Allosteric Regulation of NCLX by Mitochondrial Membrane Potential Links the Metabolic State and Ca²⁺ Signaling in Mitochondria

Calcium is a key regulator of mitochondrial function under both normal and pathological conditions. The mechanisms linking metabolic activity to mitochondrial Ca²⁺ signaling remain elusive, however. Here, by monitoring mitochondrial Ca²⁺ transients while manipulating mitochondrial membrane potential (ΔΨ_m), we found that mild fluctuations in ΔΨ_m, which do not affect Ca²⁺ influx, are sufficient to strongly regulate NCLX, the major efflux pathway of Ca²⁺ from the mitochondria. Phosphorylation of NCLX or expression of phosphomimicking mutant (S258D) rescued NCLX activity from ΔΨ_m-driven allosteric inhibition. By screening ΔΨ_m sensitivity of NCLX mutants, we also identified amino acid residues that, through functional interaction with Ser258, control NCLX regulation. Finally, we find that glucose-driven ΔΨ_m changes in pancreatic β-cells control mitochondrial Ca²⁺ signaling primarily via NCLX regulation. Our results identify a feedback control between metabolic activity and mitochondrial Ca²⁺ signaling and the “safety valve” NCLX phosphorylation that can rescue Ca²⁺ efflux in depolarized mitochondria.