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.