NCLX is an essential component of mitochondrial Na+/Ca2+ exchange.

Mitochondrial Ca(2+) efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na(+)-dependent mechanism mediates mitochondrial Ca(2+) efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na(+)/Ca(2+) exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca(2+) and Na(+) fluorescent imaging, we demonstrate that mitochondrial Na(+)-dependent Ca(2+) efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca(2+) transport was inhibited, moreover, by CGP-37157 and exhibited Li(+) dependence, both hallmarks of mitochondrial Na(+)-dependent Ca(2+) efflux. Finally, NCLX-mediated mitochondrial Ca(2+) exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na(+)/Ca(2+) exchanger.

Ratiometric-pericam-mt, a novel tool to evaluate intramitochondrial zinc.

Zn(2+) can enter mitochondria and promote a plethora of physiological and patho-physiological effects. The issue of measuring changes in intramitochondrial levels is therefore critical. Past studies have employed fluorescent Zn(2+) indicators, like Rhod-2 and RhodZin-3, however, the use of these probes is impaired by their extramitochondrial sequestration. In this study, we show that the ratiometric mitochondria-targeted pericam, RPmt, can be employed to detect changes of intramitochondrial free Zn(2+) ([Zn(2+)](m)) levels. Using RPmt in neuronal and non neuronal cell lines we demonstrate that mitochondria can take up the cation mobilized from the cytosolic pool of protein-bound Zn(2+) and that mitochondrial Zn(2+) sequestration is largely mediated by the activity of the Ca(2+) uniporter.