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

TY - JOUR

T1 - Transition metal ion FRET uncovers K(+) regulation of a neurotransmitter/sodium symporter

AU - Billesbølle, Christian B

AU - Mortensen, Jonas S

AU - Sohail, Azmat

AU - Schmidt, Solveig Gaarde

AU - Shi, Lei

AU - Sitte, Harald H

AU - Gether, Ulrik

AU - Loland, Claus J

PY - 2016/9/28

Y1 - 2016/9/28

N2 - 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 Ni(2+) 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.

AB - 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 Ni(2+) 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.

U2 - 10.1038/ncomms12755

DO - 10.1038/ncomms12755

M3 - Journal article

C2 - 27678200

SN - 2041-1723

VL - 7

JO - Nature Communications

JF - Nature Communications

M1 - 12755

ER -