Transition metal ion FRET uncovers K(+) regulation of a neurotransmitter/sodium symporter
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Transition metal ion FRET uncovers K(+) regulation of a neurotransmitter/sodium symporter. / Billesbølle, Christian B; Mortensen, Jonas S; Sohail, Azmat; Schmidt, Solveig Gaarde; Shi, Lei; Sitte, Harald H; Gether, Ulrik; Loland, Claus J.
In: Nature Communications, Vol. 7, 12755, 28.09.2016.Research output: Contribution to journal › Journal article › Research › peer-review
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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
VL - 7
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 12755
ER -
ID: 167932600