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


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.

SUBMITTER: Billesbolle CB 

PROVIDER: S-EPMC5052704 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Billesbølle Christian B CB   Mortensen Jonas S JS   Sohail Azmat A   Schmidt Solveig G SG   Shi Lei L   Sitte Harald H HH   Gether Ulrik U   Loland Claus J CJ  

Nature communications 20160928


Neurotransmitter/sodium symporters (NSSs) are responsible for Na<sup>+</sup>-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<sup>+</sup> inhibits Na<sup>+</sup>-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K<sup>+</sup>-indu  ...[more]

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