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.

Solute carrier (SLC) transporters are membrane transport proteins, which catalyse the movement of nutrients, ions, and drugs across cell membranes. Here, I will present our contribution to understanding the mechanism of the sodium/proton exchangers (NHE), belonging to the SLC9 family of membrane transporters. NHEs exchange sodium ions for protons across biological membranes, which is a critical reaction for the fine-tuning of cytoplasmic and organelle pH, sodium levels and volume homeostasis. Dysfunction of NHE members has been linked to a number of diseases and disorders, such as hypertension, heart failure, autism spectrum disorder, epilepsy and the susceptibility of long COVID. Protein structures are important for developing mechanistic models, but due to technical challenges only bacterial homologue structures of NHE proteins were previously available.

Accumulating many years of effort, we were able to determine the first structure of a mammalian Na⁺/H⁺ exchanger, the endosomal isoform NHE9 by single-particle cryo-EM. The structure of NHE9 demonstrated that NHE proteins are architecturally most similar to bacterial homologues with 13-TM segments and likely operated by a similar elevator mechanism (I). Interestingly, native MS and thermal-shift assays indicted that the NHE9 homodimer is stabilized by the binding of a rare lipid only found in late endosomes, which implies the cell may use this lipid as means to switch-on NHE9 activity once it reaches its correct functional localization. We further provided evidence that the large cytoplasmic tail in NHE proteins likely acts in an auto-inhibitory manner. It is only removed upon the binding of extrinsic proteins (II). Indeed, the first structure of a potassium specific K⁺/H⁺ exchanger KefC reveals how its cytoplasmic tail restricts movement of the ion-transporting domain to directly inhibit transport. The structure of KefC is also the first ion-bound state seen for this family and, unlike to the modeled Na⁺/H⁺ exchanger sites with a hydrated Na⁺ ion, coordinates K⁺ as a dehydrated ion (IV). Lastly, we determining the structure of a bacterial Na⁺/H⁺ exchanger NhaA to high-resolution at an active pH of 6.5. With this structure we demonstrated how a cytoplasmic “pH gate” controlled by the pH activated NhaA (III).