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 theDrosophiladopamine 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 NNSs.