Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca²⁺ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca²⁺ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry from the extracellular space. Ca²⁺ release was not affected by treatment with ST, but Ca²⁺ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca²⁺ sequestration into Ca²⁺ stores. The Ca²⁺ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca²⁺ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca²⁺ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La³⁺ and 2-APB. The large increase in Ca²⁺ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca²⁺ stores. These results show that SOCE remains activated even after the refilling of Ca²⁺ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.     

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

2-Methoxyestradiol and its derivatives inhibit store-operated Ca2+ entry in T cells: Identification of a new and potent inhibitor

T cell activation starts with formation of second messengers that release Ca²⁺ from the endoplasmic reticulum (ER) and thereby activate store-operated Ca²⁺ entry (SOCE), one of the essential signals for T cell activation. Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca²⁺ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca²⁺ entry via SOCE without affecting other ion channels and pumps involved in Ca²⁺ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.     

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca²⁺ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca²⁺ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca²⁺ levels, respectively. Histamine release and Ca²⁺ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca²⁺ increase. FcεRI-induced Ca²⁺ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca²⁺ release was both necessary and sufficient for mast cell degranulation, extracellular Ca²⁺ influx enhanced the process. Furthermore, cytosolic Ca²⁺ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca²⁺ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca²⁺ signaling in regulating histamine degranulation.     

Ca2+ signaling induced by sphingosine 1-phosphate and lysophosphatidic acid in mouse B cells

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca²⁺] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca²⁺]_c in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca²⁺ entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca²⁺]_c increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca²⁺]_c or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca²⁺]_c. Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca²⁺ was measured using Ba²⁺ as a surrogate ion. Similarly, Ba²⁺ influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca²⁺-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca²⁺ release was insignificant in primary B cells and inWEHI-231.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

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.     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca²⁺ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca²⁺ deregulation and the Ca²⁺ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca²⁺ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca²⁺ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca²⁺ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca²⁺ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca²⁺ store contributes to the disrupted Ca²⁺ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.     

Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca²⁺ signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca²⁺ signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca²⁺ and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H₂O₂ induces an initial Ca²⁺ increase, which in prostate cancer cells is blocked at high concentrations of H₂O₂. Upon depletion of intracellular Ca²⁺ stores, store-operated Ca²⁺ entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H₂O₂ compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector T_H cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca²⁺ channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca²⁺ signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.     

Role of STIM1/ORAI1-mediated store-operated Ca2+ entry in skeletal muscle physiology and disease

Store-operated Ca²⁺ entry (SOCE) is a Ca²⁺ entry mechanism activated by depletion of intracellular Ca²⁺ stores. In skeletal muscle, SOCE is mediated by an interaction between stromal-interacting molecule-1 (STIM1), the Ca²⁺ sensor of the sarcoplasmic reticulum, and ORAI1, the Ca²⁺-release-activated-Ca²⁺ (CRAC) channel located in the transverse tubule membrane. This review focuses on the molecular mechanisms and physiological role of SOCE in skeletal muscle, as well as how alterations in STIM1/ORAI1-mediated SOCE contribute to muscle disease. Recent evidence indicates that SOCE plays an important role in both muscle development/growth and fatigue. The importance of SOCE in muscle is further underscored by the discovery that loss- and gain-of-function mutations in STIM1 and ORAI1 result in an eclectic array of disorders with clinical myopathy as central defining component. Despite differences in clinical phenotype, all STIM1/ORAI1 gain-of-function mutations-linked myopathies are characterized by the abnormal accumulation of intracellular membranes, known as tubular aggregates. Finally, dysfunctional STIM1/ORAI1-mediated SOCE also contributes to the pathogenesis of muscular dystrophy, malignant hyperthermia, and sarcopenia. The picture to emerge is that tight regulation of STIM1/ORAI1-dependent Ca²⁺ signaling is critical for optimal skeletal muscle development/function such that either aberrant increases or decreases in SOCE activity result in muscle dysfunction.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.     

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle

Skeletal muscle fibres support store-operated Ca²⁺-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca²⁺ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca²⁺]_SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca²⁺-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction.Here we show that phasic SOCE is not only shaped by global [Ca²⁺]_SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca²⁺]_SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca²⁺ release. We derive a delay of 0.3 ms reflecting diffusive Ca²⁺-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca²⁺-release. Numerical simulations of Ca²⁺-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca²⁺-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca²⁺-STIM1 binding suggests a Ca²⁺ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only μs delays, because [Ca²⁺] in the terminal cisternae is just above the threshold for Ca²⁺ dissociation from STIM1 under physiological resting conditions.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

The Calmodulin Regulator Protein,PEP-19, Sensitizes ATP-induced Ca2+ Release

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.     

Interleukin-13 enhanced Ca2+ oscillations in airway smooth muscle cells

Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca²⁺ signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca²⁺ oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs.LTD4-induced Ca²⁺ oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca²⁺ entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca²⁺ oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP₃R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca²⁺ oscillations. Ca²⁺ oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP₃R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3.We conclude that IL-13 enhances frequency of LTD4-induced Ca²⁺ oscillations in human ASMCs, which may be cooperatively modulated by IP₃R, RyR systems and possibly by SOCE.