Exploring the K⁺ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT.

Abstract

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na⁺ (Na1) contributes to the substrate binding, while the other Na⁺ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the <i>Drosophila</i> dopamine transporter (dDAT), also couple substrate uptake to the antiport of K⁺ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K⁺, and could therefore serve as a model protein for the exploration of K⁺ binding in NSS proteins. Here, we characterize the impact of K⁺ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K⁺ affinities for LeuT. K⁺ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K⁺ dose-dependently increased the transport velocity of [³H]alanine, whereas extra-vesicular K⁺ had no apparent effect. K⁺ binding induced a LeuT conformation distinct from the Na⁺- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na⁺ and K⁺ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K⁺ affinity but at the same time a ~3-fold increase in Na⁺ affinity. Together, the results suggest that K⁺ binding to LeuT modulates substrate transport and that the K⁺ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K⁺ in some NSSs.