Mitochondrial Ca 2+ efflux, essential for maintaining mitochondrial calcium homeostasis, is mediated by the mitochondrial Na + /Ca 2+ exchanger and H + /Ca 2+ exchanger. NCLX has been proposed as the mitochondrial Na + /Ca 2+ exchanger, but its molecular mechanism remains poorly understood to date. We noticed that NCLX, unlike plasma-membrane Na + /Ca 2+ exchangers such as NCX1, lacks conserved serine residues required for Na + binding. Moreover, the effects of NCLX knockout on mito-NCX activity vary widely across reports. These observations raise questions about the actual function of NCLX. To pursue this further, we created four NCLX-KO cell lines (HEK, CHO, HeLa, and HCT116) and assessed mito-NCX activity. Surprisingly, NCLX-deficient cells displayed mito-NCX activity comparable to wild-type cells, a result excluding NCLX as the mito-NCX. To determine NCLX function, we expressed human NCLX in Xenopus oocytes, and measured Ca-45 flux across the membrane. Our results showed that NCLX indeed conducts Ca 2+ , but this Ca 2+ transport is independent of Na + or K + gradients. Instead, we found a proton gradient can drive energetically uphill Ca 2+ transport against the Ca 2+ electrochemical gradients, a result demonstrating that NCLX functions as a mitochondrial H + /Ca 2+ exchanger. These results, along with our recent finding that TMEM65 is the bona fide mito-NCX protein, now redefine the molecular identities of key players mediating mitochondrial Ca 2+ efflux pathway, providing a mechanistic framework to understand mitochondrial Ca 2+ homeostasis and signaling and related diseases.
Liu et al. (2026) studied this question.