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).

Na⁺/H⁺ exchangers play a vital role in maintaining intracellular pH balance, sodium (Na⁺) reabsorption, and cellular volume homeostasis. In this thesis, I explore four distinct exchangers to unravel common mechanistic themes and unique regulatory adaptations found in bacterial and eukaryotic systems. In the first paper, we investigate the bacterial exchanger. KefC, demonstrating its remarkable K+ selectivity attributed to conserved binding residues. We further reveal that its activity is modulated by C-terminal RCK domains, which detach upon binding of glutathione adduct under electrophilic stress, thereby facilitating cytosolic acidification. Papers II and III uncover that specific lipid interactions are crucial for the stability and function of the eukaryotic endosomal exchangers NHE9 and NHE6. These interactions stabilize the proteins in their homodimeric form, which is essential for effective endosomal pH regulation. Any mislocalization of NHE9 and NHE6 can disrupt dimerization, resulting in a loss of activity and impaired cellular homeostasis. Additionally, our structural studies of NHA2 in complex with a Fab fragment and the inhibitor phloretin provide insights into the molecular mechanisms behind its therapeutic potential. These findings clarify NHA2’s crucial role in insulin secretion, electrolyte balance, and blood pressure regulation, thus paving the way for targeted drug development. Overall, the goals of my thesis emphasize the central role of lipid interactions and structural adaptations in regulating ion exchanger function.