Na⁺/H⁺ exchangers (NHEs) are essential membrane transport proteins that play a fundamental role in maintaining cellular homeostasis. By exchanging intracellular H⁺ for extracellular Na⁺(K⁺), these transporters regulate intracellular pH, cell volume, and sodium concentration. They are expressed in virtually all cell types and participate in a wide range of physiological processes, including nutrient absorption, neurotransmission, organellar acidification, and cell proliferation. Dysregulation of NHE activity has been implicated in various human diseases, such as cardiovascular disorders, cancer, and neurological syndromes. Despite their physiological importance, the precise molecular mechanisms governing NHE function and regulation remain incompletely understood.

In this thesis, I have investigated both the intrinsic and extrinsic regulatory mechanisms of NHEs by combining structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) with functional assays. (1) We determined the X-ray crystal structure of E. coli NhaA in its active state, revealing a pH-gating mechanism that regulates the pH of when the protein becomes active. (2) We solved the cryo-EM structures of bison SLC9B2 (NHA2) and combined with functional analysis, demonstrated how its unique N-terminal helix and lipid interactions influence homodimerization. (3) Importantly, we have further shown that dimerization is essential for transport activity. Through further structural studies of human NHA2, with and without an bound inhibitor, we established a framework for understanding its regulatory mechanisms and for the rational design of targeted therapeutics. (4) By determining the cryo-EM structure of endosomal SLC9A6 (NHE6), we demonstrated how specific lipid interactions contribute to its allosteric regulation and dimerization, providing mechanistic insight into endosomal pH regulation and NHE6-related neurological disorders. We have also determined the first ion-bound state for an NHE transporter, which confirms the full coordination of the ion within the core domain as expected for an elevator transporter.

Together, these findings provide an integrated structural and functional understanding of NHE regulation at the molecular level, offering a conceptual framework for future investigations of ion transporters and highlighting the potential of NHEs as therapeutic targets in a broad spectrum of human diseases.

The sodium/proton exchanger NHA2, also known as SLC9B2, is important for insulin secretion, renal blood pressure regulation, and electrolyte retention. Recent structures of bison NHA2 has revealed its unique 14-transmembrane helix architecture, which is different from SLC9A/NHE members made up from 13-TM helices. Sodium/proton exchangers are functional homodimers, and the additional N-terminal helix in NHA2 was found to alter homodimer assembly. Here, we present the cryo-electron microscopy structures of apo human NHA2 in complex with a Fab fragment and also with the inhibitor phloretin bound at 2.8 and 2.9 Å resolution, respectively. We show how phosphatidic acid (PA) lipids bind to the homodimer interface of NHA2 on the extracellular side, which we propose has a regulatory role linked to cell volume regulation. The ion binding site of human NHA2 has a salt bridge interaction between the ion binding aspartate D278 and R432, an interaction previously broken in the bison NHA2 structure, and these differences suggest a possible ion coupling mechanism. Lastly, the human NHA2 structure in complex with phloretin offers a template for structure-guided drug design, potentially leading to the development of more selective and potent NHA2 inhibitors.

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. 

The Na⁺/H⁺ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na⁺/Li⁺ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na⁺/H⁺ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.