ER Calcium Research Articles

Hepatic Inositol 1,4,5 Trisphosphate Receptor Type 1 Mediates Fatty Liver.

Fatty liver is the most common type of liver disease, affecting nearly one third of the U.S. population and more than half a billion people worldwide. Abnormalities in endoplasmic reticulum (ER) calcium handling and mitochondrial function each have been implicated in abnormal lipid droplet formation. Here we show that the type 1 isoform of the inositol 1,4,5‐trisphosphate receptor (InsP3R1) specifically links ER calcium release to mitochondrial calcium signaling and lipid droplet formation in hepatocytes. Moreover, liver‐specific InsP3R1 knockout mice have impaired mitochondrial calcium signaling, decreased hepatic triglycerides, and reduced lipid droplet formation and are resistant to the development of fatty liver. Patients with nonalcoholic steatohepatitis, the most malignant form of fatty liver, have increased hepatic expression of InsP3R1, and the extent of ER–mitochondrial colocalization correlates with the degree of steatosis in human liver biopsies. Conclusion: InsP3R1 plays a central role in lipid droplet formation in hepatocytes, and the data suggest that it is involved in the development of human fatty liver disease. (Hepatology Communications 2017;1:23–35)

The sigma-1 receptor mediates the beneficial effects of pridopidine in a mouse model of Huntington disease

The tri-nucleotide repeat expansion underlying Huntington disease (HD) results in corticostriatal synaptic dysfunction and subsequent neurodegeneration of striatal medium spiny neurons (MSNs). HD is a devastating autosomal dominant disease with no disease-modifying treatments. Pridopidine, a postulated “dopamine stabilizer”, has been shown to improve motor symptoms in clinical trials of HD. However, the target(s) and mechanism of action of pridopidine remain to be fully elucidated. As binding studies identified sigma-1 receptor (S1R) as a high-affinity receptor for pridopidine, we evaluated the relevance of S1R as a therapeutic target of pridopidine in HD. S1R is an endoplasmic reticulum - (ER) resident transmembrane protein and is regulated by ER calcium homeostasis, which is perturbed in HD. Consistent with ER calcium dysregulation, we observed striatal upregulation of S1R in aged YAC128 transgenic HD mice and HD patients. We previously demonstrated that dendritic MSN spines are lost in aged corticostriatal co-cultures from YAC128 mice. We report here that pridopidine and the chemically similar S1R agonist 3-PPP prevent MSN spine loss in aging YAC128 co-cultures. Spine protection was blocked by neuronal deletion of S1R. Pridopidine treatment suppressed supranormal ER Ca2+ release, restored ER calcium levels and reduced excessive store-operated calcium (SOC) entry in spines, which may account for its synaptoprotective effects. Normalization of ER Ca2+ levels by pridopidine was prevented by S1R deletion. To evaluate long-term effects of pridopidine, we analyzed expression profiles of calcium signaling genes. Pridopidine elevated striatal expression of calbindin and homer1a, whereas their striatal expression was reduced in aged Q175KI and YAC128 HD mouse models compared to WT. Pridopidine and 3-PPP are proposed to prevent calcium dysregulation and synaptic loss in a YAC128 corticostriatal co-culture model of HD. The actions of pridopidine were mediated by S1R and led to normalization of ER Ca2+ release, ER Ca2+ levels and spine SOC entry in YAC128 MSNs. This is a new potential mechanism of action for pridopidine, highlighting S1R as a potential target for HD therapy. Upregulation of striatal proteins that regulate calcium, including calbindin and homer1a, upon chronic therapy with pridopidine, may further contribute to long-term beneficial effects of pridopidine in HD.

Reducing translation through eIF4G/IFG-1 improves survival under ER stress that depends on heat shock factor HSF-1 in Caenorhabditis elegans.

SummaryAlthough certain methods of lowering and/or altering mRNA translation are associated with increased lifespan, the mechanisms underlying this effect remain largely unknown. We previously showed that the increased lifespan conferred by reducing expression of eukaryotic translation initiation factor 4G (eIF4G/IFG‐1) enhances survival under starvation conditions while shifting protein expression toward factors involved with maintaining ER‐dependent protein and lipid balance. In this study, we investigated changes in ER homeostasis and found that lower eIF4G/IFG‐1 increased survival under conditions of ER stress. Enhanced survival required the ER stress sensor gene ire‐1 and the ER calcium ATPase gene sca‐1 and corresponded with increased translation of chaperones that mediate the ER unfolded protein response (UPRER). Surprisingly, the heat‐shock transcription factor gene hsf‐1 was also required for enhanced survival, despite having little or no influence on the ability of wild‐type animals to survive ER stress. The requirement for hsf‐1 led us to re‐evaluate the role of eIF4G/IFG‐1 on thermotolerance. Results show that lowering expression of this translation factor enhanced thermotolerance, but only after prolonged attenuation, the timing of which corresponded to increased transcription of heat‐shock factor transcriptional targets. Results indicate that restricting overall translation through eIF4G/IFG‐1 enhances ER and cytoplasmic proteostasis through a mechanism that relies heavily on hsf‐1.

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

Mephebrindole, a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2α/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells.

The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3'-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3'-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.

IRS1 deficiency protects β-cells against ER stress-induced apoptosis by modulating sXBP-1 stability and protein translation.

Endoplasmic reticulum (ER) stress is among several pathological features that underlie β-cell failure in the development of type 1 and type 2 diabetes. Adaptor proteins in the insulin/insulin-like-growth factor-1 signaling pathways, such as insulin receptor substrate-1 (IRS1) and IRS2, differentially impact β-cell survival but the underlying mechanisms remain unclear. Here we report that β-cells deficient in IRS1 (IRS1KO) are resistant, while IRS2 deficiency (IRS2KO) makes them susceptible to ER stress-mediated apoptosis. IRS1KOs exhibited low nuclear accumulation of spliced XBP-1 due to its poor stability, in contrast to elevated accumulation in IRS2KO. The reduced nuclear accumulation in IRS1KO was due to protein instability of Xbp1 secondary to proteasomal degradation. IRS1KO also demonstrated an attenuation in their general translation status in response to ER stress revealed by polyribosomal profiling. Phosphorylation of eEF2 was dramatically increased in IRS1KO enabling the β-cells to adapt to ER stress by blocking translation. Furthermore, significantly high ER calcium (Ca2+) was detected in IRS1KO β-cells even upon induction of ER stress. These observations suggest that IRS1 could be a therapeutic target for β-cell protection against ER stress-mediated cell death by modulating XBP-1 stability, protein synthesis, and Ca2+ storage in the ER.

Regulation of BMP2-induced intracellular calcium increases in osteoblasts.

Although bone morphogenetic protein-2 (BMP2) is a well-characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca2+ signaling. In this study, intracellular Ca2+ concentration ([Ca2+ ]i ) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3-E1 osteoblast cell line using fluorescence resonance energy transfer-based biosensors. Increase in [Ca2+ ]i , FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF-573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2-stimulated [Ca2+ ]i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2-induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca2+ ]i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725-1733, 2016.

ER Calcium Levels Help Regulate K(ATP) Channel Trafficking to the Plasma Membrane of Pancreatic Beta Cells

The closing of beta cell K(ATP) channels acutely triggers glucose-induced insulin secretion. However, the trafficking of K(ATP) channels to the beta cell plasma membrane has recently been shown to regulate beta cell secretory function over a longer time course, and changes in the number of K(ATP) channels can help set islet glucose sensitivity. To test whether endoplasmic reticulum free calcium level helps control K(ATP) trafficking to the cell surface, insulin-secreting INS-1 cells were treated for 16-18 h. with either palmitate (PALM; 250 µM), the SERCA inhibitor thapsigargin (TG; 200 nM) or tunicamycin (TM; 10 µg/ml); these treatments alter ER Ca. INS-1 membrane fractions were immunoblotted for SUR1, a K(ATP) channel subunit, Glucose Transporter 2 (GLUT2) or Kv2.1 channels. Isolated islets were transfected with the ER Ca sensor D4ER to monitor ER Ca, and basal ER Ca levels were expressed as FRET ratios. TM-treated cells had low levels of glycosylated SUR1 and GLUT2 on their surface membranes whereas the trafficking of Kv2.1 was unaffected. PALM or TG treatment also reduced surface K(ATP), albeit more modestly. To test if ER Ca was reduced due to Ca egress through ER IP3 receptors, we treated cells with xestospongin C (XeC) to inhibit IP3Rs. XeC prevented ER Ca reduction due to TG treatment and increased K(ATP) trafficking. Furthermore, TM or TG treated islets were more glucose sensitive. These data suggest that ER Ca contributes to the control of SUR1 trafficking to the cell surface and/or ER protein processing. Dynamic regulation of surface K(ATP) by ER Ca may provide a novel negative feedback mechanism to regulate beta cell Ca2+ influx.

ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains

Spatially and temporally controlled increases of H2O2 emerge as an intracellular signal. We hypothesized that ROS and Ca2+ interact locally, in the restricted volume of the ER-mitochondrial interface. These physically tethered structures host enrichments of ion transport proteins such as the IP3 receptor, which support elevated nanodomains of Ca2+ during signalling events and are sensitive to H2O2. We used the genetically encoded H2O2 sensor HyPer incorporated into an inducible linker system to probe the redox environment at the ER-mitochondrial interface in HepG2 cells. We found a moderately elevated H2O2 nanodomain which developes into a H2O2 transient following IP3 receptor-mediated ER Ca2+ release and mitochondrial Ca2+ uptake. Pharmacological inhibition showed that the transient was dependent upon ER Ca2+, mitochondrial membrane potential and functional electron transport chain. HyPer measurements of the mitochondrial intermembrane space revealed significantly elevated H2O2 within this volume. Using electron microscopy we found that HepG2 mitochondria possess a cohort of dilated cristae, which disappeared following IP3-linked Ca2+ release. Paxilline that inhibits mitochondrial BKCa channels blocked the cristae reshaping and also abolished the H2O2 transient at the interface. Furthermore, paxilline caused suppression of the IP3-linked calcium signal, whereas interface targeted killer red, a photoactivated H2O2 source, induced sensitization to the IP3-linked agonist. We conclude that the intermembrane/cristae volume of mitochondria represents an oxidized pool fed by the electron transport chain. Ca2+-uptake stimulates expansion of the mitochondrial matrix via K+ and concomitant water uptake, squeezing the oxidized volume of the cristae to the interface. Here, a transient H2O2 nanodomain sensitizes IP3 receptors to further stimulation. We demonstrate a physiological setting where Ca2+ release may autoregulate using mitochondrial H2O2 released from the cristae.

Anti-Cancer Effect of Cap-Translation Inhibitor 4EGI-1 in Human Glioma U87 Cells: Involvement of Mitochondrial Dysfunction and ER Stress

Background: Cancer cells are frequently addicted to deregulated oncogenic protein translation that usually arises as a consequence of increased signaling flux from eIF4F activation. The small molecule 4EG-I, a potent inhibitor of translation initiation through disrupting eIF4E/eIF4G interaction, has been shown to exert anticancer effects in animal models of human cancers. Methods: Here, we extensively investigated the anticancer activity of 4EGI-1 in human glioma U87 cells. The anti-cancer effects of 4EGI-1 were measured by cell viability, lactate dehydrogenase (LDH) release, TUNEL staining, flow cytometry and western blot analysis in vitro, and also examined in a U87 xenograft model in vivo. The potential underlying molecular mechanisms were investigated by measuring mitochondrial function and ER stress. Results: We found that 4EGI-1 impaired the assembly of the eIF4F complex and decreased the expression of the eIF4E regulated proteins. The results of TUNEL staining and flow cytometry showed that 4EGI-1 treatment induced apoptotic cell death in a dose-dependent manner. Furthermore, 4EGI-1-induced apoptosis in U87 cells was associated with mitochondrial dysfunction and activation of the intrinsic mitochondrial pathway, which was dependent on the induction of the pro-apoptotic protein Bax. In addition, 4EGI-1 treatment triggered ER stress, which was evidenced by morphological changes of ER lumen and ER calcium release, as well as the dose-dependent increases in the expression of ER stress related proteins. Moreover, knockdown of the ER chaperone GRP-78 through siRNA was shown to partially reverse the 4EGI-1-induced ER stress in U87 cells. In vivo, 4EGI-1 strongly inhibited growth of U87 glioma xenografts without any apparent organ related toxicities. Conclusion: These data indicate that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for human glioma therapy.

Intracellular sphingosine releases calcium from lysosomes.

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

GRP94 Regulates Circulating Cholesterol Levels through Blockade of PCSK9-Induced LDLR Degradation.

Clearance of circulating low-density lipoprotein cholesterol (LDLc) by hepatic LDL receptors (LDLR) is central for vascular health. Secreted by hepatocytes, PCSK9 induces the degradation of LDLR, resulting in higher plasma LDLc levels. Still, it remains unknown why LDLR and PCSK9 co-exist within the secretory pathway of hepatocytes without leading to complete degradation of LDLR. Herein, we identified the ER-resident GRP94, and more precisely its client-binding C-terminal domain, as a PCSK9-LDLR inhibitory binding protein. Depletion of GRP94 did not affect calcium homeostasis, induce ER stress, nor did it alter PCSK9 processing or its secretion but greatly increased its capacity to induce LDLR degradation. Accordingly, we found that hepatocyte-specific Grp94-deficient mice have higher plasma LDLc levels correlated with ∼ 80% reduction in hepatic LDLR protein levels. Thus, we provide evidence that, in physiological conditions, binding of PCSK9 to GRP94 protects LDLR from degradation likely by preventing early binding of PCSK9 to LDLR within the ER.

A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients.

Signal‐dependent Regulation of Transcription Factor Activity

Signal dependent regulation of transcription factor activity in the nervous system controls gene expression programs that underlie learning and memory. Transcription factor Sp4 is zinc finger DNA binding protein that regulates neuronal connectivity, brain development and function. In humans, altered Sp4 has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. We are investigating the function and regulation of Sp4 in neurons. Our recent studies have revealed two distinct calcium‐dependent signaling pathways that regulate Sp4 via altered patterns of post‐translational modification. We have found that stimulation of the NMDA receptor activates a PP1/PP2A protein phosphatase signaling pathway that dephosphorylates Sp4. Our data suggests that this phosphorylation reduces Sp4 function independent of an effect on Sp4 levels. We have also found that Sp4 protein, but not mRNA, abundance is highly regulated in response to changes in membrane potential associated with neuronal activity. In resting neurons, Sp4 is ubiquitinated and rapidly degraded by the proteasome. We have found that calcium influx via store operated calcium entry (SOCE) and the ER calcium sensor Stim1 regulates Sp4 ubiquitination. Our studies reveal new aspects of the complex mechanisms that determine signal‐dependent regulation of transcription factor activity important for neuronal plasticity, brain development and disease.

Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi’s sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of “constitutive” NFAT activation by viral GPCRs.

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca2+]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca2+]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca2+]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca2+]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca2+]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca2+]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca2+]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca2+]i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.

Inhibitory ryanodine prevents ryanodine receptor-mediated Ca2+ release without affecting endoplasmic reticulum Ca2+ content in primary hippocampal neurons

Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca2+ release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca2+-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca2+ release and endoplasmic reticulum Ca2+ depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca2+ release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca2+ release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca2+ levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca2+ release, preserves ER Ca2+ levels and thus constitutes a sound strategy to suppress neuronal RyR function.

Stoichiometry of CRAC Channel Assembly and Gating

CRAC channels are opened by binding of the ER calcium sensor STIM1 to the C-terminus of the channel subunit Orai1. Previous functional experiments suggested a tetrameric channel stoichiometry, but the crystal structure of Drosophila Orai is a trimer of dimers, with each C-terminus forming a coiled-coil with its neighbor. This raises two fundamental questions: what is the stoichiometry of the CRAC channel, and does STIM1 bind to individual or pairs of C-termini to open it? To address these questions, we constructed hexameric concatemers of Orai1. Orai1 hexamers produced currents with properties that were indistinguishable from native ICRAC, including Ca2+ selectivity, Ca2+-dependent inactivation, and modulation by 2-APB. The inhibitory effects of single L273D mutations confirmed that all 6 subunits participated equally in forming the functional channel. STIM1-Orai1 binding was studied using E-FRET between STIM1-YFP and CFP-Orai1. While the Orai1(L273D) C-terminus alone did not bind STIM1, it enhanced binding when paired with a neighboring WT C-terminus. To compare how monomer vs dimer binding are coupled to channel opening, we constructed hexamers with a single truncated or L273D C-terminus. The truncated hexamer showed significantly less activity than the L273D mutant, arguing against a pure monomeric gating mode. The relationship between STIM1 occupancy and channel activation was examined using Orai1 hexamers containing 1-3 STIM-binding mutations (producing 1-3 Orai1 heterodimers per channel). For both strong (L273D) and weak (L286S) inhibitory mutations, channel activity was well described by a model that assumes independent and equal energetic contributions from each heterodimer. In summary, we present the first functional evidence that hexameric Orai1 channels have the same properties as native CRAC channels. Our data suggest that STIM1 binds pairs of Orai C-termini and opens CRAC channels as a trimer of dimers, with each dimer contributing a constant amount of gating energy.

ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains

ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains