Cross-talk between zinc and calcium regulates ion transport: A role for the zinc receptor, ZnR/GPR39.

Zinc is essential for many physiological functions, with a major role in digestive system, skin health, and learning and memory. On the cellular level, zinc is involved in cell proliferation and cell death. A selective zinc sensing receptor, ZnR/GPR39 is a Gq-coupled receptor that acts via the inositol trisphosphate pathway to release intracellular Ca2+. The ZnR/GPR39 serves as a mediator between extracellular changes in Zn2+ concentration and cellular Ca2+ signalling. This signalling pathway regulates ion transporters activity and thereby controls the formation of transepithelial gradients or neuronal membrane potential, which play a fundamental role in the physiological function of these tissues. This review focuses on the role of Ca2+ signalling, and specifically ZnR/GPR39, with respect to the regulation of the Na+/H+ exchanger, NHE1, and of the K+/Cl- cotransporters, KCC1-3, and also describes the physiological implications of this regulation.

The ZIP3 Zinc Transporter Is Localized to Mossy Fiber Terminals and Is Required for Kainate-Induced Degeneration of CA3 Neurons.

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 expression in vivo reduced Zn2+ uptake in CA3 neurons while ZIP3 silencing reduced Zn2+ influx into dentate gyrus (DG) granule cells in acute hippocampal slices. Strikingly, in vivo silencing 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 STATEMENT Zinc 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.

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals.

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevents the cytosolic Na+ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na+ and Ca2+ signals to effect mitochondrial redox control over SOCE.

Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn2+ is driven by an active Ca2+-coupled exchange.

Manganese (Mn2+) 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 Ca2+ inward gradient for active cellular exchange of Mn2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn2+ and Ca2+ fluxes. We found that (a) application of Mn2+-containing Ca2+-free solution to ZnT10-expressing cells triggers an influx of Mn2+, (b) reintroduction of Ca2+ leads to cellular Mn2+ extrusion against an inward Mn2+ gradient, and (c) the cellular transport of Mn2+ by ZnT10 is coupled to a reciprocal movement of Ca2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn2+/Ca2+ exchange to an uncoupled channel mode, permeable to both Ca2+ and Mn2+ The findings in our study identify the first ion transporter that uses the Ca2+ 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.

Rare-variant pathogenicity triage and inclusion of synonymous variants improves analysis of disease associations of orphan G protein-coupled receptors.

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 Zn2+-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.

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins.

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 mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic ß-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration.

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.

Elucidating the H+ Coupled Zn2+ Transport Mechanism of ZIP4; Implications in Acrodermatitis Enteropathica.

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.

Identification of residues that control Li+ versus Na+ dependent Ca2+ exchange at the transport site of the mitochondrial NCLX.

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.

The Zinc Sensing Receptor, ZnR/GPR39, in Health and Disease.

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 Zn2+-sensing receptor, ZnR/GPR39, upregulates colonocytic Cl- absorption, via basolateral KCC1, and reduces fluid loss.

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 Zn2+ 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 Zn2+ we observed similar fluid accumulation in WT and ZnR/GPR39 KO mice, indicating that Zn2+ and ZnR/GPR39 are both required for a beneficial effect of Zn2+ 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 Zn2+ 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.

Amyloid ß attenuates metabotropic zinc sensing receptor, mZnR/GPR39, dependent Ca2+ , ERK1/2 and Clusterin signaling in neurons.

A hallmark of Alzheimer's disease is accumulation of amyloid beta (Aß) deposits, which are associated with neuronal dysfunction, spine loss, and impaired Ca2+ homeostasis. Amyloid beta (Aß) 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 Aß, 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 Aß 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 Aß 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 Aß treatment. In contrast, Zn2+ -dependent AKT phosphorylation is not mediated by mZnR/GPR39 and is not attenuated by Aß 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 (Aß) binds to Zn2+ thus forming oligomers that are a hallmark of Alzheimer's disease. We show that Aß attenuates Zn2+ -dependent Ca2+ -responses, abolishes ERK1/2 activation and down-regulates clusterin expression. Thus, Zn2+ signaling via mZnR/GPR39 is disrupted by Aß, a critical pathological component of Alzheimer's disease.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive decreases in pHe during acidosis. Hence, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain.

Homeostatic regulation of KCC2 activity by the zinc receptor mZnR/GPR39 during seizures.

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.

Pancreatic ß-cell Na+ channels control global Ca2+ signaling and oxidative metabolism by inducing Na+ and Ca2+ responses that are propagated into mitochondria.

Communication between the plasma membrane and mitochondria is essential for initiating the Ca(2+) and metabolic signals required for secretion in ß cells. Although voltage-dependent Na(+) channels are abundantly expressed in ß 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 ß cells.- Nita, I. I., Hershfinkel, M., Kantor, C., Rutter, G. A., Lewis, E. C., Sekler, I. Pancreatic ß-cell Na(+) channels control global Ca(2+) signaling and oxidative metabolism by inducing Na(+) and Ca(2+) responses that are propagated into mitochondria.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

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.

Synaptic Zn2+ inhibits neurotransmitter release by promoting endocannabinoid synthesis.

Although it is well established that many glutamatergic neurons sequester Zn(2+) within their synaptic vesicles, the physiological significance of synaptic Zn(2+) remains poorly understood. In experiments performed in a Zn(2+)-enriched auditory brainstem nucleus--the dorsal cochlear nucleus--we discovered that synaptic Zn(2+) and GPR39, a putative metabotropic Zn(2+)-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. Zn(2+)-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn(2+) or the mZnR. Moreover, mass spectrometry measurements of 2-AG levels reveal that Zn(2+)-mediated initiation of 2-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn(2+): synaptic Zn(2+) inhibits glutamate release by promoting 2-AG synthesis.

The ZnR/GPR39 interacts with the CaSR to enhance signaling in prostate and salivary epithelia.

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.

Impact of S100A8/A9 expression on prostate cancer progression in vitro and in vivo.

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.

NCLX controls hepatic mitochondrial Ca2+ extrusion and couples hormone-mediated mitochondrial Ca2+ oscillations with gluconeogenesis.

OBJECTIVE: Hepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+/Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics. METHODS: Primary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knock-out (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were utilized to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed first through the monitoring of glucose levels in fasted mice in vivo and by subjecting the fasted mice to a pyruvate tolerance test while monitoring blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively. RESULTS: Analysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations or level of IP3R1 expression, underscoring NCLX's pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic, and with a compromised conversion of pyruvate to glucose when provided challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX's significant contribution to hepatic glucose metabolism. CONCLUSIONS: The study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for the regulation of hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism and sustenance of hepatic gluconeogenesis.