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

Christian B Billesbølle, Jonas S Mortensen, Azmat Sohail, Solveig Gaarde Schmidt, Lei Shi, Harald H Sitte, Ulrik Gether, Claus J Loland

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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 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.

OriginalsprogEngelsk
Artikelnummer12755
TidsskriftNature Communications
Vol/bind7
Antal sider12
ISSN2041-1723
DOI
StatusUdgivet - 28 sep. 2016

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