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All metazoans depend on the consumption ofO2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production ofreactive oxygen species by modulating the fluidity ofthe inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification ofthe matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation ofthe mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition ofNa+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.
Cells and tissues produce a superoxide burst as an essential feature of several adaptive responses, including hypoxia711. Given the importance of the mitochondrial Na+/Ca2+ exchanger (NCLX) in ischaemiareperfusion injury12, we studied whether this exchanger may have a role in hypoxic redox signalling. Primary bovine aortic endothelial cells (BAECs) and mouse embryonic fibroblasts (MEFs) exposed to acute hypoxia showed an increase in cytosolic Ca2+ concentration (Ca2+cyto) and a decrease in cytosolic Na+ concentration (Na+cyto) that were prevented by NCLX knockdown with small interfering RNAs (siRNAs), NCLX genetic deletion, overexpression of a dominant negative form of NCLX (dnNCLX) or its pharmacological inhibition with CGP-37157 (Fig. 1a-e, Extended Data Figs. 1a-j, 2), an effect rescued by expression of human NCLX (pNCLX) in NCLX knockout (KO) MEFs (Fig. 1d, e, Extended Data Fig. 1e-g). Notably, NCLX inhibition did not interfere with mitochondrial membrane potential or respiration (Extended Data Fig. 1k, l). Thus, acute hypoxia induces NCLX activation. The hypoxic reactive oxygen species...