Search

Tight regulation of neuronal Zn2+is critical for physiological function. Multiple Zn2+transporters are expressed in the brain, yet their spatial distribution and distinct roles are largely unknown. Here, we show developmental regulation of the expression of Zn2+transporters ZIP1 and ZIP3 in mouse hippocampal neurons, corresponding to previously described increase in neuronal vesicular Zn2+during the first postnatal month. Rates of Zn2+uptake in cultured mouse hippocampal neurons, monitored using FluoZin-3 fluorescence, were higher in mature neurons, which express higher levels of ZIP1 and ZIP3. Zn2+uptake was attenuated by ∼50% following silencing of either ZIP1 or ZIP3. Expression of both ZIP1 and ZIP3 was ubiquitous on somas and most neuronal processes in the cultured neurons. In contrast, we observed distinct localization of the transporters in adult mouse hippocampal brain, with ZIP1 predominantly expressed in the CA3 stratum pyramidale, and ZIP3 primarily localized to the stratum lucidum. Consistent with their localization, silencing of ZIP1 expressionin vivoreduced Zn2+uptake in CA3 neurons while ZIP3 silencing reduced Zn2+influx into dentate gyrus (DG) granule cells in acute hippocampal slices. Strikingly,in vivosilencing of ZIP3, but not ZIP1, protected CA3 neurons from neurodegeneration following kainate-induced seizures. Our results indicate that distinct Zn2+transporters control Zn2+accumulation and toxicity in different neuronal populations in the hippocampus and suggest that selective regulation of Zn2+transporters can prevent seizure induced brain damage.SIGNIFICANCE STATEMENTZinc plays a major role in neuronal function and its dysregulation is associated with neurodegeneration. Multiple zinc transporters are expressed in neurons, yet little is known on their distinct roles. Here, we show that the plasma membrane ZIP1 and ZIP3 zinc transporters are expressed on distinct neuronal populations in the CA3 region of the hippocampus. We show that ZIP1 mediates zinc influx into postsynaptic cells, while ZIP3 is responsible for zinc re-uptake from this synapse into dentate granule cells. We further show that silencing of ZIP3, but not ZIP1, can rescue the postsynaptic cells from kainate-induced neurodegeneration. This suggests that neuronal zinc toxicity and degeneration can be modulated by regulation of specific zinc transporters function.

Along with the SLC30 family, SLC39 family members regulate zinc movement in cells. SLC39 metal ion transporters accumulate zinc into the cytosol. Membrane topology modelling suggests the presence of eight TM regions with both termini extracellular or in the lumen of intracellular organelles. The mechanism for zinc transport for many members is unknown but appears to involve co-transport of bicarbonate ions [3, 4].

Impaired phosphodiesterase (PDE) function and mitochondrial Ca

Manganese (Mn(2+)) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca(2+) inward gradient for active cellular exchange of Mn(2+). In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn(2+) and Ca(2+) fluxes. We found that (a) application of Mn(2+)-containing Ca(2+)-free solution to ZnT10-expressing cells triggers an influx of Mn(2+), (b) reintroduction of Ca(2+) leads to cellular Mn(2+) extrusion against an inward Mn(2+) gradient, and (c) the cellular transport of Mn(2+) by ZnT10 is coupled to a reciprocal movement of Ca(2+). Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn(2+)/Ca(2+) exchange to an uncoupled channel mode, permeable to both Ca(2+) and Mn(2+). The findings in our study identify the first ion transporter that uses the Ca(2+) gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins.

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn

Impaired phosphodiesterase (PDE) function and mitochondrial Ca2+ - [Ca2+]m signaling leads to cardiac failure, ischemic damage and dysfunctional learning and memory. Yet, a causative link between these pathways is unknown. Here, we fluorescently monitored [Ca2+]m transients in hippocampal neurons evoked by caffeine followed by depolarization. [Ca2+]m efflux was apparent in WT but diminished in neurons deficient in the mitochondrial Na+/Ca2+ exchanger NCLX. Surprisingly, neuronal depolarization-induced Ca2+ transients alone failed to evoke strong [Ca2+]m efflux in WT neurons. Caffeine is also a PDE inhibitor. Pretreatment with the PDE2 inhibitor Bay 60-7550 rescued [Ca2+]m efflux triggered by neuronal depolarization. Inhibition of PDE2 acted by diminishing the Ca2+ dependent reduction of mitochondrial cAMP, thereby promoting NCLX phosphorylation. Selective PDE2 inhibition also enhanced [Ca2+]m efflux triggered by neuromodulators. We found that protection of neurons against excitotoxic insults, conferred by PDE2 inhibition, was diminished in NCLX KO neurons, thus is NCLX dependent. Finally, administration of Bay 60-7550 enhanced new object recognition learning in WT but not in NCLX KO mice. Our results identify a long-sought link between PDE and [Ca2+]m signaling thereby providing new therapeutic targets.

Modulation of neuronal K+/Cl- cotransporter 2 (KCC2) activity, which mediates Cl- export, is considered to be regulated by phosphorylation of its C-terminus and critical to neuronal function. Here, we show that KCC2 interacts with the SNARE protein Synaptosome Associated Protein 23, SNAP23, an essential component of membrane insertion machinery. Using KCC2 truncated mutants, we show that KCC2 C-terminal domain is essential for the Zn2+-sensitive receptor mZnR/GPR39 upregulation of KCC2 activity and membrane targeting in HEK293 cells. Surprisingly, we found that SNAP23 is essential for ZnR/GPR39-dependent upregulation of transport mediated by KCC2 and its epithelial isoform KCC3. Moreover, IκB kinase (IKK)-dependent phosphorylation of SNAP23 is essential for ZnR/GPR39 upregulation of KCC2 activity in mouse hippocampal neurons. Finally, we found that interaction of SNAP23 and Syntaxin 1A with KCC2 enhances its insertion into the neuronal membrane. Our results elucidate a G-protein coupled receptor dependent pathway for regulation of KCC activity that via interaction with SNARE proteins.

Consumption of coffee and caffeine enhances learning efficiency and reduces the risk of stroke and neurodegenerative syndromes by a molecular pathway that is poorly understood. Here we fluorescently monitored mitochondrial Ca2+ transients in hippocampal neurons evoked by caffeine. Surprisingly, caffeine also had a long-lasting potentiation effect on mitochondrial Ca2+ ([Ca2+]m) efflux evoked by depolarization, in WT but not in neurons deficient in the mitochondrial Na+/Ca2+ exchanger NCLX. We reasoned that the caffeine effect is linked to phosphodiesterase inhibition, and focused on PDE2 that has the highest affinity to caffeine and is targeted to the mitochondria. Pretreatment of neurons with the selective PDE2 inhibitor Bay 60-7550 mimicked caffeine and rescued [Ca2+]m efflux triggered by neuronal depolarization. PDE2 inhibition also enhanced [Ca2+]m efflux triggered by neuromodulators. Inhibition of PDE2 acted by increasing mitochondrial cAMP thereby promoting NCLX phosphorylation. We found that protection of neurons against excitotoxic insults, conferred by PDE2 inhibition was diminished in NCLX KO neurons, thus is NCLX dependent. Finally, administration of Bay 60-7550 enhanced new object recognition learning in WT but not in NCLX KO mice. Our results identify a pathway linking caffeine and PDE to [Ca2+]m signaling, thereby providing new therapeutic targets for treating cognitive impairment and excitotoxicity.

Presynaptic zinc release and a postsynaptic transporter organize zinc into spatial and functional microdomains., The NMDA receptor (NMDAR) is inhibited by synaptically released zinc. This inhibition is thought to be the result of zinc diffusion across the synaptic cleft and subsequent binding to the extracellular domain of the NMDAR. However, this model fails to incorporate the observed association of the highly zinc-sensitive NMDAR subunit GluN2A with the postsynaptic zinc transporter ZnT1, which moves intracellular zinc to the extracellular space. Here, we report that disruption of ZnT1-GluN2A association by a cell-permeant peptide strongly reduced NMDAR inhibition by synaptic zinc in mouse dorsal cochlear nucleus synapses. Moreover, synaptic zinc inhibition of NMDARs required postsynaptic intracellular zinc, suggesting that cytoplasmic zinc is transported by ZnT1 to the extracellular space in close proximity to the NMDAR. These results challenge a decades-old dogma on how zinc inhibits synaptic NMDARs and demonstrate that presynaptic release and a postsynaptic transporter organize zinc into distinct microdomains to modulate NMDAR neurotransmission.

Zinc is an essential micronutrient affecting many aspects of human health. Cellular Zn2+ homeostasis is critical for cell function and survival. Zn2+, acting as a first or second messenger, triggers signaling pathways that mediate the physiological roles of Zn2+. Transient changes in Zn2+ concentrations within the cell or in the extracellular region occur following its release from Zn2+ binding metallothioneins, its transport across membranes by the ZnT or ZIP transporters, or release of vesicular Zn2+. These transients activate a distinct Zn2+ sensing receptor, ZnR/GPR39, or modulate numerous proteins and signaling pathways. Importantly, Zn2+ signaling regulates cellular physiological functions such as: proliferation, differentiation, ion transport and secretion. Indeed, novel therapeutic approaches aimed to maintain Zn2+ homeostasis and signaling are evolving. This review focuses on recent findings describing roles of Zn2+ and its transporters in regulating physiological or pathological processes.

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn2+ neurotoxicity. The mechanism mediating Zn2+ transport by ZnT1, however, has remained elusive. Here, we investigated ZnT1-dependent Zn2+ transport by measuring intracellular changes of this ion using the fluorescent indicator FluoZin-3. In primary mouse cortical neurons, which express ZnT1, transient addition of extracellular Zn2+ triggered a rise in cytosolic Zn2+, followed by its removal. Knockdown of ZnT1 by adeno associated viral (AAV)-short hairpin RNA (shZnT1) markedly increased rates of Zn2+ rise, and decreased rates of its removal, suggesting that ZnT1 is a primary route for Zn2+ efflux in neurons. Although Zn2+ transport by other members of the SLC30A family is dependent on pH gradients across cellular membranes, altered H+ gradients were not coupled to ZnT1-dependent transport. Removal of cytoplasmic Zn2+, against a large inward gradient during the initial loading phase, suggests that Zn2+ efflux requires a large driving force. We therefore asked if Ca2+ gradients across the membrane can facilitate Zn2+ efflux. Elimination of extracellular Ca2+ abolished Zn2+ efflux, while increased extracellular Ca2+ levels enhanced Zn2+ efflux. Intracellular Ca2+ rises, measured in GCaMP6 expressing neurons, closely paralleled cytoplasmic Zn2+ removal. Taken together, these results strongly suggest that ZnT1 functions as a Zn2+/Ca2+ exchanger, thereby regulating the transport of two ions of fundamental importance in neuronal signaling.

Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that accumulates in humans and affects the nervous system. To elucidate the mechanisms of HBCD neurotoxicity, we used transcriptomic profiling in brains of female mice exposed through their diet to HBCD (199 mg/kg body weight per day) for 28 days and compared with those of neuronal N2A and NSC-19 cell lines exposed to 1 or 2 µM HBCD. Similar pathways and functions were affected both in vivo and in vitro, including Ca2+ and Zn2+ signalling, glutamatergic neuron activity, apoptosis, and oxidative stress. Release of cytosolic free Zn2+ by HBCD was confirmed in N2A cells. This Zn2+ release was partially quenched by the antioxidant N-acetyl cysteine indicating that, in accordance with transcriptomic analysis, free radical formation is involved in HBCD toxicity. To investigate the effects of HBCD in excitable cells, we isolated mouse hippocampal neurons and monitored Ca2+ signalling triggered by extracellular glutamate or zinc, which are co-released pre-synaptically to trigger postsynaptic signalling. In control cells application of zinc or glutamate triggered a rapid rise of intracellular [Ca2+]. Treatment of the cultures with 1 µM of HBCD was sufficient to reduce the glutamate-dependent Ca2+ signal by 50%. The effect of HBCD on zinc-dependent Ca2+ signalling was even more pronounced, resulting in the reduction of the Ca2+ signal with 86% inhibition at 1 µM HBCD. Our results show that low concentrations of HBCD affect neural signalling in mouse brain acting through dysregulation of Ca2+ and Zn2+ homeostasis. Electronic supplementary material The online version of this article (doi:10.1007/s00204-017-2119-2) contains supplementary material, which is available to authorized users.

Background The Na+/Ca2 +/Li+ exchanger (NCLX) is a member of the Na+/Ca2 + exchanger family. NCLX is unique in its capacity to transport both Na+ and Li+, unlike other members, which are Na+ selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li+ or Na+ selective Ca2 + transport and map their putative location on NCLX cation transport site. Method We combined molecular modeling to map transport site of NCLX with euryarchaeal H+/Ca2 + exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2 + efflux of transport site mutants of NCLX in permeabilized cells. Result Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca2 + exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2 + exchange activity but a reduced Na+/Ca2 + exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2 + exchange, but Na+/Ca2 + exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2 + binding residues was required to completely abolish both Na+/Ca2 + and Li+/Ca2 + exchange activities. Conclusions We identified distinct Na+ and Li+ selective residues in the NCLX transport site. We propose that functional segregation in Li+ and Na+ sites reflects the functional properties of NCLX required for Ca2 + exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. General significance The results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Administration of zinc, as a complement to oral rehydration solutions, effectively diminishes duration and severity of diarrhea, but it is not known whether it merely fulfills a nutritional deficiency, or if zinc has a direct role of regulating solute absorption. We show that Zn 2+ acts via a specific receptor, ZnR/GPR39, to reduce fluid loss. Intestinal fluid secretion triggered by cholera toxin (CTx) was lower in WT mice compared to ZnR/GPR39 KO. In the absence of dietary Zn 2+ we observed similar fluid accumulation in WT and ZnR/GPR39 KO mice, indicating that Zn 2+ and ZnR/GPR39 are both required for a beneficial effect of Zn 2+ in diarrhea. In primary colonocytes and in Caco-2 colonocytic cells, activation of ZnR/GPR39 enhanced Cl − transport, a critical factor in diarrhea, by upregulating K + /Cl − cotransporter (KCC1) activity. Importantly, we show basolateral expression of KCC1 in mouse and human colonocytes, thus identifying a novel Cl − absorption pathway. Finally, inhibition of KCC-dependent Cl − transport enhanced CTx-induced fluid loss. Altogether, our data indicate that Zn 2+ acting via ZnR/GPR39 has a direct role in controlling Cl − absorption via upregulation of basolateral KCC1 in the colon. Moreover, colonocytic ZnR/GPR39 and KCC1 reduce water loss during diarrhea and may therefore serve as effective drug targets.

The pace of deorphanization of G protein–coupled receptors (GPCRs) has slowed, and new approaches are required. Small molecule targeting of orphan GPCRs can potentially be of clinical benefit even if the endogenous receptor ligand has not been identified. Many GPCRs lack common variants that lead to reproducible genome-wide disease associations, and rare-variant approaches have emerged as a viable alternative to identify disease associations for such genes. Therefore, our goal was to prioritize orphan GPCRs by determining their associations with human diseases in a large clinical population. We used sequence kernel association tests to assess the disease associations of 85 orphan or understudied GPCRs in an unselected cohort of 51,289 individuals. Using rare loss-of-function variants, missense variants predicted to be pathogenic or likely pathogenic, and a subset of rare synonymous variants that cause large changes in local codon bias as independent data sets, we found strong, phenome-wide disease associations shared by two or more variant categories for 39% of the GPCRs. To validate the bioinformatics and sequence kernel association test analyses, we functionally characterized rare missense and synonymous variants of GPR39, a family A GPCR, revealing altered expression or Zn(2+)-mediated signaling for members of both variant classes. These results support the utility of rare variant analyses for identifying disease associations for GPCRs that lack impactful common variants. We highlight the importance of rare synonymous variants in human physiology and argue for their routine inclusion in any comprehensive analysis of genomic variants as potential causes of disease.

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca2+ signaling leading to tumor progression. The K+/Cl- co-transporters (KCC), activated by intracellular signaling, enhance breast cancer cell migration and invasion. We asked if ZnR/GPR39 enhances breast cancer cell malignancy by activating KCC. Activation of ZnR/GPR39 by Zn2+ upregulated K+/Cl- co-transport activity, measured using NH4+ as a surrogate to K+ while monitoring intracellular pH. Upregulation of NH4+ transport was monitored in tamoxifen resistant cells with functional ZnR/GPR39-dependent Ca2+ signaling but not in MCF-7 cells lacking this response. The NH4+ transport was Na+-independent, and we therefore focused on KCC family members. Silencing of KCC3, but not KCC4, expression abolished Zn2+-dependent K+/Cl- co-transport, suggesting that KCC3 is mediating upregulated NH4+ transport. The ZnR/GPR39-dependent KCC3 activation accelerated scratch closure rate, which was abolished by inhibiting KCC transport with [(DihydroIndenyl) Oxy] Alkanoic acid (DIOA). Importantly, silencing of either ZnR/GPR39 or KCC3 attenuated Zn2+-dependent scratch closure. Thus, a novel link between KCC3 and Zn2+, via ZnR/GPR39, promotes breast cancer cell migration and proliferation.

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca

Actin re-organization and degradation of extracellular matrix by metalloproteases (MMPs) facilitate formation of cellular protrusions that are required for cell proliferation and migration. We find that Zn2+ activation of the Gq-coupled receptor ZnR/GPR39 controls these processes by regulating K+/Cl− co-transporter KCC3, which modulates cell volume. Silencing of KCC3 expression or activity reverses ZnR/GPR39 enhancement of cell proliferation, migration and invasion through Matrigel. Activation of ZnR/GPR39 recruits KCC3 into F-actin rich membrane protrusions, suggesting that it can locally control volume changes. Immunofluorescence analysis indicates that Zn2+ activation of ZnR/GPR39 and KCC3 are required to enhance formation of F-actin stress fibers and cellular protrusions. In addition, ZnR/GPR39 upregulation of KCC3-dependent transport increases the activity of matrix metalloproteases MMP2 and MMP9. Our study establishes a mechanism in which ZnR/GPR39 orchestrates localization and activation of KCC3, formation of F-actin rich cell protrusions and activation of MMPs, and thereby controls cell proliferation and migration.

A hallmark of Alzheimer's disease is accumulation of amyloid beta (Aβ) deposits, which are associated with neuronal dysfunction, spine loss, and impaired Ca2+ homeostasis. Amyloid beta (Aβ) binds to and is aggregated by Zn2+ , a metal released from synaptic glutamatergic vesicles during neuronal activity. Synaptically released Zn2+ activates a metabotropic Gq-coupled Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling in post-synaptic neurons. We examined if Aβ, as a Zn2+ binding protein, regulates neuronal Zn2+ -signaling mediated by mZnR/GPR39 using SHSY-5Y cells and cortical neurons from GPR39 wild-type and knockout mice. Following acute or chronic treatment with Aβ neuronal Zn2+ -dependent Ca2+ release via mZnR/GPR39 is significantly reduced. This impairment is overcome when excess Zn2+ is applied, suggesting that impaired Ca2+ -signaling results from Aβ binding of Zn2+ . The Zn2+ -dependent mZnR/GPR39 activation triggers phosphorylation of extracellular regulated kinase and up-regulates expression of the chaperone protein clusterin (Clu). Importantly, neuronal Zn2+ -dependent extracellular regulated kinase1/2 phosphorylation and up-regulation of Clu are attenuated by silencing mZnR/GPR39 as well as by Aβ treatment. In contrast, Zn2+ -dependent AKT phosphorylation is not mediated by mZnR/GPR39 and is not attenuated by Aβ treatment. Thus, Zn2+ signaling via mZnR/GPR39 is distinctively disrupted by a critical pathological component of Alzheimer's disease. Synaptically released Zn2+ activates a Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling, followed by ERK1/2 MAPK activation and up-regulation of clusterin. Amyloid beta (Aβ) binds to Zn2+ thus forming oligomers that are a hallmark of Alzheimer's disease. We show that Aβ attenuates Zn2+ -dependent Ca2+ -responses, abolishes ERK1/2 activation and down-regulates clusterin expression. Thus, Zn2+ signaling via mZnR/GPR39 is disrupted by Aβ, a critical pathological component of Alzheimer's disease.

Zinc plays a well-documented role in intestinal function, perturbations in zinc homeostasis are associated with impaired barrier function and disease. A large repertoire of zinc transport proteins ( ZnTs, ZIPs), TRP channels, and a distinct zinc receptor, ZnR/GPR39, are expressed along the intestinal epithelial cells. We will discuss the cellular roles of zinc ions, Zn2+, in activating signaling and physiological function of multiple cells along the intestinal epithelium. We will further describe the network of Zn2+-homeostatic proteins that are responsible for maintaining Zn2+ concentrations within the cytosol and their role in maintaining cellular functions. This review will reveal the tip of the iceberg that is currently known regarding the role of intestinal Zn2+ and will elucidate the need for extensive studies that will expound the complete map and physiological roles of Zn2+-homeostatic proteins.

Many G protein-coupled receptors (GPCRs) lack common variants that lead to reproducible genome-wide disease associations. Here we used rare variant approaches to assess the disease associations of 85 orphan or understudied GPCRs in an unselected cohort of 51,289 individuals. Rare loss-of-function variants, missense variants predicted to be pathogenic or likely pathogenic, and a subset of rare synonymous variants were used as independent data sets for sequence kernel association testing (SKAT). Strong, phenome-wide disease associations shared by two or more variant categories were found for 39% of the GPCRs. Validating the bioinformatics and SKAT analyses, functional characterization of rare missense and synonymous variants of GPR39, a Family A GPCR, showed altered expression and/or Zn2+-mediated signaling for members of both variant classes. Results support the utility of rare variant analyses for identifying disease associations for genes that lack common variants, while also highlighting the functional importance of rare synonymous variants.Author summaryRare variant approaches have emerged as a viable way to identify disease associations for genes without clinically important common variants. Rare synonymous variants are generally considered benign. We demonstrate that rare synonymous variants represent a potentially important dataset for deriving disease associations, here applied to analysis of a set of orphan or understudied GPCRs. Synonymous variants yielded disease associations in common with loss-of-function or missense variants in the same gene. We rationalize their associations with disease by confirming their impact on expression and agonist activation of a representative example, GPR39. This study highlights the importance of rare synonymous variants in human physiology, and argues for their routine inclusion in any comprehensive analysis of genomic variants as potential causes of disease.

Glucose-dependent cytosolic Na(+) influx in pancreatic islet β cells is mediated by TTX-sensitive Na(+) channels and is propagated into the mitochondria through the mitochondrial Na(+)/Ca(2+) exchanger, NCLX. Mitochondrial Na(+) transients are also controlled by the mitochondrial Na(+)/H(+) exchanger, NHE, while cytosolic Na(+) changes are governed by Na(+)/K(+) ATPase pump. The functional interaction between the Na(+) channels, Na(+)/K(+) ATPase pump and mitochondrial Na(+) transporters, NCLX and NHE, in mediating Na(+) signaling is poorly understood. Here, we combine fluorescent Na(+) imaging, pharmacological inhibition by TTX, ouabain and EIPA, with molecular control of NCLX expression, so as to investigate the crosstalk between Na(+) transporters on both the plasma membrane and the mitochondria. According to our results, glucose-dependent cytosolic Na(+) response was enhanced by ouabain and was followed by a rise in mitochondrial Na(+) signal. Silencing of NCLX expression using siNCLX, did not affect the glucose- or ouabain-dependent cytosolic rise in Na(+). In contrast, the ouabain-dependent rise in mitochondrial Na(+) was strongly suppressed by siNCLX. Furthermore, mitochondrial Na(+) influx rates were accelerated in cells treated with the Na(+)/H(+) exchanger inhibitor, EIPA or by combination of EIPA and ouabain. Similarly, TTX blocked the cytosolic and mitochondrial Na(+) responses, which were enhanced by ouabain or EIPA, respectively. Our results suggest that Na(+)/K(+) ATPase pump controls cytosolic glucose-dependent Na(+) rise, in a manner that is mediated by TTX-sensitive Na(+) channels and subsequent mitochondrial Na(+) uptake via NCLX. Furthermore, these results indicate that mitochondrial Na(+) influx via NCLX is antagonized by Na(+) efflux, which is mediated by the mitochondrial NHE; thus, the duration of mitochondrial Na(+) transients is set by the interplay between these pivotal transporters.

A distinct G-protein coupled receptor that senses changes in extracellular Zn2+, ZnR/GPR39, was found in cells from tissues in which Zn2+ plays a physiological role. Most prominently, ZnR/GPR39 activity was described in prostate cancer, skin keratinocytes, and colon epithelial cells, where zinc is essential for cell growth, wound closure, and barrier formation. ZnR/GPR39 activity was also described in neurons that are postsynaptic to vesicular Zn2+ release. Activation of ZnR/GPR39 triggers Gαq-dependent signaling and subsequent cellular pathways associated with cell growth and survival. Furthermore, ZnR/GPR39 was shown to regulate the activity of ion transport mechanisms that are essential for the physiological function of epithelial and neuronal cells. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

The 2014 ISZB meeting provided an exciting forum for presenting zinc biology-related research across the full spectrum. ISZB meetings are among the few scientific gatherings that are truly interdisciplinary, bringing together scientists from widely diverging fields, all having in common the desire to study the role of zinc in biological systems. Thus membrane transporters and cell signaling are discussed with relevance to structure/ function relationships, while physiological and pathophysiological studies are described in many species and across most organ systems. Novel tools and techniques are presented to researchers and potential users to allow dissemination of zinc research-related information for the benefit of a wide scientific community studying various aspects of zinc in biology. This is one meeting that should not be missed by biochemists, cell and molecular biologists, physiologists, or anyone interested in the rich and exciting biology of zinc. We all look forward to our next gathering in Istanbul, Turkey, September 25-29, 2016, http://iszb2016turkey.medicine.Ankara.edu.tr. The ISZB would like to thank Qiagen, Pfizer, Teva Pharmaceutical Industries, Novartis Pharma AG, Shino Test Corporation, Strem Chemicals, Genostaff and Hamari Chemicals for their generous donations that helped support the meeting. Book prizes for excellent talks were kindly provided by The Royal Society of Chemistry, Springer and IOS Press. Travel awards were supported in part by NIH grant GM112419. The authors would also like to thank Wolfgang Maret, Daren L. Knoell, Robert A. Colvin and David I. Soybel for their invaluable contributions to the organization of the meeting.

Mitochondrial functions depend on the steep H+ electrochemical gradient (ΔμH+) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H+ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca2+ signaling.

Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH+) across the inner membrane. Although several drugs can modulate ΔμH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondriatargeted opsinswere used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

While zinc has had a well-established structural role for many years, it is only during the last two decades that its role as a signaling molecule has been recognized. Ionic zinc, Zn2+, that is endogenously released during physiological activity acts as a first messenger, triggering the activity of a distinct Zn2+-sensing-receptor, ZnR. The ZnR is a member of the Gq-coupled receptor family, and the molecular moiety mediating its activity is GPR39. In this review, we will discuss the role of the ZnR/GPR39 in mediating Zn2+-dependent signaling in epithelial tissues and in neurons, where Zn2+ homeostasis plays physiological as well as pathological roles. Importantly, ZnR/GPR39 activates signaling that regulates a remarkably wide range of cell functions, including proliferation, differentiation and survival, as well as modulation of ion transport, and thereby, regulation of Na+, H+ and Cl- homeostasis. Moreover, signaling activated by ZnR/GPR39 plays a key role in mediating effects of Zn2+ in health and disease. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

Communication between the plasma membrane and mitochondria is essential for initiating the Ca(2+) and metabolic signals required for secretion in β cells. Although voltage-dependent Na(+) channels are abundantly expressed in β cells and activated by glucose, their role in communicating with mitochondria is unresolved. Here, we combined fluorescent Na(+), Ca(2+), and ATP imaging, electrophysiological analysis with tetrodotoxin (TTX)-dependent block of the Na(+) channel, and molecular manipulation of mitochondrial Ca(2+) transporters to study the communication between Na(+) channels and mitochondria. We show that TTX inhibits glucose-dependent depolarization and blocks cytosolic Na(+) and Ca(2+) responses and their propagation into mitochondria. TTX-sensitive mitochondrial Ca(2+) influx was largely blocked by knockdown of the mitochondrial Ca(2+) uniporter (MCU) expression. Knockdown of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) and Na(+) dose response analysis demonstrated that NCLX mediates the mitochondrial Na(+) influx and is tuned to sense the TTX-sensitive cytosolic Na(+) responses. Finally, TTX blocked glucose-dependent mitochondrial Ca(2+) rise, mitochondrial metabolic activity, and ATP production. Our results show that communication of the Na(+) channels with mitochondria shape both global Ca(2+) and metabolism signals linked to insulin secretion in β cells.- Nita, I. I., Hershfinkel, M., Kantor, C., Rutter, G. A., Lewis, E. C., Sekler, I. Pancreatic β-cell Na(+) channels control global Ca(2+) signaling and oxidative metabolism by inducing Na(+) and Ca(2+) responses that are propagated into mitochondria.

Zinc signaling is mediated by the zinc sensing receptor, ZnR, recently suggested to be the same receptor as G-protein coupled receptor 39, GPR39. However, it is unknown if GPR39 is mediating Zn(2+) -dependent signaling in prostate and salivary tissue where changes in zinc concentrations are frequent and of physiological significance. Here, we show that GPR39 is mediating Zn(2+) -dependent Ca(2+) responses and is regulating activity of MAP and PI3 pathways in prostate cancer cells, PC3, and ductal salivary gland cells, HSY. We next ask whether ZnR/GPR39 interacts with other GPCR family members. We find that endogenous ZnR/GPR39 activity is regulated by the expression and activity of another cation sensing GPCR, the Ca(2+) -sensing receptor (CaSR). Although CaSR is not activated by Zn(2+), co-expression of CaSR and ZnR/GPR39 synergistically enhances Ca(2+) responses in PC3 and HSY cells. Silencing of the CaSR using siRNA or a dominant negative construct reduces the Zn(2+) -dependent signaling. Importantly, overexpression of GPR39 in HEK293 cells is sufficient to trigger Zn(2+) -dependent responses. Nevertheless, application of the CaSR agonist spermine, at concentration below its threshold, enhanced Zn(2+) -dependent Ca(2+) response. Our results suggest that the CaSR interacts with ZnR/GPR39 and thereby regulates its activity. Finally, we show that in PC3 cells ZnR/GPR39 is required for mediating the Zn(2+) -dependent activation of MAPK and PI3K, pathways leading to enhanced cell growth. Importantly, Zn(2+) -dependent activation of ZnR/GPR39 also enhances the expression of the Ca(2+) -binding protein S100A4 that is linked to invasion of prostate cancer cells.

The proinflammatory S100A8/A9 proteins, which are expressed in myeloid cells under physiological conditions, are strongly expressed in human prostate cancer epithelial cells. Their role in the tumor cells and in tumor progression is largely unclear. We established a prostate cancer epithelial cell line (PC-3 TO-A8/A9) expressing S100A8 and S100A9 simultaneously under doxycycline control, to study the role of S100A8/A9 on tumor growth and infiltration of immune cells in subcutaneous xenografts in male NMRI nu/nu mice. Colonization of distant organs was studied after intracardial injection of the tumor cells in male NOD/SCID mice. PC-3 TO-A8/A9 cells grown in vitro and subcutaneous xenografts in mice not treated with doxycycline expressed high levels of S100A8/A9 mRNA and protein, whereas doxycycline treatment suppressed S100A8/A9 expression. S100A8/A9 expression did not significantly alter growth rate and invasion of the subcutaneous tumors into surrounding tissues. However, S100A8/A9 expression caused increased infiltration of immune cells, especially neutrophils. In intracardially injected mice sporadic tumor settlement was observed in muscle and lymph nodes. Colonies of tumor cells and micro-metastases were observed in the lung of 64.3% (9 out of 14) of mice not treated with doxycycline and in 33.3% (5 out of 15) of mice treated with doxycycline. Our data demonstrate for the first time that S100A8/A9 expression in epithelial cancer cells causes enhanced infiltration of immune cells, especially neutrophils, and stimulates settlement of the cancer cells in the lung.

Although it is well established that many glutamatergic neurons sequester Zn2+within their synaptic vesicles, the physiological significance of synaptic Zn2+remains poorly understood. In experiments performed in a Zn2+-enriched auditory brainstem nucleus—the dorsal cochlear nucleus—we discovered that synaptic Zn2+and GPR39, a putative metabotropic Zn2+-sensing receptor (mZnR), are necessary for triggering the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The postsynaptic production of 2-AG, in turn, inhibits presynaptic probability of neurotransmitter release, thus shaping synaptic strength and short-term synaptic plasticity. Zn2+-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn2+or the mZnR. Moreover, mass spectrometry measurements of 2-AG levels reveal that Zn2+-mediated initiation of 2-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn2+: synaptic Zn2+inhibits glutamate release by promoting 2-AG synthesis.

Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

The NaWe combined molecular modeling to map transport site of NCLX with euryarchaeal HMutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial CaWe identified distinct NaThe results of this study provide functional insights into the unique Li

A hallmark of Alzheimer's disease is accumulation of amyloid beta (Aβ) deposits, which are associated with neuronal dysfunction, spine loss, and impaired Ca

Impaired epithelial barrier function is a hallmark of inflammatory bowel diseases, such as colitis, contributing to diarrhoea and perpetuating inflammation. We show that the zinc sensing receptor, ZnR/GPR39, triggers intracellular Ca 2+ signalling in colonocytes thereby inducing occludin expression. Moreover, ZnR/GPR39 is essential for epithelial barrier recovery in the dextran sodium sulfate (DSS) ulcerative colitis model. Loss of ZnR/GPR39 results in increased susceptibility to DSS-induced inflammation, owing to low expression of the tight junction protein occludin and impaired epithelial barrier. Recovery of wild-type (WT) mice from the DSS insult was faster than that of ZnR/GPR39 knockout (KO) mice. Enhanced recovery of the epithelial layer and increased crypt regeneration were observed in WT mice compared with ZnR/GPR39 KO, suggesting that ZnR/GPR39 is promoting epithelial barrier integrity following DSS insult. Indeed, cell proliferation and apical expression of occludin, following the DSS-induced epithelial erosion, were increased in WT tissue but not in ZnR/GPR39 KO tissue. Importantly, survival following DSS treatment was higher in WT mice compared with ZnR/GPR39 KO mice. Our results support a direct role for ZnR/GPR39 in promoting epithelial renewal and barrier function following DSS treatment, thereby affecting the severity of the disease. We suggest ZnR/GPR39 as a novel therapeutic target that can improve epithelial barrier function in colitis. This article is part of the themed issue ‘Evolution brings Ca 2+ and ATP together to control life and death’.

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABA(A) receptor channels. In a recent study, we showed that Zn(2+) regulates GABA(A) reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn(2+) initiates this process by activating the Gq-coupled metabotropic Zn(2+) receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca(2+) indicator and exposing cells to Zn(2+), which triggered consistent Ca(2+) responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn(2+) treatment under these conditions did not increase the intracellular concentrations of Zn(2+) itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH(4)(+) influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn(2+) pretreatment induced a Ca(2+)-dependent increase in KCC2 activity. The effects of Zn(2+) on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39(-/-) mice, suggesting that Zn(2+) acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn(2+)-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn(2+). These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn(2+) stimulation of mZnR/GPR39.