Mitochondrial Ca²⁺efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na⁺-dependent mechanism mediates mitochondrial Ca²⁺efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na⁺/Ca²⁺exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca²⁺and Na⁺fluorescent imaging, we demonstrate that mitochondrial Na⁺-dependent Ca²⁺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 Ca²⁺transport was inhibited, moreover, by CGP-37157 and exhibited Li⁺dependence, both hallmarks of mitochondrial Na⁺-dependent Ca²⁺efflux. Finally, NCLX-mediated mitochondrial Ca²⁺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⁺/Ca²⁺exchanger.

Mitochondrial Ca(2+) overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na(+)/Ca(2+) exchanger (NCLX), leading to impaired mitochondrial Ca(2+) extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLX(S258D)) prevents mitochondrial Ca(2+) overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca(2+) transport regulatory pathway that protects against mitochondrial Ca(2+) overload. Because mitochondrial Ca(2+) dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2+ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn2+ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp599 and His451, with alanine was sufficient to block Zn2+ transport. These findings indicate, for the first time, that Zn2+ transport mediated by a mammalian ZnT is catalyzed by H+/Zn2+ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.

Zinc is an essential cofactor for the activity and folding of up to ten percent of mammalian proteins and can modulate the function of many others. Because

Zinc and cadmium are similar metal ions, but though Zn ²⁺ is an essential nutrient, Cd ²⁺ is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn ²⁺ vs. Cd ²⁺ suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn ²⁺ transport, but reject Cd ²⁺ , thus constituting the first mammalian metal transporter with a refined selectivity against Cd ²⁺ . Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn ²⁺ and Cd ²⁺ . A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn ²⁺ transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd ²⁺ by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn ²⁺ and Cd ²⁺ , and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd ²⁺ binding.