Na⁺/H⁺ exchangers (NHEs) are ancient membrane-bound nanoma- chines that work to regulate intracellular pH, sodium levels and cell volume. NHE activities contribute to the control of the cell cycle, cell proliferation, cell migration and vesicle trafficking. NHE dysfunction has been linked to many diseases, and they are targets of pharma- ceutical drugs. Despite their fundamental importance to cell home- ostasis and human physiology, structural information for the mammalian NHEs was lacking. Here, we report the cryogenic elec- tron microscopy structure of NHE isoform 9 (SLC9A9) from Equus caballus at 3.2 Å resolution, an endosomal isoform highly expressed in the brain and associated with autism spectrum (ASD) and atten- tion deficit hyperactivity (ADHD) disorders. Despite low sequence identity, the NHE9 architecture and ion-binding site are remarkably most similar to distantly related bacterial Na⁺/H⁺ antiporters with 13 transmembrane segments. Collectively, we reveal the conserved architecture of the NHE ion-binding site, their elevator-like structural transitions, the functional implications of autism disease mutations and the role of phosphoinositide lipids to promote homodimerization that, together, have important physiological ramifications.

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.