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.