Intracellular potassium (K⁺) homeostasis is fundamental to cell viability. In addition to channels, K⁺ levels are maintained by various ion transporters. One major family is the proton-driven K⁺ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K⁺-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K⁺ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na⁺/H⁺ antiporter NapA. By structural assignment of a coordinated K⁺ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K⁺ ion. KefC harbors C-terminal regulator of K⁺ conductance (RCK) domains, as present in some bacterial K⁺-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K⁺-ion-channels.

The strict exchange of Na⁺ for H⁺ ions across cell membranes is a reaction carried out in almost every cell. Na⁺/H⁺ exchangers that perform this task are physiological homodimers, and whilst the ion transporting domain is highly conserved, their dimerization differs. The Na⁺/H⁺ exchanger NhaA from Escherichia coli has a weak dimerization interface mediated by a β-hairpin domain and with dimer retention dependent on cardiolipin. Similarly, organellar Na⁺/H⁺ exchangers NHE6, NHE7 and NHE9 also contain β-hairpin domains and recent analysis of Equus caballus NHE9 indicated PIP₂ lipids could bind at the dimer interface. However, structural validation of the predicted lipid-mediated oligomerization has been lacking. Here, we report cryo-EM structures of E. coli NhaA and E. caballus NHE9 in complex with cardiolipin and phosphatidylinositol-3,5-bisphosphate PI(3,5)P₂ lipids binding at their respective dimer interfaces. We further show how the endosomal specific PI(3,5)P₂ lipid stabilizes the NHE9 homodimer and enhances transport activity. Indeed, we show that NHE9 is active in endosomes, but not at the plasma membrane where the PI(3,5)P₂ lipid is absent. Thus, specific lipids can regulate Na⁺/H⁺ exchange activity by stabilizing dimerization in response to either cell specific cues or upon trafficking to their correct membrane location.