biostudies-literatureVetralla MTelethon923https://www.ebi.ac.uk/biostudies/studies/S-EPMC12830682biostudies-literatureUnknown17(1)The bidirectional transport of Ca²⁺ into and out of mitochondria regulates metabolism, signaling, and cell fate. While influx is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, efflux mechanisms are more diversified, involving Na⁺ or H⁺ exchange pathways. We here demonstrate that TMEM65 is a fundamental component of the Ca²⁺ efflux machinery of mitochondria. Its overexpression specifically enhances Na⁺- and Li⁺-dependent mitochondrial Ca²⁺ extrusion. This effect is inhibited by CGP-37157 and does not depends on NCLX, currently considered the bona fide mitochondrial Na⁺/Ca²⁺ exchanger. Its downregulation chronically elevates basal [Ca²⁺]_mt and impairs efflux upon stimulation. In Caenorhabditis elegans, deletion of TMEM65 Nature communicationsTMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis.PMC12830682GJC21054Ehninger DDe Stefani DHabert MSKahsay ACadenelli VSbrissa MWischhof LScifo ERizzuto RBano DVetralla MXie BfalseTMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis.The bidirectional transport of Ca²⁺ into and out of mitochondria regulates metabolism, signaling, and cell fate. While influx is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, efflux mechanisms are more diversified, involving Na⁺ or H⁺ exchange pathways. We here demonstrate that TMEM65 is a fundamental component of the Ca²⁺ efflux machinery of mitochondria. Its overexpression specifically enhances Na⁺- and Li⁺-dependent mitochondrial Ca²⁺ extrusion. This effect is inhibited by CGP-37157 and does not depends on NCLX, currently considered the bona fide mitochondrial Na⁺/Ca²⁺ exchanger. Its downregulation chronically elevates basal [Ca²⁺]_mt and impairs efflux upon stimulation. In Caenorhabditis elegans, deletion of TMEM65 2025-01-01T00:00:00Z2025 Dec2026-06-13T05:08:07.346Z2026-06-13T03:08:59.922ZS-EPMC128306824140804510.1038/s41467-025-67647-y