The strict exchange of protons for sodium ions across cell membranes by Na⁺/H⁺ exchangers is a fundamental mechanism for cell homeostasis. At active pH, Na⁺/H⁺ exchange can be modelled as competition between H⁺ and Na⁺ to an ion-binding site, harbouring either one or two aspartic-acid residues. Nevertheless, extensive analysis on the model Na⁺/H⁺ antiporter NhaA from Escherichia coli, has shown that residues on the cytoplasmic surface, termed the pH sensor, shifts the pH at which NhaA becomes active. It was unclear how to incorporate the pH senor model into an alternating-access mechanism based on the NhaA structure at inactive pH 4. Here, we report the crystal structure of NhaA at active pH 6.5, and to an improved resolution of 2.2 angstrom. We show that at pH 6.5, residues in the pH sensor rearrange to form new salt-bridge interactions involving key histidine residues that widen the inward-facing cavity. What we now refer to as a pH gate, triggers a conformational change that enables water and Na⁺ to access the ion-binding site, as supported by molecular dynamics (MD) simulations. Our work highlights a unique, channel-like switch prior to substrate translocation in a secondary-active transporter. 

Sodium/proton exchangers (NHEs) are secondary active transporters that are ubiquitously found in all kingdoms of life. They facilitate the exchange of protons for sodium ions or other inorganic ions across biological membranes, regulating pH, sodium levels, and osmotic pressure. They are therefore involved in many fundamental cellular processes such as cell migration and proliferation, and trafficking and turnover of vesicles. Their dysfunction consequently implicates them in a number of diseases and disorders, including hypertension, heart failure, epilepsy, autism spectrum disorders, and brain cancer, making them potential targets for drug development. It is therefore crucial to clearly establish their structure, the molecular basis of ion translocation and their regulation.

In this thesis I discuss the key findings of four publications I contributed to with my research. As part of these findings, we could confirm that sodium/proton exchangers operate according to the now broadly accepted elevator transport mechanism. We identified the residues of the ion-binding site that enable electrogenic ion transport in bacterial sodium/proton antiporters, and how they are potentially linked to adaption to different temperature environments. We provide a structural link between two models of regulation by pH of bacterial NhaA; suggesting a channel-like activation of a secondary active transporter. We determined the structure of endosomal NHE9, the first structure of a mammalian sodium/proton exchanger. The structure shows that these transporters, exemplified here by NHE9, share the same topology, fold, and transport mechanism as was observed in bacterial antiporters. In addition, we showed that NHE9 preferentially binds phosphatidylinositol phosphates, a class of lipids enriched in endosomes and involved in a number of signaling and regulation pathways, suggesting a potential regulatory mechanism for NHE9. Taken together, this research contributes to the growing understanding of sodium/proton exchangers and provides direction for future research.

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.

Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+ antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+ antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by Delta pH, but no longer respond to either an electrochemical potential (psi) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+ exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+ antiporters.

To fully understand the transport mechanism of Na+/H+ exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog Glt(ph). Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na+/H+ antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na+/H+ antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.