The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na⁺ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K⁺, but investigations of K⁺-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K⁺, and the conformational dynamics of the Drosophila dopamine transporter in K⁺ are divergent from the apo- and Na⁺-states. Furthermore, we find that K⁺ increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na⁺ and K⁺ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K⁺ on other transporters in this pharmacologically important family.

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.

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na⁺ and Cl⁻ binding on [³H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (K_d) for [³H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.