Michal Hershfinkel

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

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.

The Neurophysiology and Pathology of Brain Zinc

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 Alzheimers disease.

A zinc-sensing receptor triggers the release of intracellular Ca2+ and regulates ion transport

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 Cai2+ release was zinc specific and could not be triggered by other heavy metals. Furthermore, zinc failed to activate the Ca2+-sensing receptor heterologously expressed in HEK293 cells. The zinc-induced Cai2+ rise stimulated the activity of the Na+/H+ exchanger in HT29 cells. Our results indicate that a previously uncharacterized extracellular, G protein-coupled, Zn2+-sensing receptor is functional in colonocytes. Because Cai2+ 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.

Synaptically Released Zinc Triggers Metabotropic Signaling via a Zinc-Sensing Receptor in the Hippocampus

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.

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

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.

Synaptic release of zinc from brain slices : Factors governing release, imaging, and accurate calculation of concentration

Cerebrocortical neurons that store and release zinc synaptically are widely recognized as critical in maintenance of cortical excitability and in certain forms of brain injury and disease. Through the last 20 years, this synaptic release has been observed directly or indirectly and reported in more than a score of publications from over a dozen laboratories in eight countries. However, the concentration of zinc released synaptically has not been established with final certainty. In the present work we have considered six aspects of the methods for studying release that can affect the magnitude of zinc release, the imaging of the release, and the calculated concentration of released zinc. We present original data on four of the issues and review published data on two others. We show that common errors can cause up to a 3000-fold underestimation of the concentration of released zinc. The results should help bring consistency to the study of synaptic release of zinc.

Upregulation of KCC2 Activity by Zinc-Mediated Neurotransmission via the mZnR/GPR39 Receptor

Vesicular Zn2+ regulates postsynaptic neuronal excitability upon its corelease with glutamate. We previously demonstrated that synaptic Zn2+ acts via a distinct metabotropic zinc-sensing receptor (mZnR) in neurons to trigger Ca2+ responses in the hippocampus. Here, we show that physiological activation of mZnR signaling induces enhanced K+/Cl− cotransporter 2 (KCC2) activity and surface expression. As KCC2 is the major Cl− outward transporter in neurons, Zn2+ also triggers a pronounced hyperpolarizing shift in the GABAA reversal potential. Mossy fiber stimulation-dependent upregulation of KCC2 activity is eliminated in slices from Zn2+ transporter 3-deficient animals, which lack synaptic Zn2+. Importantly, activity-dependent ZnR signaling and subsequent enhancement of KCC2 activity are also absent in slices from mice lacking the G-protein-coupled receptor GPR39, identifying this protein as the functional neuronal mZnR. Our work elucidates a fundamentally important role for synaptically released Zn2+ acting as a neurotransmitter signal via activation of a mZnR to increase Cl− transport, thereby enhancing inhibitory tone in postsynaptic cells.

Distribution of the zinc transporter ZnT-1 in comparison with chelatable zinc in the mouse brain

Zinc maintains a diverse array of functions in the mammalian central nervous system as a key component of numerous enzymes, via its role in the activation of transcription factors, and as a neuroregulator, modulating neuronal receptors such as N‐methyl‐D‐aspartate and γ‐aminobutyric acid. Zinc has a dark side, however, with massive influx of Zn2+ to neurons considered to be a key factor in neuronal death secondary to ischemia and seizure. Several different putative zinc transporters, ZnT‐1–4, have recently been identified and characterized. Among them, ZnT‐1 has been suggested to play a key role in reducing cellular Zn2+ toxicity. In the present study, we describe the regional and cellular distribution of ZnT‐1 in the adult mouse brain using an antibody raised against the C‐terminal domain of mouse ZnT‐1. The distribution of ZnT‐1 was compared to that of chelatable Zn2+, visualized by means of neoTimm histochemistry or N‐(6‐methoxy‐8‐quinolyl)‐p‐toluene‐sulfonamide (TSQ) histofluorescence. Extracts from various brain regions specifically stained a 60‐kDa peptide corresponding to the expected molecular weight of ZnT‐1. The expression of ZnT‐1 was highest in the cerebral cortex and cerebellum, moderate in the hippocampus, hypothalamus, and olfactory bulb, and lowest in the striatum and septum. In brain sections, ZnT‐1‐immunoreactive neurons, in particular principle neurons, in the somatosensory cortex, hippocampus, and olfactory bulb, were closely related to synaptic Zn2+. Robust ZnT‐1 immunoreactivity was also observed in cerebellar Purkinje cells. Although the function of the protein in these cells is unclear, in the forebrain, ZnT‐1 is strikingly present in cells and regions where significant Zn2+ homeostasis is required. This finding suggests a protective role for neuronal ZnT‐1 in the context of both normal and pathophysiological activity. J. Comp. Neurol. 447:201–209, 2002.

Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

ZnT-1 expression in astroglial cells protects against zinc toxicity and slows the accumulation of intracellular zinc

Zinc ions are emerging as an important factor in the etiology of neurodegenerative disorders and in brain damage resulting from ischemia or seizure activity. High intracellular levels of zinc are toxic not only to neurons but also to astrocytes, the major population of glial cells in the brain. In the present study, the role of ZnT‐1 in reducing zinc‐dependent cell damage in astrocytes was assessed. Zinc‐dependent cell damage was apparent within 2 h of exposure to zinc, and occurred within a narrow range of ∼200 μM. Pretreatment with sublethal concentrations of zinc rendered astrocytes less sensitive to toxic zinc levels, indicating that preconditioning protects astrocytes from zinc toxicity. Fluorescence cell imaging revealed a steep reduction in intracellular zinc accumulation for the zinc‐pretreated cells mediated by L‐type calcium channels. Heterologous expression of ZnT‐1 had similar effects; intracellular zinc accumulation was slowed down and the sensitivity of astrocytes to toxic zinc levels was reduced, indicating that this is specifically mediated by ZnT‐1 expression. Immunohistochemical analysis demonstrated endogenous ZnT‐1 expression in cultured astroglia, microglia, and oligodendrocytes. Pretreatment with zinc induced a 4‐fold increase in the expression of the putative zinc transporter ZnT‐1 in astroglia as shown by immunoblot analysis. The elevated ZnT‐1 expression following zinc priming or after heterologous expression of ZnT‐1 may explain the reduced zinc accumulation and the subsequent reduction in sensitivity toward toxic zinc levels. Induction of ZnT‐1 may play a protective role when mild episodes of stroke or seizures are followed by a massive brain insult.