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

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 Drosophila 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.