3807
U
Gether
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark.
Claus J
http://orcid.org/0000-0002-1773-1446
CJ
Loland
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