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.

Our understanding of the roles played by zinc in the physiological and pathological functioning of the brain is rapidly expanding. The increased availability of genetically modified animal models, selective zinc-sensitive fluorescent probes, and novel chelators is producing a remarkable body of exciting new data that clearly establishes this metal ion as a key modulator of intracellular and intercellular neuronal signaling. In this Mini-Symposium, we will review and discuss the most recent findings that link zinc to synaptic function as well as the injurious effects of zinc dyshomeostasis within the context of neuronal death associated with major human neurological disorders, including stroke, epilepsy, and Alzheimer's disease.

Zn²⁺is coreleased with glutamate from mossy fiber terminals and can influence synaptic function. Here, we demonstrate that synaptically released Zn²⁺activates a selective postsynaptic Zn²⁺-sensing receptor (ZnR) in the CA3 region of the hippocampus. ZnR activation induced intracellular release of Ca²⁺, as well as phosphorylation of extracellular-regulated kinase and Ca²⁺/calmodulin kinase II. Blockade of synaptic transmission by tetrodotoxin or CdCl inhibited the ZnR-mediated Ca²⁺rises. The responses mediated by ZnR were largely attenuated by the extracellular Zn²⁺chelator, CaEDTA, and in slices from mice lacking vesicular Zn²⁺, suggesting that synaptically released Zn²⁺triggers the metabotropic activity. Knockdown of the expression of the orphan G-protein-coupled receptor 39 (GPR39) attenuated ZnR activity in a neuronal cell line. Importantly, we observed widespread GPR39 labeling in CA3 neurons, suggesting a role for this receptor in mediating ZnR signaling in the hippocampus. Our results describe a unique role for synaptic Zn²⁺acting as the physiological ligand of a metabotropic receptor and provide a novel pathway by which synaptic Zn²⁺can regulate neuronal function.

Changes in extracellular zinc concentration participate in modulating fundamental cellular processes such as proliferation, secretion, and ion transport in a mechanism that is not well understood. Here, we show that a micromolar concentration of extracellular zinc triggers a massive release of calcium from thapsigargin-sensitive intracellular pools in the colonocytic cell line HT29. Calcium release was blocked by a phospholipase-C inhibitor, indicating that formation of inositol 1,4,5-triphosphate is required for zinc-dependent calcium release. Zinc influx was not observed, indicating that extracellular zinc triggered the release. The Ca _i ²⁺ release was zinc specific and could not be triggered by other heavy metals. Furthermore, zinc failed to activate the Ca ²⁺ -sensing receptor heterologously expressed in HEK293 cells. The zinc-induced Ca _i ²⁺ rise stimulated the activity of the Na ⁺ /H ⁺ exchanger in HT29 cells. Our results indicate that a previously uncharacterized extracellular, G protein-coupled, Zn ²⁺ -sensing receptor is functional in colonocytes. Because Ca _i ²⁺ rise is known to regulate key cellular and signal-transduction processes, the zinc-sensing receptor may provide the missing link between extracellular zinc concentration changes and the regulation of cellular processes.

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.