ER Ca2 Research Articles

Rectifying Attenuated Store-Operated Calcium Entry as a Therapeutic Approach for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Although the pathological hallmarks of AD have been identified, the derived therapies cannot effectively slow down or stop disease progression; hence, it is likely that other pathogenic mechanisms are involved in AD pathogenesis. Intracellular calcium (Ca2+) dyshomeostasis has been consistently observed in AD patients and numerous AD models and may emerge prior to the development of amyloid plaques and neurofibrillary tangles. Thus, intracellular Ca2+ disruptions are believed to play an important role in AD development and could serve as promising therapeutic intervention targets. One of the disrupted intracellular Ca2+ signaling pathways manifested in AD is attenuated storeoperated Ca2+ entry (SOCE). SOCE is an extracellular Ca2+ entry mechanism mainly triggered by intracellular Ca2+ store depletion. Maintaining normal SOCE function not only provides a means for the cell to replenish ER Ca2+ stores but also serves as a cellular signal that maintains normal neuronal functions, including excitability, neurogenesis, neurotransmission, synaptic plasticity, and gene expression. However, normal SOCE function is diminished in AD, resulting in disrupted neuronal spine stability and synaptic plasticity and the promotion of amyloidogenesis. Mounting evidence suggests that rectifying diminished SOCE in neurons may intervene with the progression of AD. In this review, the mechanisms of SOCE disruption and the associated pathogenic impacts on AD will be discussed. We will also highlight the potential therapeutic targets or approaches that may help ameliorate SOCE deficits for AD treatment.

D,l-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+

The search continues for improved therapy for acute lymphoblastic leukemia (aLL), the most common malignancy in children. Recently, d,l-methadone was put forth as sensitizer for aLL chemotherapy. However, the specific target of d,l-methadone in leukemic cells and the mechanism by which it induces leukemic cell apoptosis remain to be defined. Here, we demonstrate that d,l-methadone induces leukemic cell apoptosis through activation of the mu1 subtype of opioid receptors (OPRM1). d,l-Methadone evokes IP3R-mediated ER Ca2+ release that is inhibited by OPRM1 loss. In addition, the rate of Ca2+ extrusion following d,l-methadone treatment is reduced, but is accelerated by loss of OPRM1. These d,l-methadone effects cause a lethal rise in [Ca2+]i that is again inhibited by OPRM1 loss, which then prevents d,l-methadone-induced apoptosis that is associated with activation of calpain-1, truncation of Bid, cytochrome C release, and proteolysis of caspase-3/12. Chelating intracellular Ca2+ with BAPTA-AM reverses d,l-methadone-induced apoptosis, establishing a link between the rise in [Ca2+]i and d,l-methadone-induced apoptosis. Altogether, our findings point to OPRM1 as a specific target of d,l-methadone in leukemic cells, and that OPRM1 activation by d,l-methadone disrupts IP3R-mediated ER Ca2+ release and rate of Ca2+ efflux, causing a rise in [Ca2+]i that upregulates the calpain-1-Bid-cytochrome C-caspase-3/12 apoptotic pathway.

Soluble \u03b1Klotho downregulates Orai1-mediated store-operated Ca2+ entry via PI3K-dependent signaling

αKlotho is a type 1 transmembrane anti-aging protein. αKlotho-deficient mice have premature aging phenotypes and an imbalance of ion homeostasis including Ca2+ and phosphate. Soluble αKlotho is known to regulate multiple ion channels and growth factor-mediated phosphoinositide-3-kinase (PI3K) signaling. Store-operated Ca2+ entry (SOCE) mediated by pore-forming subunit Orai1 and ER Ca2+ sensor STIM1 is a ubiquitous Ca2+ influx mechanism and has been implicated in multiple diseases. However, it is currently unknown whether soluble αKlotho regulates Orai1-mediated SOCE via PI3K-dependent signaling. Among the Klotho family, αKlotho downregulates SOCE while βKlotho or γKlotho does not affect SOCE. Soluble αKlotho suppresses serum-stimulated SOCE and Ca2+ release-activated Ca2+ (CRAC) channel currents. Serum increases the cell-surface abundance of Orai1 via stimulating vesicular exocytosis of the channel. The serum-stimulated SOCE and cell-surface abundance of Orai1 are inhibited by the preincubation of αKlotho protein or PI3K inhibitors. Moreover, the inhibition of SOCE and cell-surface abundance of Orai1 by pretreatment of brefeldin A or tetanus toxin or PI3K inhibitors prevents further inhibition by αKlotho. Functionally, we further show that soluble αKlotho ameliorates serum-stimulated SOCE and cell migration in breast and lung cancer cells. These results demonstrate that soluble αKlotho downregulates SOCE by inhibiting PI3K-driven vesicular exocytosis of the Orai1 channel and contributes to the suppression of SOCE-mediated tumor cell migration.

Regulation of Store-Operated Ca <sup>2+</sup> Entry by IP <sub>3</sub> Receptors Independent of Their Ability to Release Ca <sup>2+</sup>

Loss of ER Ca2+ activates store-operated Ca2+ entry (SOCE) by causing STIM1 to unfurl domains that activate Orai1 channels in the plasma membrane at membrane contact sites (MCS). In human neurons, SOCE evoked by thapsigargin to deplete ER Ca2+ is attenuated by loss of IP3Rs, and restored by expression of IP3Rs even when they cannot releaseCa2+, but only if they can bind IP3. In cells expressing pore-dead IP3Rs, IP3 enhances SOCE evoked by partial store depletion without enhancing Ca2+ release. Proximity ligation assays establish that IP3Rs enhance association of STIM1 with Orai1 in cells with empty stores; this requires an IP3-binding site, but not a pore. Over-expressing STIM1, or extended synaptotagmin-1 to exaggerate MCS, circumvents the need for IP3Rs. Thus, IP3 binding to IP3Rs stimulates SOCE by both mediating ER Ca2+ release and promoting STIM1-Orai1 interactions. Convergent regulation by IP3Rs may tune SOCE to respond selectively to IP3.

Interactome Analysis of the ER Stress Sensor Perk Uncovers Key Components of ER-Mitochondria Contact Sites and Ca2+ Signalling.

We recently reported that the ER stress kinase PERK regulates ER-mitochondria appositions and ER- plasma membrane (ER-PM) contact sites, independent of its canonical role in the unfolded protein response. PERK regulation of ER-PM contacts was revealed by a proximity biotinylation (BioID) approach and involved a dynamic PERK-Filamin A interaction supporting the formation of ER-PM contacts by actin-cytoskeleton remodeling in response to depletion of ER-Ca2+ stores. In this report, we further interrogated the PERK BioID interactome by validating through co-IP experiments the interaction between PERK and two proteins involved in Ca2+ handling and ER-mitochondria contact sites. These included the vesicle associated membrane (VAMP)-associated proteins (VAPA/B) and the main ER Ca2+ pump sarcoplasmic/endoplasmic reticulum Ca ATPase 2 (SERCA2). These data identify new putative PERK interacting proteins with a crucial role in membrane contact sites and Ca2+ signaling further supporting the uncanonical role of PERK in Ca2+ signaling through membrane contact sites (MCSs).

Phosphodiesterase 1C integrates store-operated calcium entry and cAMP signaling in leading-edge protrusions of migrating human arterial myocytes

In addition to maintaining cellular ER Ca2+ stores, store-operated Ca2+ entry (SOCE) regulates several Ca2+-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin–mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

Emodin-Induced Oxidative Inhibition of Mitochondrial Function Assists BiP/IRE1α/CHOP Signaling-Mediated ER-Related Apoptosis.

Cassiae Semen is a widely used herbal medicine and a popular edible variety in many dietary or health beverage. Emerging evidence disclosed that improper administration of Cassiae Semen could induce obvious liver injury, which is possibly attributed to emodin, one of the bioactive anthraquinone compounds in Cassiae Semen, which caused hepatotoxicity, but the underlying mechanisms are not completely understood. Hence, the present study firstly explored the possible role of oxidative stress-mediated mitochondrial dysfunction and ER stress in emodin-cause apoptosis of L02 cells, aiming to elaborate possible toxic mechanisms involved in emodin-induced hepatotoxicity. Our results showed that emodin-induced ROS activated ER stress and the UPR via the BiP/IRE1α/CHOP signaling pathway, followed by ER Ca2+ release and cytoplasmic Ca2+ overloading. At the same time, emodin-caused redox imbalance increased mtROS while decreased MMP and mitochondrial function, resulting in the leaks of mitochondrial-related proapoptotic factors. Interestingly, blocking Ca2+ release from ER by 2-APB could inhibit emodin-induced apoptosis of L02, but the restored mitochondrial function did not reduce the apoptosis rates of emodin-treated cells. Besides, tunicamycin (TM) and doxorubicin (DOX) were used to activate ER stress and mitochondrial injury at a dosage where obvious apoptosis was not observed, respectively. We found that cotreatment with TM and DOX significantly induced apoptosis of L02 cells. Thus, all the results indicated that emodin-induced excessive ROS generation and redox imbalance promoted apoptosis, which was mainly associated with BiP/IRE1α/CHOP signaling-mediated ER stress and would be enhanced by oxidative stress-mediated mitochondrial dysfunction. Altogether, this finding has implicated that redox imbalance-mediated ER stress could be an alternative target for the treatment of Cassiae Semen or other medicine-food homologous varieties containing emodin-induced liver injury.

Characterization of novel RyR1 selective inhibitors identified by ER Ca<sup>2+</sup> measurements

Characterization of novel RyR1 selective inhibitors identified by ER Ca<sup>2+</sup> measurements

Small intestinal glucose and sodium absorption through calcium‐induced calcium release and store‐operated Ca2+ entry mechanisms

Background and PurposeLuminal glucose enhances intestinal Ca2+ absorption through apical Cav1.3 channels necessary for GLUT2‐mediated glucose absorption. As these reciprocal mechanisms are not well understood, we investigated the regulatory mechanisms of intestinal [Ca2+]cyt and SGLT1‐mediated Na+‐glucose co‐transports.Experimental ApproachGlucose absorption and channel expression were examined in mouse upper jejunal epithelium using an Ussing chamber, immunocytochemistry and Ca2+ and Na+ imaging in single intestinal epithelial cells.Key ResultsGlucose induced jejunal Isc via Na+‐glucose cotransporter 1 (SGLT1) operated more efficiently in the presence of extracellular Ca2+. A crosstalk between luminal Ca2+ entry via plasma Cav1.3 channels and the ER Ca2+ release through ryanodine receptor (RYR) activation in small intestinal epithelial cell (IEC) or Ca2+‐induced Ca2+ release (CICR) mechanism was involve in Ca2+‐mediated jejunal glucose absorption. The ER Ca2+ release through RyR triggered basolateral Ca2+ entry or store‐operated Ca2+ entry (SOCE) mechanism and the subsequent Ca2+ entry via Na+/Ca2+ exchanger 1 (NCX1) were found to be critical in Na+‐glucose cotransporter‐mediated glucose absorption. Blocking RyR, SOCE and NCX1 inhibited glucose induced [Na+]cyt and [Ca2+]cyt in single IEC and protein expression and co‐localization of STIM1/Orai1, RyR1 and NCX1 were detected in IEC and jejunal mucosa.Conclusion and ImplicationsLuminal Ca2+ influx through Cav1.3 triggers the CICR through RyR1 to deplete the ER Ca2+, which induces the basolateral STIM1/Orai1‐mediated SOCE mechanism and the subsequent Ca2+ entry via NCX1 to regulate intestinal glucose uptake via Ca2+ signalling. Targeting these mechanisms in IEC may help to modulate blood glucose and sodium in the metabolic disease.

Abstract 17415: Mitochondrial Ca2+/Calmodulin-Dependent Kinase II Inhibition Changes the Calcium Homeostasis in the Endothelium and Decreases the Vascular Relaxation in Mesenteric Arteries

Introduction: The Ca2+/Calmodulin-dependent Kinase II (CaMKII) is present in mitochondria and cytosol. In mitochondria, it regulates the mitochondrial Ca 2+ uptake via the mitochondrial Ca2+ uniporter. Since endothelial nitric oxide synthase activity is regulated by intracellular [Ca2+], we hypothesized that it affects cytosolic Ca2+, NO production and ACh-dependent vasodilation. Hypothesis: Inhibition of mitochondrial CaMKII in endothelium increases the cytosolic [Ca2+], and decreases vasorelaxation by Acetylcholine. Methods: CaMKII in mitochondria was inhibited through expression of the mitochondria-targeted CaMKII inhibitor peptide (mito-CaMKIIN) in a novel transgenic mouse model (endo-mtCaMKIIN) in endothelial cells only or delivered by adenoviral transduction (Ad-mtCaMKIIN) in human Aortic Endothelium cells (HAEC). In HAEC, cytosolic Ca2+ levels (by FURA-2 AM), eNOS activation and NOx levels were measured. Results: The basal Ca2+ levels were higher in the cytosol of mitoCaMKIIN cells (1.08 ± 0.02 Fura-2 ratio normalized by control, p<0.05). Thapsigargin-induced ER Ca 2+ release was significantly higher with mitoCaMKIIN (AUC 0.252 ± 0.027 versus 0.112 ± 0.01275, p<0.05), whereas cytosolic Ca 2+ levels after ACh were reduced (AUC 0.191 ± 0.025 versus 0.435 ± 0.054). Higher levels of phosphorylation of eNOS at Ser1177 and Thr495 sites were seen at baseline. The concentration-response curve of vascular relaxation to acetylcholine and SNP shifted to the right (p<0.05) in mesenteric resistance artery of mitoCaMKIIN mice. Conclusions: The inhibition of mitochondrial CaMKII in the endothelium increases the cytosolic levels, endoplasmic reticulum storage of calcium and eNOS phosphorylation. However, there are lower calcium release and lower sensitivity to acetylcholine and SNP.

STIM1 a calcium sensor promotes the assembly of an ECM that contains Extracellular vesicles and factors that modulate mineralization

Osteoblasts and odontoblasts, are non-excitable cells and facilitate mass calcium transport during matrix mineralization. A sophisticated Ca2+ sensing mechanism is used to maintain Ca2+ homeostasis. STIM1 (Stromal interaction molecule 1) is a calcium sensor protein localized in the ER membrane and maintains calcium homeostasis by initiating the store-operated Ca2+ entry (SOCE) process, following store depletion. The role of STIM1 in dentin mineralization is yet to be elucidated. Therefore, transgenic DPSCs were generated in which overexpression or knockdown of STIM1 was achieved to study its function in matrix mineralization. Gene expression analysis and Alizarin Red staining assay demonstrated upregulation of genes involved in odontogenic differentiation and matrix mineralization with increased calcium deposition with STIM1 overexpression. Topology of the ECM examined by Field Emission Scanning Electron Microscopy (FESEM) showed the presence of large amounts of extracellular microvesicles with mineral deposits. Interestingly, silencing STIM1 resulted in fewer vesicles and less mineral deposits in the ECM. Analysis of the dentin-pulp complex of STIM1- deficient mice by micro-CT show reduced dentin thickness, malformed and highly porous alveolar bone, suggesting a cell intrinsic role for STIM1 in dentin mineralization. Confocal microscopy showed that DMP1-mediated depletion of store Ca2+ resulted in aggregation or “puncta-formation” of STIM1 at the plasma membrane indicative of a gating arrangement with Orai1 for Ca2+ influx. Together, our data provide evidence for an important role for STIM1 in dentin and alveolar bone mineralization by influencing intracellular Ca2+ oscillations that could provide signals for a wide array of cellular functions. Statement of SignificanceCalcium signaling and transport are fundamental to bone and dentin mineralization. Osteoblasts and odontoblasts transport large amounts of Ca2+ to the extracellular matrix. These cells maintain calcium homeostasis by spatially distributed calcium pumps and channels at the plasma membrane. STIM1 an ER Ca2+ sensor protein is an important component of the store-operated calcium entry (SOCE) process. In this study, we examined the role of STIM1 during the differentiation of dental pulp stem cells into functional odontoblasts and formation of mineralized dentin matrix. Stimulation of these cells with DMP1, a key regulatory protein in matrix mineralization, stimulates STIM1-mediated release of ER Ca2+ and SOCE activation. Silencing of STIM1 impairs signaling events, release of exosomes containing matrix proteins and matrix mineralization.

Differential modulation of short-term plasticity at hippocampal mossy fiber and Schaffer collateral synapses by mitochondrial Ca2.

Presynaptic mitochondrial Ca2+ plays a critical role in the regulation of synaptic transmission and plasticity. The presynaptic bouton of the hippocampal mossy fiber (MF) is much larger in size than that of the Schaffer collateral (SC) synapse. Here we compare the structural and physiological characteristics of MF and SC presynaptic boutons to reveal functional and mechanistic differences between these two synapses. Our quantitative ultrastructural analysis using electron microscopy show many more mitochondria in MF presynaptic bouton cross-section profiles compared to SC boutons. Consistent with these results, post-tetanic potentiation (PTP), a form of presynaptic short-term plasticity dependent on mitochondrial Ca2+, is reduced by inhibition of mitochondrial Ca2+ release at MF synapses but not at SC synapses. However, blockade of mitochondrial Ca2+ release results in reduction of PTP at SC synapses by disynaptic MF stimulation. Furthermore, inhibition of mitochondrial Ca2+ release selectively decreases frequency facilitation evoked by short trains of presynaptic stimulation at MF synapses, while having no effect at SC synapses. Moreover, depletion of ER Ca2+ stores leads to reduction of PTP at MF synapses, but PTP is unaffected by ER Ca2+ depletion at SC synapses. These findings show that MF and SC synapses differ in presynaptic mitochondrial content as well as mitochondrial Ca2+ dependent synaptic plasticity, highlighting differential regulatory mechanisms of presynaptic plasticity at MF and SC synapses.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.

Dimerization of SERCA2a Enhances Transport Rate and Improves Energetic Efficiency in Living Cells

The type 2a sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA2a) plays a key role in intracellular Ca2+ regulation in the heart. We have previously shown evidence of stable homodimers of SERCA2a in heterologous cells and cardiomyocytes. However, the functional significance of the pump dimerization remains unclear. Here, we analyzed how SERCA2a dimerization affects ER Ca2+ transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca2+ pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca2+ uptake was measured with the ER-targeted Ca2+ sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca2+ uptake rate and the maximal Ca2+ load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca2+ uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca2+ load also increased with an increase in the pump expression level, suggesting improved catalytic efficiency of the dimeric species. Reciprocally, thapsigargin inhibition of a fraction of the population of SERCA2a reduced not only the maximal ER Ca2+ uptake rate but also the maximal Ca2+ load. These data suggest that SERCA2a dimerization regulates Ca2+ transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca2+ uptake in cells cotransfected with human wild-type SERCA2a (SERCA2aWT) and SERCA2a mutants with different catalytic activity revealed that an intact catalytic cycle in both protomers is required for enhancing the efficacy of Ca2+ transport by a dimer. The data are consistent with the hypothesis of functional coupling of two SERCA2a protomers in a dimer that reduces the energy barrier of rate-limiting steps of the catalytic cycle of Ca2+ transport.

NAADP‐regulated two‐pore channels drive phagocytosis through endo‐lysosomal Ca2+ nanodomains, calcineurin and dynamin

Macrophages clear pathogens by phagocytosis and lysosomes that fuse with phagosomes are traditionally regarded as to a source of membranes and luminal degradative enzymes. Here, we reveal that endo‐lysosomes act as platforms for a new phagocytic signalling pathway in which FcγR activation recruits the second messenger NAADP and thereby promotes the opening of Ca2+‐permeable two‐pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo‐lysosomal Ca2+ nanodomains rather than global cytoplasmic or ER Ca2+ signals. Motile endolysosomes contact nascent phagosomes to promote phagocytosis, whereas endo‐lysosome immobilization prevents it. We show that TPC‐released Ca2+ rapidly activates calcineurin, which in turn dephosphorylates and activates the GTPase dynamin‐2. Finally, we find that different endo‐lysosomal Ca2+ channels play diverse roles, with TPCs providing a universal phagocytic signal for a wide range of particles and TRPML1 being only required for phagocytosis of large targets.

Klotho rewires cellular metabolism of breast cancer cells through alteration of calcium shuttling and mitochondrial activity.

Klotho is a transmembrane protein, which can be shed and act as a circulating hormone and is involved in regulating cellular calcium levels and inhibition of the PI3K/AKT pathway. As a longevity hormone, it protects normal cells from oxidative stress, and as a tumor suppressor it inhibits growth of cancer cells. Mechanisms governing these differential activities have not been addressed. Altered cellular metabolism is a hallmark of cancer and dysregulation of mitochondrial activity is a hallmark of aging. We hypothesized that klotho exerts its differential effects through regulation of these two hallmarks. Treatment with klotho inhibited glycolysis, reduced mitochondrial activity and membrane potential only in cancer cells. Accordingly, global metabolic screen revealed that klotho altered pivotal metabolic pathways, amongst them glycolysis and tricarboxylic acid cycle in breast cancer cells. Alteration of metabolic activity and increased AMP/ATP ratio lead to LKB1-dependent AMPK activation. Indeed, klotho induced AMPK phosphorylation; furthermore, inhibition of LKB1 partially abolished klotho's tumor suppressor activity. By diminishing deltapsi (Δψ) klotho also inhibited mitochondria Ca2+ shuttling thereby impairing mitochondria communication with SOCE leading to reduced Ca2+ influx by SOCE channels. The reduced SOCE was followed by ER Ca2+ depletion and stress. These data delineate mechanisms mediating the differential effects of klotho toward cancer versus normal cells, and indicate klotho as a potent regulator of metabolic activity.

IP3 mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release.

The 'building-block' model of inositol trisphosphate (IP3)-mediated Ca2+ liberation posits that cell-wide cytosolic Ca2+ signals arise through coordinated activation of localized Ca2+ puffs generated by stationary clusters of IP3 receptors (IP3Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca2+ signaling. Puffs make only small, transient contributions to global Ca2+ signals, which are sustained by diffuse release of Ca2+ through a functionally distinct process. These two modes of IP3-mediated Ca2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca2+-dependent effector functions and Ca2+ transport.

Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels.

ANO1 (TMEM16A) is a Ca2+-activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca2+-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca2+ dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca2+ stores reduced capsaicin-induced Ca2+ increases and CaCC activation, suggesting that ER Ca2+ release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane-localized TRPV1 channels was demonstrated with an ER-localized Ca2+ sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1, and the IP3 receptor IP3R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multichannel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca2+ release, which may be necessary to enhance ANO1 activation.

Loss‐ And Gain of Function Mutations in the Type 1 IP3 Receptor (IP3R1) Modify Agonist‐Evoked Vasoactive Responses in Cerebral and Skeletal Muscle Resistance Arteries From Adult Mice

Calcium mobilization within vascular smooth muscle and endothelial cells of small resistance arteries plays a critical role in agonist‐evoked vasoconstriction and dilation, respectively. Despite the recognized contribution of IP3‐mediated intracellular Ca2+ release for agonist‐evoked vasoactive responses, it has been difficult to study this process acutely in native arteries, due to the lack of selective pharmacological agents or experimental strategies to target the predominant IP3R1. In humans, heterozygous mutations in the IP3R1 are known to produce either loss‐of‐function (LOF) or gain‐of‐function phenotypes for Ca2+ release that impact cerebellar activity (e.g. ataxia), yet it remains unclear how these global mutations affect the cardiovascular system. To facilitate such investigation, we have engineered novel genetic knock‐in mice harbouring a single amino acid mutation at position Asp2594 in the transmembrane S6 helix of the endogenous IP3R1 Ca2+ release channel that confers either a LOF (Asp2594Ala) or GOF (Asp2594Lys) phenotype, respectively. In permeabilized HEK293 cells expressing these mutant receptors, the LOF mutation shifted IP3‐mediated ER Ca2+ release ~2‐fold to the right, whereas the GOF mutation produced a 2.3‐fold leftward shift compared with wild‐type (WT) IP3R1. In endothelium‐denuded cerebral arteries pressurized to 10 mmHg, the sensitivity of serotonin‐evoked vasoconstriction was reduced in vessels from heterozygous LOF mice compared with WT, whereas agonist responsiveness was enhanced in arteries from heterozygous GOF mice. Vasoconstriction in response to the thromboxane receptor agonist U46619 was modestly enhanced by the GOF mutation, but largely unaffected in arteries from LOF mice. In cannulated cremaster skeletal muscle arteries from LOF and GOF mice pressurized to 70 mmHg, the sensitivity of endothelium‐dependent, acetylcholine‐mediated inhibition of myogenic tone was right shifted (3.7‐fold) and left shifted (2‐fold), respectively, compared with vessels from WT. Comparable changes in functional sensitivity were observed in response to the endothelium‐dependent vasodilator bradykinin. In contrast, dilation evoked by the NO donor sodium nitroprusside was not different among the three genotypes. Collectively, these data demonstrate that genetic modification of IP3R1‐mediated Ca2+ mobilization in small resistance arteries functionally impacts agonist‐evoked vasoactive responses. These observations highlight the importance of IP3R1 activity in the vascular wall, and further implicate vascular impairment as a contributing factor to the neurological dysfunction observed in patients with LOF and GOF IP3R1 mutations.Support or Funding InformationThis work was funded by the following research grants from the Canadian Institutes of Health Research: MOP‐97988 (WCC), MOP‐142467 (APB) and PJT‐155963 (WC, WCC and APB).

Loss‐ and Gain‐of‐Function Mutations in the Type 1 IP3 Receptor (IP3R1) Affect Myogenic Constriction of Cerebral Resistance Arteries in Response to Intraluminal Pressure

The constriction and relaxation of resistance arteries/arterioles in response to changes in intraluminal pressure, referred to as the myogenic response, is a key physiological mechanism for the regulation of blood pressure and flow. Despite the recognized contribution of phospholipase C activity and IP3‐mediated intracellular Ca2+ release to the myogenic response, a lack of selective pharmacological agents to target the predominant IP3R1 Ca2+ release channel has prevented interrogation of this key mechanism. Mutations in the IP3R1 are known to produce either loss‐of‐function (LOF) or gain‐of‐function (GOF) phenotypes in humans that impact cerebellar activity (e.g. ataxia), yet the effect of these mutations on the cardiovascular system is not established. Here, we employed a novel genetic knock‐in mouse model with a single point mutation in the transmembrane S6 helix of IP3R1 that confers either a LOF (D2594A) or GOF (D2594K) phenotype. In preliminary experiments, the LOF and GOF mutations shifted IP3‐mediated ER Ca2+ release ~2‐fold to the right and left, respectively, compared to wild‐type (WT) IP3R1 in permeabilized HEK293 cells. In endothelium‐denuded posterior cerebral arteries of 3–4 month old WT mice, myogenic vasoconstriction was evident at ≥40 mmHg, maximal at ~120 mmHg, and declined at >120 mmHg (n=7). In contrast, myogenic constriction was initiated at a higher pressure (≥50 mmHg) and was smaller in amplitude in arteries of heterozygous IP3R1 LOF mice (n=8). In IP3R1 GOF vessels (n=7), myogenic tone developed at a lower pressure (≥20 mmHg), was maximal at ~80 mmHg, and declined at >90 mmHg. Relative levels of phosphorylation of myosin regulatory light chain (LC20) and myosin phosphatase targeting subunit (MYPT1) at 10, 40, 80 and 120 mmHg, and of globular (G‐) actin content at 10, 60 and 120 mmHg, in arteries from WT, LOF and GOF mice were assessed by ultra‐high sensitivity immunoblotting. An identical pressure‐dependent increase in phospho‐LC20 content was detected in WT, LOF and GOF vessels (n=5 each). Although phospho‐MYPT1‐T855 content increased with intraluminal pressure in arteries of WT and LOF mice, it did not change in vessels of GOF mice. Elevation of intraluminal pressure decreased in G‐actin content in arteries of WT mice, did not affect G‐actin content in LOF vessels, and caused a significantly greater decrease in GOF arteries. Taken together, these data demonstrate that genetic modification of IP3R1‐mediated Ca2+ mobilization affects the myogenic response of cerebral arteries to intraluminal pressure, a vascular impairment that may contribute to neurological dysfunction in patients with LOF and GOF IP3R1 mutations.Support or Funding InformationThis work was funded by the following research grants from the Canadian Institutes of Health Research: MOP‐97988 (WCC) MOP‐142467 (APB) and PJT‐155963 (APB, WC and WCC).