Mitochondrial Ca2+ transient is the earliest discovered organellar Ca2+ signaling pathway. It consist of a Ca2+ influx, mediated by mitochondrial Ca2+ uniporter (MCU), and mitochondrial Ca2+ efflux ...mediated by a Na+/Ca2+ exchanger (NCLX). Mitochondrial Ca2+ signaling machinery plays a fundamental role in linking metabolic activity to cellular Ca2+ signaling, and in controlling local Ca2+ concertation in distinct cellular compartments. Impaired balance between mitochondrial Ca2+ influx and efflux leads to mitochondrial Ca2+ overload, an early and key event in ischemic or neurodegenerative syndromes. Molecular identification of NCLX and MCU happened only recently. Surprisingly, MCU knockout yielded a relatively mild phenotype while conditional knockout of NCLX led to a rapid fatal heart failure. Here we will focus on recent functional and molecular studies on NCLX structure and its mode of regulation. We will describe the unique crosstalk of this exchanger with Na+ and Ca2+ signaling pathways in the cell membrane and the endoplasmic reticulum, and with protein kinases that posttranslationally modulate NCLX activity. We will critically compare selectivity of pharmacological blockers versus molecular control of NCLX expression and activity. Finally we will discuss why this exchanger is essential for survival and can serve as an attractive therapeutic target.
Calcium is a key regulator of mitochondrial function under both normal and pathological conditions. The mechanisms linking metabolic activity to mitochondrial Ca2+ signaling remain elusive, however. ...Here, by monitoring mitochondrial Ca2+ transients while manipulating mitochondrial membrane potential (ΔΨm), we found that mild fluctuations in ΔΨm, which do not affect Ca2+ influx, are sufficient to strongly regulate NCLX, the major efflux pathway of Ca2+ from the mitochondria. Phosphorylation of NCLX or expression of phosphomimicking mutant (S258D) rescued NCLX activity from ΔΨm-driven allosteric inhibition. By screening ΔΨm sensitivity of NCLX mutants, we also identified amino acid residues that, through functional interaction with Ser258, control NCLX regulation. Finally, we find that glucose-driven ΔΨm changes in pancreatic β-cells control mitochondrial Ca2+ signaling primarily via NCLX regulation. Our results identify a feedback control between metabolic activity and mitochondrial Ca2+ signaling and the “safety valve” NCLX phosphorylation that can rescue Ca2+ efflux in depolarized mitochondria.
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•Mitochondrial membrane potential (ΔΨm) allosterically inhibits NCLX•Phosphorylation of NCLX at Ser258 can override ΔΨm-driven NCLX regulation•ΔΨm regulation of NCLX requires Ser258 interaction with positively charged residues•NCLX regulation by ΔΨm links mitochondrial metabolism and Ca2+ signaling
Kostic et al. report that mild mitochondrial depolarization is sufficient to allosterically regulate Ca2+mito efflux by NCLX. Downregulation of NCLX is mediated by a cluster of positively charged residues and NCLX activity is rescued by phosphorylation of Ser258. Regulation of NCLX by ΔΨm links metabolic activity and Ca2+mito signaling.
The past few years have witnessed dramatic progress on all frontiers of zinc neurobiology. The recent development of powerful tools, including zinc-sensitive fluorescent probes, selective chelators ...and genetically modified animal models, has brought a deeper understanding of the roles of this cation as a crucial intra- and intercellular signalling ion of the CNS, and hence of the neurophysiological importance of zinc-dependent pathways and the injurious effects of zinc dyshomeostasis. The development of some innovative therapeutic strategies is aimed at controlling and preventing the damaging effects of this cation in neurological conditions such as stroke and Alzheimer's disease.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Cellular Zn2+ homeostasis is tightly regulated and primarily mediated by designated Zn2+ transport proteins, namely zinc transporters (ZnTs; SLC30) that shuttle Zn2+ efflux, and ZRT-IRT-like proteins ...(ZIPs; SLC39) that mediate Zn2+ influx. While the functional determinants of ZnT-mediated Zn2+ efflux are elucidated, those of ZIP transporters are lesser understood. Previous work has suggested three distinct molecular mechanisms: (I) HCO3− or (II) H+ coupled Zn2+ transport, or (III) a pH regulated electrodiffusional mode of transport. Here, using live-cell fluorescent imaging of Zn2+ and H+, in cells expressing ZIP4, we set out to interrogate its function. Intracellular pH changes or the presence of HCO3− failed to induce Zn2+ influx. In contrast, extracellular acidification stimulated ZIP4 dependent Zn2+ uptake. Furthermore, Zn2+ uptake was coupled to enhanced H+ influx in cells expressing ZIP4, thus indicating that ZIP4 is not acting as a pH regulated channel but rather as an H+ powered Zn2+ co-transporter. We further illustrate how this functional mechanism is affected by genetic variants in SLC39A4 that in turn lead to Acrodermatitis enteropathica, a rare condition of Zn2+ deficiency.
A sharp increase in mitochondrial Ca
marks the activation of brown adipose tissue (BAT) thermogenesis, yet the mechanisms preventing Ca
deleterious effects are poorly understood. Here, we show that ...adrenergic stimulation of BAT activates a PKA-dependent mitochondrial Ca
extrusion via the mitochondrial Na
/Ca
exchanger, NCLX. Adrenergic stimulation of NCLX-null brown adipocytes (BA) induces a profound mitochondrial Ca
overload and impaired uncoupled respiration. Core body temperature, PET imaging of glucose uptake and VO
measurements confirm a thermogenic defect in NCLX-null mice. We show that Ca
overload induced by adrenergic stimulation of NCLX-null BAT, triggers the mitochondrial permeability transition pore (mPTP) opening, leading to a remarkable mitochondrial swelling and cell death. Treatment with mPTP inhibitors rescue mitochondrial function and thermogenesis in NCLX-null BAT, while calcium overload persists. Our findings identify a key pathway through which BA evade apoptosis during adrenergic stimulation of uncoupling. NCLX deletion transforms the adrenergic pathway responsible for thermogenesis activation into a death pathway.
Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction ...indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis.
Cognitive dysfunction and dementia are common features of Parkinson's disease (PD), causing significant disability. Mutations in LRRK2 represent the most common known genetic cause of PD. We found that PD-linked LRRK2 mutations increased dendritic and mitochondrial calcium uptake in cortical neurons and familial PD patient fibroblasts, accompanied by increased expression of the mitochondrial calcium transporter MCU. Blocking the ERK1/2-dependent upregulation of MCU conferred protection against mutant LRRK2-elicited dendrite shortening, as did inhibiting MCU-mediated calcium import. Conversely, stimulating the export of calcium from mitochondria was also neuroprotective. These results implicate increased susceptibility to mitochondrial calcium overload in LRRK2-driven neurodegeneration, and suggest possible interventions that may slow the progression of cognitive dysfunction in PD.
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.
Abstract The aim of this study was to investigate the role of the synaptic metabotropic zinc receptor mZnR/GPR39 in physiological adaptation to epileptic seizures. We previously demonstrated that ...synaptic activation of mZnR/GPR39 enhances inhibitory drive in the hippocampus by upregulating neuronal K+ /Cl− co-transporter 2 (KCC2) activity. Here, we first show that mZnR/GPR39 knockout (KO) adult mice have dramatically enhanced susceptibility to seizures triggered by a single intraperitoneal injection of kainic acid, when compared to wild type (WT) littermates. Kainate also substantially enhances seizure-associated gamma oscillatory activity in juvenile mZnR/GPR39 KO hippocampal slices, a phenomenon that can be reproduced in WT tissue by extracellular Zn 2 + chelation. Importantly, kainate-induced synaptic Zn 2 + release enhances surface expression and transport activity of KCC2 in WT, but not mZnR/GPR39 KO hippocampal neurons. Kainate-dependent upregulation of KCC2 requires mZnR/GPR39 activation of the Gαq/phospholipase C/extracellular regulated kinase (ERK1/2) signaling cascade. We suggest that mZnR/GPR39-dependent upregulation of KCC2 activity provides homeostatic adaptation to an excitotoxic stimulus by increasing inhibition. As such, mZnR/GPR39 may provide a novel pharmacological target for dampening epileptic seizure activity.
Zinc and cadmium are similar metal ions, but though Zn2+ is an essential nutrient, Cd2+ is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution ...of Zn2+ vs. Cd2+ suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn2+ transport, but reject Cd2+, thus constituting the first mammalian metal transporter with a refined selectivity against Cd2+. Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn2+ and Cd2+. 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 Zn2+ transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd2+ by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn2+ and Cd2+, and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd2+ binding.
In the absence of the electrical signaling for which neurons are so highly specialized, GLIA rely on the slow propagation of ionic signals to mediate network events such as Ca2+ and Na+ waves. Glia ...differ from neurons in another important way, they are replete with a high density of ionic‐transport proteins that are essential for them to fulfil their basic functions as guardians of the intra and extra‐cellular milieux. Both the signaling and the homeostatic properties of glial cells are therefore particularly dependent upon the regulation of the two principle physiological metal cations, Ca2+ and Na+. For both ions, glia express high‐affinity/low capacity ATP‐fuelled pumps that can rapidly move small numbers of ions against an electro‐chemical gradient. For both Ca2+ and Na+ regulation, a single transporter family, the Na+‐Ca2+ exchanger (NCX), is used to maintain cellular ion homeostasis over the longer term and under conditions of prolonged or acute ionic dysregulation in astrocytes, oligodendroglia and microglia. Our understanding of glial NCX, both plasmalemmal and mitochondrial, is undergoing the kind of transformation that our understanding of glial cells, in general, has undergone in recent decades. These exchange proteins are becoming increasingly recognized for their essential roles in intracellular homeostasis while their signaling functions are starting to come to light. This review summarizes these key aspects and highlights the many areas where work has yet to begin in this rapidly evolving field. GLIA 2016;64:1646–1654
Main points
Na+ −Ca2+ exchange activity in cell membranes and mitochondria of glial cell has a profound influence on the homeostatic regulation of both ions.
While in its infancy, our understanding of the signalling properties of this pathway is evolving rapidly.
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