KIT D816 mutations (KIT D816mut) are strongly associated with systemic mastocytosis (SM) but are also detectable in acute myeloid leukemia (AML), where they represent an adverse prognostic factor in combination with core binding factor (CBF) fusion genes. Here, we evaluated the clinical and molecular features of KIT D816mut/CBF-negative (CBFneg) AML, a previously uncharacterized combination. All KIT D816mut/CBFneg cases (n = 40) had histologically proven SM with associated AML (SM-AML). Molecular analyses revealed at least one additional somatic mutation  beside KIT D816  in 32/32 (100%) patients. Secondary AML evolved in 29/40 (73%) patients from SM ± associated myeloid neoplasm. Longitudinal molecular and cytogenetic analyses revealed the acquisition of new mutations and/or karyotype evolution in 15/16 (94%) patients at the time of SM-AML. Median overall survival (OS) was 5.4 months. A screen of two independent AML databases (AMLdatabases) revealed remarkable similarities between KIT D816mut/CBFneg SM-AML and KIT D816mut/CBFneg AMLdatabases (n = 69) with regard to KIT D816mut variant allele frequency, mutation profile, aberrant karyotype, and OS suggesting underlying SM in a significant proportion of AMLdatabases patients. Bone marrow histology and reclassification as SM-AML has important clinical implications regarding prognosis and potential inclusion of KIT inhibitors in treatment concepts. According to the World Health Organization (WHO) classification, advanced systemic mastocytosis (advSM) comprises aggressive SM, SM with an associated hematologic neoplasm (SM-AHN), and mast cell leukemia –3. SM-AHKIT  are detectable in >90% of patients with SM, acknowledged to be most relevant for disease pathogenesis , mitoxantrone/cytarabine [S-HAM]) ± non-intensive therapy . The complete response (CR) rates after intensive induction chemotherapy and non-intensive therapy were 40% and 0, respectively. Two patients had cytarabine-based consolidation  and are alive 91 and 15 months, retrospectively, after diagnosis of SM-AML. Allogeneic SCT was performed in 12/40 (30%) patients with 4 patients being in CR prior to allogeneic SCT. A durable CR was achieved by 6/12 patients (50%). Nine of 40 (22%) patients received only best supportive care due to advanced age ± comorbidity.Thirty-one of 40 (78%) patients were treated with intensive (induction) chemotherapy  with 10/11 (91%) non-responders presenting with S/A/Rpos ± poor-risk karyotype. On the other hand, 4/8 (50%) responders were S/A/Rpos ± poor-risk karyotype (P = 0.04) indicating that intensive treatment should not be withheld in this subgroup.S/A/RKIT D816mut/CBFneg AML represents a distinct subtype which is associated with SM and poor prognosis, two independent AML databases (AMLdatabases) were retrospectively screened for KIT D816mut/CBFneg AML patients. Overall, 69 KIT D816mut/CBFneg AMLdatabases patients could be identified. Mutation profile and karyotype were available from all patients, detailed clinical characteristics from 17/69 patients  and 29% (range 3–93), respectively, (b) with the exception of SRSF2 (38% vs. 18%), the frequency of the most frequently somatic mutations  was highly similar between the two groups, (c) in contrast to de novo AML, the frequency of FLT3 aberrations was very low , and (d) the frequency of an aberrant karyotype was 52% and 42, respectively, with a comparable rate of intermediate-risk and poor-risk karyotype.This comparison revealed remarkable molecular and karyotype similarities between the ort Fig. : (a) TheKIT D816mut/CBFneg SM-AML and 17 evaluable KIT D816mut/CBFneg AMLdatabases patients was 5.4  and 26.4 (95% CI [0–61.0]) months (P = 0.015), respectively. In the KIT D816mut/CBFneg SM-AML cohort, 16 patients received non-intensive therapy only, with a median OS of 2.7 months (95% CI [1.5–3.9]), while all 17 KIT D816mut/CBFneg AMLdatabases patients received intensive chemotherapy. Median OS was not significantly different  between KIT D816mut/CBFneg SM-AML and KIT D816mut/CBFneg AMLdatabases patients who received intensive chemotherapy  only vs. intensive chemotherapy followed by allogeneic SCT (n = 19), 10.2 months (95% CI [5.9–14.4]) vs. 26.5 months (95% CI [0–58.5]), respectively (P = 0.3)  Fig. . With ex CI 5.9–1.4) vs. 2pos) and a poor-risk karyotype were identified as poor prognostic variables regarding OS. In multivariate analysis, S/A/Rpos and a poor-risk karyotype remained the only independent adverse factors with regard to OS. Accordingly, a weighted score (based on the HR) of 1 was assigned to S/A/Rpos and poor-risk karyotype. Significantly different OS probabilities were observed for the comparisons S/A/Rneg + normal-/intermediate-risk karyotype , S/A/Rpos or poor-risk karyotype , and S/A/Rpos + poor-risk karyotype  with median OS not reached vs. 14.0 [6.2–21.8] vs. 7.0 months [4.5–9.6] (P = 0.001). These results were independent of treatment modalities , only age >60 years, at least one additional somatic mutation in the S/A/R gene panel , the poor median OS of 26 months in 17 KIT D816mut/CBFneg AML patients from the two independent AML databases adds further evidence that KIT D816mut/CBFneg AML may in fact represent SM-AML in the vast majority, if not all patients. Independently of treatment modalities and consistent with previous reports on other advSM subtypes, e.g., mast cell leukemia, mutations in S/A/R and a poor-risk karyotype conferred an adverse impact on response to treatment, disease progression, and OS [The median OS of the 40 SM-AML patients was 5.4 months and thus even worse as compared to patients with mast cell leukemia, which is defined by the presence of ≥20% mast cells in a bone marrow smear . No pati, and OS , 29, 30.FLT3pos AML and achieves overall response rates of 60% in patients with advSM [KIT D816V VAF at month 6 [KIT D816mut/CBFneg AML by bone marrow histology and elevated serum tryptase, KIT inhibitors  in combination with intensive chemotherapy and allogeneic SCT may help to improve the poor prognosis of this distinct AML subtype [Midostaurin, an orally administered multi-kinase/FLT3-/KIT-inhibitor improves survival in th advSM , 47. Bet month 6 , 46–49.  subtype , 51.KIT D816mut SM-AHN is frequently observed and may be triggered by the acquisition of additional somatic mutations with or without karyotype evolution, (b) KIT D816mut/CBFneg AML is a distinct subtype with remarkable similarities compared to SM-AML cases concerning KIT D816 VAF mutation profile, aberrant karyotype, and poor prognosis, suggesting that a significant proportion of these AML patients may in fact have SM-AML, which is a strong argument to propose a new evaluation, (c) with its very high positive and negative predictive value, serum tryptase is an excellent screening marker for SM and should therefore be part of the diagnostic workflow in all AML patients. Cases with an elevated serum tryptase level should subsequently be screened for KIT D816mut, and (d) bone marrow histology is mandatory in KIT D816mut patients. This simple diagnostic procedure will allow reclassification to SM-AML and thus allow inclusion of KIT inhibitors in established treatment modalities of AML.We conclude that (a) progression to secondary AML from a preceding Supplementary Material


Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H2O2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H2O2 on Ca2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca2+ signals and acetylcholine evoked Ca2+ increases were inhibited by H2O2. H2O2 inhibition of acetylcholine-evoked Ca2+ signals was reversed by catalase. H2O2 exerts its inhibition on the IP3 receptor as Ca2+ release evoked by photolysis of caged IP3 was supressed by H2O2. H2O2 suppression of IP3-evoked Ca2+ signalling may be mediated by mitochondria. H2O2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP3-evoked Ca2+ release by H2O2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca2+ overload during IP3-linked Ca2+ signals by a H2O2 mediated negative feedback depolarization of the organelle and inhibition of IP3-evoked Ca2+ release.Hydrogen peroxide (H These vasoactive factors allow the endothelium to regulate almost all cardiovascular activities including vascular tone, immune responses, angiogenesis and vascular remodelling . Strathclyde BPU is a conventional unit which undertakes FELASA quarterly heath monitoring. Male Sprague-Dawley rats (10–12 weeks old), from an in-house colony, were used in the study. Animals were housed 3 per cage , provided with enrichment (aspen wood chew sticks and hanging huts), nesting material , and fresh water and chow  were available 2.2en face preparation). The endothelium was then loaded with acetoxymethyl ester form of the Ca2+ indicator, Cal-520 (5 μM) and 0.02% pluronic F-127 in DMSO, for 30 min at 37 °C  that results in Ca2+oscillations occurring at basal [IP3] in unstimulated cells [3may be a general mechanism responsible for the action of other oxidants, it has also been suggested that they (oxidants) may alter the sensitivity of IP3R to Ca2+ [2O2 on endothelial mitochondria may dominate control of IP3R. Since H2O2 may increase IP3R activity, these findings buttress the proposal that inhibition of Ca2+ release by H2O2 is indirect.While the present results suggest that Hto occur . In suppal store ,37,77. Vimerosal ,16,42, timerosal , and diaimerosal ,46 each ed cells ,40. Alth to Ca2+ ,45. Our 2O2 regulates several key modulators of cell activity including cell proliferation, migration, and differentiation, and because H2O2 is membrane permeable, the ROS may exert widespread control across many endothelial cells to regulate cardiovascular function. H2O2 mediates at least part of its effects through changes in Ca2+ signalling. Ca2+ influx in native and in cultured endothelial cell lines is evoked by H2O2. In the native endothelium of superior epigastric arteries, H2O2 evoked Ca2+ increases result from Ca2+ influx via TRPV4 channels [2O2 evokes Ca2+ influx via TRPV4 or TRPM2 [2O2. H2O2 may inhibit IP3-evoked Ca2+ release in native endothelial cells. The inhibition of IP3 evoked Ca2+ by H2O2 may be an indirect and mediated via depolarization of the mitochondrial membrane potential. This process may serve as a negative feedback modulation of mitochondrial function. Ca2+ increases associated with cell activity may initially stimulate mitochondrial ATP production. However, an increase in electron transport activity will result in increased ROS production, with a decrease in mitochondrial membrane potential and inhibition of IP3-evoked Ca2+ release as a result. H2O2 mediated mitochondrial depolarization may be a mechanism by which the ogranelles inhibit IP3-evoked Ca2+ signalling to protect themselves against Ca2+ overload .Hchannels . In mousor TRPM2 ,71. Our XZ, MDL,CW & JGM developed the concept. XZ performed the experiments. JGM & XZ drafted the manuscript. JGM, ZX, CW & MDL edited and revised the manuscript. CW & JGM sourced funding. All authors approved the final version of the manuscript.None.


The present study tests the hypothesis that Ca2+ oscillations play a vital role in maintaining the stemness of liver cancer stem cells (CSCs), which are postulated to be responsible for cancer initiation and progression. We found that niche factor-stimulated Ca2+ oscillation is a signature feature of CSC-enriched Hep-12 cells and purified α2δ1+ CSC fractions from hepatocellular carcinoma cell lines. In Hep-12 cells, the Ca2+ oscillation frequency positively correlated with the self-renewal potential. Using a newly developed high signal, endoplasmic reticulum (ER) localized Ca2+ sensor GCaMP-ER2, we demonstrated CSC-distinctive oscillatory ER Ca2+ release controlled by the type 2 inositol 1,4,5-trisphosphate receptor (IP3R2). Knockdown of IP3R2 severely suppressed the self-renewal capacity of liver CSCs. We propose that targeting the IP3R2-mediated Ca2+ oscillation in CSCs might afford a novel, physiologically inspired anti-tumor strategy for liver cancer.Ca Recent studies have shown that dysregulation of Ca2+ homeostasis emerges as an important hallmark of tumor cells because remodeling of Ca2+ channels and transporters is common to tumor progression2 and Ca2+ oscillation orchestrates invadopodium formation and invasion during cancer metastasis3.Ca2+/calmodulin-dependent protein kinase IIγ prompts the stem-like properties of lung cancer cells4, while calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, represses keratinocyte CSC potential5. Recently, we have shown that spontaneous Ca2+ oscillation occurs in the CSC-enriched liver cell line Hep-126, and modulates the efficiency of self-renewal7. Ca2+ oscillation decodes the stimulations not only by its amplitude, but also by its frequency and duration, thereby expanding the capabilities of the signaling pathway10.Cancer stem cells (CSCs) are thought to be the origin of cancer initiation and progression. It has been reported that Ca2+ store. However, investigation of ER Ca2+ signaling has been limited by a lack of ER-localized, high-signal sensors that can report store Ca2+ dynamics in real time. Circularly permutated EGFP variants fused with calmodulin and M13 motif, termed GCaMPs or G-GECOs are robust Ca2+ sensors that have undergone progressive improvements15, and for which the structural basis of Ca2+-dependent fluorescence has been determined16. While GCaMPs have been optimized for the detection of rapid cytosolic Ca2+ signaling, there has been limited success in creating variants with high dynamic range within the micromolar Ca2+ endoplasmic/sarcoplasmic environment. Recent progress has been made in this area using new classes of fluorescent protein sensors18, or GCaMP variants with mutated calmodulin moieties19. However, the brightness and dynamic range of these proteins, have been limited.The endoplasmic reticulum (ER), which extends over the entire cytoplasm as an elaborated nanotubular network, acts as the most important intracellular Ca2+ oscillation might play a vital role in maintaining the stemness of CSCs. We investigated the Ca2+ phenotypes, their subcellular and molecular mechanisms, their significance in cancer biology and potential strategies for intervening CSCs through manipulating Ca2+ signaling in CSC-enriched Hep-12 cells. In addition, we developed GCaMP-ER2, a novel ER localized Ca2+ sensor, to probe the store Ca2+ dynamics simultaneously. Our comprehensive approaches allow us to propose that targeting the IP3R2-mediated Ca2+ oscillation in CSCs might afford a novel, physiologically inspired anti-tumor strategy for liver cancer.In the present study, we aimed to test the hypothesis that Ca2+ oscillation is a hallmark of CSCs, we applied a panel of potential niche factors, including ATP, epidermal growth factor (EGF)/basic fibroblast growth factor (FGFb)/B27, and interleukin (IL)-6, and assessed the Ca2+ response in the liver CSC line Hep-12 and its matching hepatocellular cancer (HCC) line Hep-11. ATP is frequently released from cells to facilitate metastasis20. EGF is a well-accepted niche factor and EGF/FGFb/B27 are commonly used in media for in vitro spheroid formation assays, the most popular end-point experiment for self-renewal determination in solid tumors22. IL-6 has been reported to elevate or maintain the self-renewal of liver CSCs23. Besides these, we opted to include methacholine, a muscarinic receptor agonist capable of inducing dynamic Ca2+ changes in many types of cells24. All of these stimulants are linked to the inositol 1,4,5-trisphosphate (IP3) signaling pathway and thereby trigger intracellular Ca2+ release from the ER via IP3 receptors (IP3Rs)27.To test the hypothesis that Ca2+ transient that lasted several minutes in Hep-12 cells. Remarkably, Ca2+ oscillations of spiky appearance overlaid the evoked Ca2+ transient, and often continued even in the tail of the transient, when the cytosolic Ca2+ level returned to baseline , or exhibited only a smooth, monophasic Ca2+ transient in response to ATP . Briefly, we screened a series of random and structure–based mutations in the four calmodulin EF-hand loops of G-GECO1.213, and identified a D129A mutant within the EF-hand of loop IV , and a Kd for Ca2+ of 106 μM. This variant was systematically mutated in EF-hand loops, resulting in an array of molecules with Ca2+ affinities ranging from 106 μM to 5.9 mM (Table SFmax/F0) of 40, a marked improvement over the previously highest signal strength ER reporter, GCaMPer/GCaMP3 (10.19)19 (∆Fmax/F0 = 14). As the Kd of GCaMP-L2 (388 μM) is well below the resting level of ER Ca2+, the indicator would be expected to operate near maximum brightness prior to ER Ca2+ release events.To measure ER Ca6 linker between CaM and KDEL improved GCaMP-L2 brightness in HeLa cells, resulting in GCaMP-ER2 ER2 Fig. , which e 2+ Fig. . Figure 2+ oscillation is an emergent property of the entire cellular Ca2+-handling system. To investigate the cellular mechanism underlying the distinctive Ca2+ response of CSCs, we measured the resting cytosolic Ca2+ concentration ([Ca2+]c) and the capacity of the releasable ER Ca2+ store. The latter was indexed as the peak cytosolic Ca2+ concentration during release stimulated by the Ca2+ ionophore A23187 (10 μM) ([Ca2+]release)      Fig. . We founlls Fig. , excludiol Fig. S. In Hep-ent Fig. . Subsequ77) Fig.  even thovel Fig. . This re34) Fig. , and str16) Fig. . The pea2) Fig. S. Close oion Fig.  inset. Tor Fig. S. Taken t2+ entry (SOCE), the main Ca2+ entry pathway in nonexcitable cells, is activated upon depletion of the ER Ca2+ and plays pivotal roles in the timely replenishment of the internal Ca2+ store. In both cell types, inhibition of ER Ca2+ uptake by thapsigargin (4 μM) caused a biphasic cytosolic Ca2+ increase, with the second peak reflecting the SOCE capacity  had additive effects   , ER Ca2+ release channels , and the ER Ca2+ uptake pump (SERCA3), as well as transient receptor potential TRPC4 channels. Notably, α2δ1, which helps to anchor VOCCs in the plasma membrane, has been previously reported by us as a functional liver CSC marker7.Next, we sought to identify the key molecular players underlying the Calls Fig. . Among ted) Fig. . Intereslls Fig. . These g2+ regulators, we targeted the top five genes on the list, CACNA2D1, CACNA2D2, ITPR1, ITPR2, and ATP2A3, and determine the consequences of the Ca2+ oscillation phenotype. We used α2δ1 as a positive control on some occasions, and included ITPR3 (IP3R3 gene) alongside ITPR1 (IP3R1 gene) and ITPR2 (IP3R2 gene) to acquire a comprehensive view of the IP3R family. We established stable cell lines harboring shRNAs against these genes  or unchanged   Fig. S. These rery Fig. , resembl2+ oscillation in CSC biology, we assessed the efficiency of spheroid formation, which reflects the self-renewal capacity, in relation to Ca2+ oscillation frequency over a broad range of experimental conditions. The experimental groups included: (i) knockdown of the VOCC subunits α2δ1 and α2δ2; (ii) knockdown of the ER release channels IP3R1, IP3R2, and IP3R3; (iii) removal of extracellular Ca2+; (iv) inhibition of SOCE; and (v) treatment with BAPTA-AM or EGTA-AM to retard Ca2+ changes. We found that, while inhibiting Ca2+ oscillation, both α2δ1 and α2δ2 knockdown significantly compromised the self-renewal   Fig. . This re2+ oscillation and spheroid formation, we hypothesized that targeting the Ca2+ oscillation machinery might provide an effective therapeutic strategy to limit CSC expansion and therefore retard or even prevent the genesis of tumors. Among the aforementioned molecular participants in the genesis and regulation of Ca2+ oscillation, IP3R2 is particularly noteworthy, not only because it is greatly upregulated in liver CSCs, but also its cyclic opening may serve as the intrinsic oscillator30. To further evaluate the impact of IP3R2 on the self-renewal of Hep-12 cells, we injected 103 or 102 scrambled control cells and IP3R2-knockdown cells subcutaneously into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to test their tumorigenicity potential. Tumors were dissected and weighed 42 or 52 days after injection of 103 or 102 cells, respectively. As shown in Fig. 3R2 knockdown. To further confirm the role of IP3R2, we purified α2δ1+ CSCs from Huh7 cells and assayed spheroid formation and tumorigenicity with IP3R2 knockdown. Both the efficiency of spheroid formation and the frequency of tumorigenic cells were markedly inhibited , robust Ca2+ oscillation occurred in liver CSCs, including CSC-enriched Hep-12 cells, the small portion of α2δ1+ Hep-11 cells, and α2δ1+ Huh7 cells. This Ca2+ phenotype is CSC-specific because the vast majority of matching Hep-11 and Huh7 cancer cells did not show similar Ca2+ oscillation, and neither did HCC LM3, MHCC97-L, and the immortalized liver cell line MIHA. This finding is in general agreement with previous reports that Ca2+ oscillation occurs in a portion of highly metastatic, but not in weakly/non-metastatic human prostate and breast cancer cells31. Such CSC Ca2+ oscillation plays important roles in cancer biology. The efficiency of spheroid formation was significantly decreased by suppressing Ca2+ oscillation by removing extracellular Ca2+, inhibiting SOCE, buffering cytosolic Ca2+, or knocking down key Ca2+ regulators (IP3R2 or α2δ1). Strikingly, there was a positive correlation between the Ca2+ oscillation frequency and spheroid formation efficiency under a broad range of conditions. More importantly, we showed that CSCs with defective Ca2+ oscillation, i.e., knockdown of IP3R2, exhibited a greatly compromised ability to form tumors in NOD/SCID mice.In this study, we demonstrate that Ca2+ oscillation reflects a system-level, emergent behavior of the complex Ca2+-handling machinery. To sustain a persistent cyclic Ca2+ oscillation, the ER store must be kept replenished. With our newly developed ER Ca2+ sensor GCaMP-ER2, we disclosed a surprising new insight at the cellular level. In CSCs undergoing niche factor-stimulated Ca2+ oscillation, initial ER depletion was followed by a complete restoration and oftentimes an overshoot of the ER Ca2+ content. In HCCs displaying no Ca2+ oscillation, however, stimulated ER Ca2+ release was followed by a deep ER depletion without any sign of recovery over the period of observation (15 min). The distinctive ER Ca2+ dynamics are in part attributable to higher activity of SOCE and ER Ca2+ recycling in CSCs (this study) as well as Ca2+ entry through VOCCs as we reported previously6. More importantly, these results strongly suggest that the ER release channels in CSCs undergo cyclic, brief openings, allowing for ER Ca2+ retention and supporting the Ca2+ oscillation behavior, whereas those in HCCs may be kept open after stimulation, preventing replenishment of the ER Ca2+. Through RNA-seq and bioinformatics analysis confirmed by functional validation, we identified the ER release channel IP3R2 as the intrinsic oscillator driving the CSC Ca2+ phenotype, while the other Ca2+ proteins examined serve largely permissive roles .Ca3R family consists of three subtypes, IP3R1, IP3R2, and IP3R3, which share 60–80% homology of amino acid sequence, and the subtype expression is differentially regulated in response to physiological and pathological stimuli. There is a bell-shaped Ca2+-dependence of the channel activity: activation via the CICR mechanism at low [Ca2+]i, inhibition via Ca2+-dependent inactivation at higher [Ca2+]i32. These properties lay the foundation for Ca2+ oscillation in nonexcitable cells. All three IP3R subtypes are similar in terms of permeability, but their high-Ca2+ inactivation properties are variable33. Functional experiments have shown that IP3R2 is required for long-lasting and regular Ca2+ oscillation30, IP3R1 is responsible for short and irregular Ca2+ oscillation28, and IP3R3 produces a monophasic Ca2+ spike28 and even has anti-oscillatory actions34. More interestingly, IP3R2 co-expression with IP3R1 or IP3R3 facilitates Ca2+ oscillation28. In this regard, we found that IP3R2 was specifically expressed in liver CSCs but not HCCs; and knockdown of IP3R2, but not IP3R1 and IP3R3, repressed Ca2+ oscillation  supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 ℃ under 5% CO2. The LM3 and MHCC97-L cell lines were gifted by Dongqin Yang , and the MIHA cell line was gifted by Jinying Ning . The HeLa cell line was kept in our labs. All four cell lines were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS. To generate stable Hep-12 cell lines with knockdown of target genes, packaging plasmids pLP1, pLP2, and pVSVG (Invitrogen) were transfected with expression plasmids harboring shRNA targeting the specific genes into 293FT cells at the ratio 1:1:1:3, then the lentivirus was collected and cells were infected. The puromycin-resistant cells were cultured and stable target gene-knockdown cell lines were acquired. ShRNAs (Sigma) were gifted by Guoqiang Bi (University of Science and Technology of China) and their sequences are listed in Table SHep-12, Hep-11, and Huh7 cells2+ dynamics. When ER Ca2+ was measured simultaneously using GCaMP-ER2, Rhod-4 AM (AAT Bioquest) was used for cytosolic Ca2+ detection. Briefly, cells were incubated in the presence of Fluo-4 AM (5 μM) or Rhod-4 AM (5 μM) at room temperature for 30 min. After washing with Tyrode’s solution consisting of (in mM) 137 NaCl, 5.4 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1.8 CaCl2, 10 glucose, and 20 HEPES , Fluo-4 or Rhod-4 fluorescence was measured with a Zeiss LSM 710 confocal microscope equipped with a 40×, 1.3 NA oil-immersion objective. GCaMP-ER2 and RCaMP1.01 plasmids were co-transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) 48–72 h before imaging and measurement. For ER Ca2+ measurement in CSCs and HCCs, adenovirus expressing GCaMP-ER2 was used to infect the cells 48 h before measurement. For Fluo-4 fluorescence measurement, cells were excited at 488 nm and emission collected at 493–622 nm. For GCaMP-ER2 and Rhod-4 dual indicator measurement, images were captured in multi-track mode with excitation at 488 or 543 nm and emission collection at 490–516 or 556–733 nm, respectively.Fluo-4 AM (Invitrogen) was used to monitor cytosolic Ca2+ level was quantified using the equation [Ca2+] = Kd (F − Fmin)/(Fmax − F). After measurement of fluorescence at rest (F0), cells were exposed to Tyrode’s solution (zero Ca2+) with 4 mM EGTA, 5 μM thapsigargin, and 10 μM A23187. Store Ca2+ release was induced and cytosolic Ca2+ increased abruptly and reached the peak in ~10 s (Frelease) followed by a gradual decline. When stabilized, 100 μM BAPTA-AM was added to further chelate cytosolic Ca2+ to measure the minimum fluorescence level (Fmin). Then, for measurement of the maximal level (Fmax), cells were exposed to Tyrode’s solution containing 10 mM Ca2+, 5 μM thapsigargin, 12 μM A23187, 3 μM FCCP, and 20 mM 2-DG. Assuming that Kd = 1 μM for Fluo-4 in intact cells38, the resting Ca2+ [Ca2+]c, and the releasable store Ca2+ indexed by [Ca2+]release were obtained with the above equation.The cytosolic CaNheI/HindIII sites of pRSET-A expression vector (Invitrogen). The mutant proteins were expressed in E. coli BL21 Star (DE3) pLysS cells and purified using Ni-charged resins as previously described39. After elution, the buffer was changed to 30 mM MOPS (pH 7.2) with 100 mM KCl using an Amicon Ultra-4 filter unit (Millipore). Protein concentration was measured using BCA Protein Assay (Pierce).Mutations of CaM were introduced into the G-GECO1.2 sequence by overlap extension PCR. The amplified product was inserted into the 2+]free buffer were made in 1 mM EGTA, 50 mM MOPS, and 100 mM KCl (pH 7.2) and [Ca2+]free concentrations were calculated using WEBMAXC EXTENDED program (maxchelator.stanford.edu). The fluorescence of 1 µM purified protein in various [Ca2+]free buffers were measured with excitation at 485/20 nm and emission at 516/20 nm using a Synergy 2 Microplate Reader (Biotek).Calcium titration of G-GECO1.2 was performed by Calcium Calibration Buffer Kit #1 (Invitrogen). For calcium titration of low affinity mutants, a series of zero to 10 mM [Ca6 between CaM and retention signal. The final construct was generated by PCR with primers containing described coding sequences and GCaMP-L2 template. The PCR product was cloned into the pEGFP-N1 mammalian expression vector (replacing EGFP) using BglII and NotI restriction sites.The GCaMP-L2 was targeted to and retained in the ER via the N-terminal calreticulin ER targeting sequence MLLSVPLLLGLLGLAVA and the C-terminal ER retention signal KDEL, respectively, with a linker KL(AP)40.The α2δ1 antibody (Abcam) was directly labeled with the Lightning-Link PE-Cy5 Labeling Kit following the vendor’s protocol (Innova Biosciences). For flow cytometry, cells were digested, dispersed, labeled, and analyzed as previously describedCells were plated in ultra low attachment 96-well plates (Corning) and cultured in Dulbecco’s modified Eagle’s medium/F12 (Invitrogen) supplemented with B27 (Invitrogen), 40 ng/ml epidermal growth factor (Invitrogen), 40 ng/ml basic fibroblast growth factor (Peprotech), and 1% methylcellulose (Sigma). Cells were incubated for 2–3 weeks, and spheres were counted under a stereomicroscope (Olympus).Cells were suspended in 50 μL of a 1:1 mixture of plain RPMI 1640 and Matrigel (BD Biosciences) and injected subcutaneously into the back of 4- to 6-week-old NOD/SCID mice . Tumor formation was monitored weekly.+ Huh7 cells with IP3R2 knockdown, sorted cells were first infected with shRNA-IP3R2 or scrambled control lentivirus for 4 h at 37 ℃.To assess the tumorigenicity detection of α2δ1All animals were treated in compliance with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health  and approved by the Animal Care and Use Committee of Peking University .®UltraTM RNA Library Prep Kit for Illumina® (New England Biolabs) following the manufacturer’s recommendations. Deep sequencing was then performed on an Illumina Hiseq 4000 platform and 150-bp paired-end reads were generated. To obtain the differentially-expressed genes, RNA-seq reads were first aligned to the human genome (hg19) with TopHat (v 1.2.0) and all the mapping results were evaluated as previously described41. Cuffdiff software (v 2.0.2) was used to obtain differentially expressed genes with a cutoff of fold-change > 2 and q value < 0.05. By searching Gene Ontology (http://www.geneontology.org/) we found Ca2+-related genes distributed in process, function, and component.Using TRIzol reagent (Invitrogen), total RNA was isolated from cells according to the manufacturer’s protocol. The quality of the extracted RNA was controlled using Agilent 2100. A sequencing library was prepared using the NEBNext3R expression) or 8% SDS-PAGE gel  and transferred to a 0.45-μm polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% bovine serum albumin (for IP3R expression) or 5% nonfat dry milk  and incubated with primary antibody overnight at 4 °C. Primary antibodies against IP3R1 , IP3R2 , IP3R3 , α2δ1 , α2δ2 , SERCA3 , and tubulin  were used.Cells lysates were obtained by incubating cells directly with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. After ultrasonicating 5 times (5 s each), lysates were heated at 100 °C for 10 min. Proteins were separated on 6% SDS-PAGE gel (for IPt test was applied to determine statistical significance. P < 0.05 was considered statistically significant.The data are expressed as the mean ± SEM and, when appropriate, Student’s Supplementary materialsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Table 1Supplementary Table 2Supplementary Movie 1Supplementary Movie 2


This study investigated whether bradykinin‐mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ (Cai2+) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store‐operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free Cai2+ via ER‐IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit+ progenitor cells.Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit They reconstitute the myocardium with new vessels and myocytes after being injected into the ischaemic heart.+ progenitor cell is believed to be a cell source for transplantation to treat ischaemic cardiomyopathy. A clinical trial showed that the cardiac c‐Kit+ progenitor cells isolated from patients with ischaemic cardiomyopathy may significantly improve heart function and the quality of life when transplanted back into the patients via intracoronary injection;+ progenitor cells for ischaemic cardiomyopathy is limited by the poor survival and retention in the heart and the lack of sufficient de novo differentiation of transplanted stem cells into mature cardiac cell types,+ progenitor cells.Cardiac progenitor cells, also known as cardiac stem cells, are self‐renewing, clonogenic and pluripotent and can differentiate into a minimum of three cell types including cardiomyocytes, vascular smooth muscle cells and vascular endothelial cells.2+ (Cai2+) play an important role in regulating human cardiac c‐Kit+ progenitor cell growth.+ progenitor cells.2+ signalling in human cardiac c‐Kit+ progenitor cells. This study was therefore designed to investigate the detailed molecular mechanisms of whether/how Ca2+ signalling‐related channels/receptors are involved in bradykinin‐induced increase in cell growth and migration in human cardiac c‐Kit+ progenitor cells with multiple biochemical/molecular biological approaches. Our results demonstrated that bradykinin‐induced Cai2+ increase by activating Ca2+ release via inositol triphosphate receptor 3 (IP3R3) followed by Ca2+ influx through store‐operated Ca2+ entry (SOCE) channel, which plays a crucial role in regulating cell cycle progression and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit+ progenitor cells.An earlier study reported that spontaneous oscillations of cytosolic free Ca22.1The experimental solutions and reagents used in this study were described in online Supporting Information .2.2+ progenitor cells were isolated from human atrial specimens from patients undergoing coronary artery bypass surgery as described previously.−1l‐glutamine, 5 ng/mL bFGF, 5 ng/mL EGF, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The cells at 3‐6 passages used in this study were from 2 female patients (54 and 56 years old) and 2 male patients (48 and 61 years old).Human cardiac c‐Kit2.32+ (Cai2+) was measured with a laser scanning confocal microscopy technique in human cardiac c‐Kit+ progenitor cells loaded with the free Ca2+ indicator Fluo‐4 AM (Thermo Fisher Scientific Inc.) following manufacturer's instruction.4 cells for 24 hours, and then loaded with 1 μmol L−1 Fluo‐4 AM in FBS‐free α‐MEM for 30 minutes at 37°C in the dark. Afterwards, the cells were washed three times with Tyrode's solution to remove the residual fluorescent dye. Cai2+ was monitored every 5 seconds using the laser scanning confocal microscope Leica SP5‐II at room temperature (23‐25°C).Cytosolic free Ca2.4+ progenitor cells were seeded in six‐well plates or 96‐well plates at a confluence of 60%‐80% overnight. Then the cells were transfected with different siRNA molecules (Santa Cruz Biotech) at 10 or 40 nmol L−1 using Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 48‐72 hours. The control siRNA, which had no known target in the human genome, was used as negative control.Gene silencing was conducted with small interfering RNA (siRNA) technique as described previously.2.5+ progenitor cells transfected with corresponding siRNA for 48 hours using TRIzol reagent. The amount of total RNA was quantified by spectrophotometry, and reverse transcription reaction was performed using 2 μg of total RNA to transcribe into complementary DNA with Advantage® RT‐for‐PCR Kit  following manufacturer's instruction. Primers for the corresponding targets are shown online in Supporting Information ‐2,5‐diphenyltetrazolium bromide (MTT) and 5‐bromo‐2‐deoxy uridine (BrdU) in human cardiac c‐Kit2.7+ progenitor cells as described previously.The cell cycle distribution involved in the proliferation process was detected by flow cytometry in human cardiac c‐Kit2.8+ cells transfected with corresponding siRNA were determined with wound‐healing and transwell assay as described previouslyThe effects of bradykinin on human cardiac c‐Kit2.9+ progenitor cells as described previously.https://imagej.nih.gov/ij/).Western blot was conducted to determine the protein expression in human cardiac c‐Kit2.10t test was used as appropriate to evaluate the statistical significance of differences between two group means. One‐way ANOVA followed by Tukey's test was applied for multiple group comparison. A value of P < .05 was considered to indicate statistical significance.All data were presented as mean ± SEM. Unpaired Student's 33.1−1 remarkably stimulates cell growth and migration.−1 bradykinin in this study to investigate whether the stimulation of cell growth and migration is linked to Ca2+ signalling induced by bradykinin.In our previous study, bradykinin at 10 nmol LCai2+ in human cardiac c‐Kit+ progenitor cells. Changes in Cai2+ induced by bradykinin show a quick transient increase followed by a sustained low level increase in Cai2+ , but not the B1R antagonist R715 (300 nmol L−1)  abolished the sustained component without affecting the transient Cai2+ increase by bradykinin  or the inositol 1,4,5‐trisphosphate (IP3) receptor (IP3R) inhibitor araguspongin B−1), but not ryanodine receptor (RyR) inhibitor ryanodine (10 μmol L−1). In addition, we found that cardiac RyR2 protein was expressed in human atria, but not in human cardiac c‐Kit+ progenitor cells  . The bradykinin‐induced Cai2+ increase was also not affected in these cells  or the TRPV4 blocker RN1734 (10 μmol L−1)  or the non‐selective TRPC channel blocker SKF96365 (50 μmol L−1), bradykinin‐induced a transient Cai2+ increase  nor ML204 (10 μmol L−1) affected the Ca2+ activity induced by bradykinin  and Orai1 . Genes and proteins of B2R, IP3R1, IP3R2, IP3R3, TRPC1, TRPC3, TRPC4, Orai1 and STIM1 were remarkably reduced in cells transfected with 10 and 40 nmol L−1 of the corresponding siRNAs   but had no such effect in cells with silenced IP3R3, TRPC1, Orai1 or STIM1 . These results indicate that the enhancement of cell growth by bradykinin in human cardiac c‐Kit+ progenitor cells is related to the Ca2+ signalling.Figure −1), but not in cells transfected with IP3R3, TRPC1, STIM1 or Orai1 siRNA. These results indicate that bradykinin‐induced promotion of cell cycling progression is related to Cai2+ increase mediated by IP3R3 and SOCE channel in human cardiac c‐Kit+ progenitor cells.The cell cycling progression was then determined by flow cytometry analysis in cells with silenced IP3Rs, TRPCs, STIM1 or Orai1 in the absence or presence of bradykinin Figure . Bradyki−1 control, IP3R1, IP3R2, IP3R3, TRPC1, TRPC3, TRPC4, Orai1 or STIM1 siRNA molecules. The cells migrated to the acellular area or the lower membrane surface of transwell were increased by bradykinin in cells transfected with control, IP3R1, IP3R2, TRPC3 or TRPC4 siRNA, but not in cells transfected with IP3R3, TRPC1, Orai1 or STIM1 siRNA. These results indicate that cell migration induced by bradykinin is related to Cai2+ increase via both IP3R3 and SOCE channel in human cardiac c‐Kit+ progenitor cells.The effects of bradykinin on cell migration were determined with wound‐healing assay Figure A,B and t3.5+ progenitor cells.Cai2+ increase, these signal molecules were determined in cells with silenced IP3Rs, TRPCs and SOCE components. Figure Cai2+ increase in human cardiac c‐Kit+ progenitor cells.Our recent study has showed that activation of pAkt, pERK1/2 and cyclin D1 is involved in regulation of proliferation and/or migration by bradykinin in human cardiac c‐Kit4+ progenitor cells is a promising cell source for repairing ischaemic cardiomyopathy,+ progenitor cells.Although human cardiac c‐KitCai2+ increase via Ca2+ release‐induced Ca2+ influx mechanism: (1) bradykinin binds to B2R to stimulate PLC and generate IP3, (2) IP3 activates ER IP3R3 to release Ca2+ and (3) Ca2+ release induces a reduced ER Ca2+ level and activates SOCE channel to mediate Ca2+ influx. The bradykinin‐induced Ca2+ signalling plays a crucial role in regulating cell cycling progression and mobility via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit+ progenitor cells  signalling, but can also control apoptosis, intracellular pH, the initiation and regulation of neuronal Ca2+ signalling, exocytosis and gene expression by coupling with modulatory protein.2+ release from ER/SR in different types of cells. Although the three types of IP3Rs are expressed in human cardiac c‐Kit+ progenitor cells, we found that only IP3R3 mediates the bradykinin‐induced Ca2+ release and the subsequent Ca2+ influx through SOCE channel.Inositol 1,4,5‐trisphosphate receptors (IP3Rs) play a key role in intracellular calcium signalling.2+ sensor, and Orai1 is a pore subunit located on the plasma membrane.+ progenitor cells2+ signals and regulate specific cellular functions.SOCE channel is a complex composed of STIM1, Orai1 and also TRPC1. STIM1 functions as an ER Ca2+ influx in c‐Kit+ progenitor cells results from PLC‐IP3R3 ER‐Ca2+ release and activates the STIM1‐Orai1‐TRPC1 complex of SOCE channel to mediate extracellular Ca2+ entry. The involvement of TRPC1 in SOCE channel is demonstrated by the fact that inhibition of TRPC1 with the silencing TRPC1 abolishes the bradykinin‐induced Ca2+ influx. Similar results were observed in cells with silenced STIM1 or Orai1, in which bradykinin only induces a Ca2+ transient release without subsequent Ca2+ influx. This indicates that bradykinin‐induced Ca2+ influx relies on the SOCE channel as a STIM1‐Orai1‐TRPC1 complex in human cardiac c‐Kit+ progenitor cells.The present study shows that bradykinin‐induced Ca2+ signalling, very low cytoplasmic Ca2+ concentration can increase in a specific manner to trigger downstream cellular events, and every cell type in the human body utilizes some form of Ca2+ signalling to function or survive.2+ oscillations promote human cardiac c‐Kit+ progenitor cell proliferation.2+ signalling‐related channels  are involved in cell cycling progression and migration in human cardiac c‐Kit+ progenitor cells.+ cardiac stem cells in the mouse is promoted by spontaneous Ca2+ spikes, which dictate the pattern of stem cell replication and the generation of myocyte progeny at all phases of prenatal life and up to 1 day after birth.In Ca+ progenitor cells.2+ signalling mediated by PLC‐IP3R3 ER‐Ca2+ release followed by Ca2+ influx through SOCE channel (STIM1‐Orai1‐TRPC1 complex); the cell growth, migration as well as pAkt, pERK1/2 and cyclin D1 are no longer increased by bradykinin in cells with silenced IP3R3, STIM1, Orai1 or TRPC1.The endogenous peptide bradykinin has an extensive range of biological activities in different organs, including regulation of mitogenesis in different types of cells.2+ followed by Ca2+ influx through SOCE channel in human cardiac c‐Kit+ progenitor cells.In the present study, we did not explore why IP3R3s, but not IP3R1s and IP3R2s, are activated by bradykinin. However, this limitation would not affect the conclusion that bradykinin is coupled with B2R to activate PLC and generate IP3, which then stimulates ER IP3R3 to release Ca+ progenitor cells are an essential cellular function for the heart development2+ signalling via stimulating ER IP3R3 to release Ca2+ followed by Ca2+ influx through SOCE channel initiates Akt and ERK1/2 phosphorylation and cyclin D1 expression and promotes cell proliferation and migration, suggesting that bradykinin may be useful for preconditioning cardiac progenitor cells to promote myocardial repair.Collectively, cell proliferation and migration of cardiac c‐KitThe authors confirm that there are no conflict of interests. Click here for additional data file.


Although the roles of Ca2+ signal transduction related to photosynthesis have been discussed, the relationship between calcium and photosynthesis is seldom systematically summarized. In this review, we provide an overview of current knowledge of calcium’s role in photosynthesis.As a secondary messenger, calcium participates in various physiological and biochemical reactions in plants. Photosynthesis is the most extensive biosynthesis process on Earth. To date, researchers have found that some chloroplast proteins have Ca Calcium is involved in many pathways in plant cells, including plant growth and development, resistance to environmental stress ,2, hormo2+-binding proteins have been consistently found to reside in chloroplasts, but some are located in the chloroplast membrane, such as s-adenosylmethionine transporter-like (SAMTL) cyt) concentration decreases and stomatal closure is prevented [2+]cyt oscillation [2O2 treatment [2O2 affects stomatal movement by activating [Ca2+]cyt channels cyt oscillation by activating [Ca2+]cyt channels and then causes stomatal closure. Some reports showed that [Ca2+]cyt oscillations can also be induced by the inositol 1,4,5-trisphosphate (IP3) under external stress  is an important regulator in chloroplast function . Researcthaliana . Genome synthase . Chloropnd motif . Thus,  and the 10 kDa chloroplast co-chaperonin (ch-CPN10) have been widely studied ,108. In involved . Moreoveproteins .2+, such as SAMTL, which is located on the inner membrane of the chloroplast envelope and contains the EF-hand structure of Ca2+-binding sites [2+ ATPase that resides in the inner envelope of A. thaliana chloroplast [Many other chloroplast proteins are also regulated by Cang sites . Chloroporoplast . ATPase oroplast . These p2+-related proteins located in the chloroplast outer membrane may directly link the cytoplasmic signal with the chloroplast signal. They are involved in multiple pathways responding to environmental stimulus  and regulate photosynthesis. The complex signal network related to Ca2+ needs further systematic research.Calcium plays an important role in multiple photosynthetic pathways. It can affect gas exchange related to photosynthesis by regulating stomatal movement. Several photosynthetic proteins are regulated directly or indirectly by calcium. In addition to the proteins mentioned in this paper, a variety of Ca


We examined alterations in expression, phosphorylation status, Ca2+ sensitivity, and in situ function (by measuring Ca2+ sparks and Ca2+ transients) of RyR2; alterations in these characteristics could help to explain the Ca2+ handling disturbances in MetS cardiomyocytes. MetS was induced in rats by adding commercially refined sugar (30% sucrose) to their drinking water for 24 weeks. Cardiomyocytes of MetS rats displayed decreased Ca2+ transient amplitude and cell contractility at all stimulation frequencies. Quiescent MetS cardiomyocytes showed a decrease in Ca2+ spark frequency, amplitude, and spark-mediated Ca2+ leak. The [3H]-ryanodine binding data showed that functionally active RyRs are significantly diminished in MetS heart microsomes; and exhibited rapid Ca2+-induced inactivation. The phosphorylation of corresponding Ser2814  of the hRyR2 was significantly diminished. RyR2 protein expression and Ser2808 phosphorylation level were both unchanged. Further, we demonstrated that cardiomyocyte Ca2+ mishandling was associated with reduced SERCA pump activity due to decreased Thr17-PLN phosphorylation, suggesting a downregulation of CaMKII in MetS hearts, though the SR Ca2+ load remained unchanged. The reduction in the phosphorylation level of RyR2 at Ser2814 decreases RyR2 availability for activation during ECC. In conclusion, the impaired in situ activity of RyR2 may also account for the poor overall cardiac outcome reported in MetS patients; hence, the SERCA pump and RyR2 are both attractive potential targets for future therapies.Metabolic syndrome (MetS) has become a global epidemic. MetS is a serious health problem because of its related cardiovascular complications, which include hypertension and delayed heart rate recovery after exercise. The molecular bases of cardiac dysfunction in MetS are still under scrutiny and may be related to anomalies in the activity and expression of key proteins involved in the cardiac excitation–contraction coupling (ECC). The cardiac Ca Ca2+ entry is not of sufficient magnitude to activate the contractile machinery but triggers a larger Ca2+ release from the stores in the sarcoplasmic reticulum (SR) via the activation of the intracellular Ca2+ channel/ryanodine receptor (RyR2), thus promoting cell contraction. This process is known as Ca2+-induced Ca2+ release (CICR). Relaxation takes place after Ca2+ removal, primarily through its recapture into the intracellular Ca2+ stores by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA pump) and its extrusion by the action of both the Na+/Ca2+ exchanger (NCX1) and the plasma membrane calcium ATPase (PMCA) (2+ clearing have been documented in cardiomyocytes of the prediabetic sucrose-fed rat model at early stages of MetS development (6–18 weeks of sucrose treatment) (Metabolic syndrome (MetS) is a cluster of biochemical and physiological risk factors for cardiovascular disease and diabetes mellitus type 2 (DM2); it represents a severe public health problem around the world . Risk fae (PMCA) . ECC defeatment) , but the2+/Calmodulin-dependent protein kinase II- (CaMKII) mediated RyR phosphorylation have been reported in cardiomyocytes from prediabetic rats, resulting in defective RyR2 regulation  to quantify functional RyR2 and to measure the Ca2+ sensitivity, protein expression, and phosphorylation status at Ser2808 and Ser2814 of RyR2 in heart homogenates and SR-enriched membranes.Alterations in the Wistar rats, aged 25 days, were divided into two groups and maintained under a dark–light cycle of 12 h and controlled temperature of 22 ± 2°C. The first experimental group (MetS) received 30% sucrose  in their drinking water and commercial rat chow  ad libitum for 24 weeks. The second group  received water and commercial rat chow ad libitum during the same 24 weeks. Measurement of systolic blood pressure (SBP) was carried out by the tail-cuff method with a sensor connected to a pressure transducer and a PC equipped with special software (Grass PolyView) for data capture and processing. Recordings were taken in quadruplicate and were compared with those from invasive techniques: no difference has been found, in agreement with previous reports  and anesthetized with sodium pentobarbital . Anesthetized animals were subjected to a thoracotomy, during which the heart was exposed and excised rapidly, and blood samples were collected immediately from the sectioned aorta. The serum levels of glucose, triglycerides, total cholesterol, and HDL-cholesterol were measured using glucose and lipid panel strips with the CardioCheck PA analyzer .2PO4 0.4, MgCl2 0.5, glucose 22, Hepes 25 and insulin 10–3 (pH 7.4 with NaOH). The aorta was cannulated above the aortic valve and heart was perfused by gravity with warm (37°C) Tyrode solution supplemented with 0.1 mM EGTA for 5 min. Enzyme solution containing 0.8 g/L collagenase Type II  in Tyrode solution supplemented with 0.1 mM CaCl2 was then perfused until the aortic valve was digested (confirmed by the increased outflow of perfusate). The heart was transferred to a Petri dish containing enzyme solution supplemented with 1 mg/mL bovine serum albumin (BSA) and gently shaken for 2–3 min at 37°C to disperse individual myocytes. The resulting cell suspension was centrifuged for 3 min at 170 × g. The cell pellet was suspended in Tyrode solution supplemented with 0.5 mM CaCl2 and was centrifuged again at the same speed. Finally, the cell pellet was suspended in storage solution containing Tyrode solution supplemented with 1 mM CaCl2.Left ventricle myocytes were enzymatically isolated from MetS and control rat hearts following a previously reported protocol . Animals2+ sparks and field-stimulated Ca2+ transients were imaged in Fluo-3 loaded cardiomyocytes as previously reported  and 2D-images were obtained with a confocal microscope  in the line-scan mode (1.9 ms/line). To obtain electrically-stimulated Ca2+ transients, cells were paced at different frequencies using a Grass stimulator set at 70 V and pulse length of 20 ms; 5 images of the cell were taken for each stimulation frequency. The Y-axis of the image indicates the length of the cell (in μm) and the X-axis indicates the scan time (in s). Fluo-3 was excited at 488 nm using an argon laser at 2% intensity. The emitted fluorescence of the dye was measured at 510 nm. To determined SR Ca2+ load, myocytes were paced at 1 Hz for at least 1 min to restore steady state SR Ca2+ load, then confocal laser scanning was initiated and four electrically-evoked Ca2+ transients were followed by rapid application of caffeine (10 mM)  .0) . Cell shortening was reported in % respect to total cell length. Cells with cell shortening values below 4.5% were not considered in the analysis. Decay Time (in ms) was determined at 50% of recovery of the Ca2+ transient peak. Parameters such as amplitude (F/F0), frequency (events/s*100 μm), full duration at half maximum (FDHM in ms), full width at half maximum (FWHM in μm), time-to-peak (in ms) and decay time constant (in ms), were analyzed for Ca2+ sparks  running a custom protocol written by Ana Maria Gómez , including baseline fluorescence correction and normalizing fluorescence levels (F) with respect to basal fluorescence (F0) . The flu+ sparks . The spaon*width .2. Tissue powder was suspended in homogenization buffer  and homogenized with a Potter-Elvehjem homogenizer  and spun at 2000 × g for 10 min; the resulting supernatant (homogenate) was spun at 8000 × g for 10 min; SR-enriched fractions (or microsomes) were isolated using the second supernatant by ultracentrifugation at 40000 × g for 30 min at 4°C. The resulting pellets were suspended in homogenization buffer and protein concentration was determined using the Lowry method as previously reported (Heart homogenates and SR-enriched fractions were prepared from left-ventricle tissue pulverized to a fine powder with a mortar and pestle over liquid Nreported .2+ uptake accordingly to a previously described method (2+ uptake was measured in a buffer containing (in mM): KCl 100, MgCl2 4, MOPS 20, potassium oxalate 10, ATP-Mg 1.25, creatine phosphate 1.5, pH 7.4 with KOH, and 0.3 U/ml creatine phosphokinase. 10 μM Ruthenium Red was added to inhibit Ca2+ release through RyRs. Fura-4F was added to a final concentration of 0.5 μM. 600 μg of protein from SR-enriched vesicles were added to a cuvette with uptake buffer and equilibrated at 37°C during 5 min with stirring. Fluorescence was measured in a QM-8 spectrofluorometer . The addition of 5 μM CaCl2 initiated Ca2+ uptake reactions; the fluorescence was recorded up to 900 s. The experiment was finished by adding Thapsigargin (1 μM). To determine Rmax, 200 μM CaCl2 was added; to measure Rmin, 250 μM EGTA was added. Ratio values of fluorescence (F340/F380) were subsequently transformed into Ca2+ concentrations using the Grynkiewicz equation  was added outside SR-membrane vesicles and used to monitor the SERCA-dependent Cad method . SR Ca2+equation . Ca2+ up3H]-ryanodine binding experiments were performed as previously described using SR-enriched fractions ; and [3H]-ryanodine in the range of 0.625 to 20 nM. Equilibrium binding data were fitted to the one-site model (Hill equation), apparent dissociation constant (Kd) and total functional RyRs (Bmax) were calculated from the fitted curve. The Ca2+ dependence of [3H]-ryanodine binding was assessed in incubation medium containing 0.2 mM KCl, 20 HEPES (pH 7.2 with KOH), 50 μg of SR-enriched fractions, 10 nM [3H]-ryanodine, 1 mM EGTA, and CaCl2 necessary to set free Ca2+ in the range of 10 nM to 10 mM . Ca2+-EGTA ratios were calculated using the MaxChelator program1  to the following equation: B = Bmax([Ca2+]na/([Ca2+]na+Kana))(1–[Ca2+]ni/([Ca2+]ni+Kini))+C, modified from Meissner’s group   .2PO4 2.9, Na2HPO4 10.07, NaCl2 150.58 and Tween 20, 0.1%) for 1 h, before incubation with primary antibody against RyR2 , pSer2808RyR2 , pSer2814RyR2 , SERCA2a , PLN , pSer16PLN , pThr17PLN , CaMKII , and GAPDH  for 2 h at room temperature. After washing, the membranes were incubated with corresponding secondary peroxidase-conjugated antibodies  for 1 h. The membranes were washed . Proteins were visualized by chemiluminescent reaction  and the relative amount of protein was determined by densitometric analysis using Kodak MI SE Software (v.5.01.30. Molecular Imaging Software).SDS-PAGE and Western Blots were performed as a modification of previously reported protocols . Gradien2+ release events, or experiments. Statistical significance was evaluated by Student’s t-test or one-way ANOVA followed by Tukey post hoc test, when appropriated . Significance was defined at P < 0.05.Data are presented as the mean ± SEM of indicated independent determinations, where “N” was used to designate the number of animals or hearts; and “n” to specify the number of cells, CaMale rats that received 30% sucrose in the drinking water for 24 weeks developed several of the hallmark characteristics of MetS: for instance, a significant increase in body weight (32%) and visceral fat accumulation (2.4-fold) compared to the control group . Additio2+ transient (2+ transients and associated cell shortening in single cardiomyocytes at three different stimulation frequencies. line scan mode) of Fluo-3-loaded cardiomyocytes from control (top) and MetS (bottom) rats at 0.5 Hz, with their corresponding Ca2+ transient profiles (F/F0), cellular shortening (% of resting cell size), and Ca2+ transient decay time (ms). Cardiomyocytes of both experimental groups exhibited a rate-dependent fall in the Ca2+ transient amplitude; however, in the MetS condition, the magnitude of the Ca2+ transient was consistently lower than in control cells  was slightly increased in MetS cells at all frequencies  and MetS (bottom) cardiomyocytes in line scan mode. Analyses of Ca2+ spark characteristics are summarized in 2+ sparks recorded in MetS cells showed significantly reduced frequency  by subtracting the rate constant of decay of the caffeine-evoked Ca2+ transient from that of the systolic Ca2+ transients. These calculations demonstrated that k SERCA was significantly reduced in MetS cardiomyocytes . These data enabled us to hypothesize impaired SERCA pump function in MetS cardiomyocytes.The amount of Ca+ uptake . Thus, wh groups , in agreh groups . Unchangg others . Therefod method . We calc2+ uptake assay in cardiac SR-enriched membranes . This result agrees with our finding of unchanged SR Ca2+ load despite the reduced diastolic Ca2+ leak observed in MetS cardiomyocytes.We therefore used a more direct approach to investigate SERCA pump enzymatic activity by performing a SERCA pump-mediated Caembranes . These ecrosomes . SERCA-p2+ uptake, changes in PLN expression level and/or phosphorylation status would be expected modify SERCA activity . Western Blot data showed a slight but not significant decrease in the phosphorylation level of Ser16-PLN  than those from control hearts. Importantly, the Kd for [3H]-ryanodine was found in the nanomolar range and remained unchanged . Nevertheless, we found a significant reduction (46%) in the phosphorylation status of Ser2814  and Ser2814  in RyR2, in light of the finding that modifications of RyR phosphorylation level account for the dysfunctional activity of cardiac RyRs in insulin-resistance and prediabetes . Figure 2+ handling are altered and the function of RyR2 is clearly compromised; the latter has been associated with a decrease in RyR2 protein expression and reduced SR Ca2+ load  for 24 weeks successfully generated key features of MetS in rats, characterized by the presence of central obesity, hypertension, hypertriglyceridemia, and increased TG/HDL-C ratio . Some of these are excluded when k SERCA is calculated as reported by In light of the fact that 2+ release events (Ca2+ sparks) constitute the building blocks of Ca2+ transients . Although an augmentation in diastolic [Ca2+]i might participate in the impairment of the relaxation speed in MetS cells, previous reports in related insulin resistant models showed no changes in basal Ca2+ in cardiac cells  and indirect  approaches  in Ca2+ dependent RyR activation/inactivation curves were revealed. We therefore examined RyR2 phosphorylation status at Serines 2808 and 2814 (in human and mouse nomenclature). These post-translational modifications, induced by kinases such as PKA and CaMKII, have been defined as a critical regulatory mechanism for this intracellular Ca2+ channel , may account for the poor overall cardiac outcome reported in MetS patients and could be targeted for future therapies.Principal findings of this work are that abnormal CaThis study was carried out in accordance with the ethical guidelines of the Mexican Official Norm (NOM-062-ZOO-1999) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication updated in 2011); and was approved by the Institutional Bioethical Committee for Care and Handling of Laboratory Animals at the CINVESTAV-IPN (approved CICUAL Protocol No. 0105-14).2+ sparks, electrically stimulated Ca2+ transients, determination of SR Ca2+ load; [3H]-ryanodine bindings, Ca2+ uptake assays and data analysis. M-MM contributed towards SDS-PAGE, Western Blots, and densitometric analysis. AR contributed towards the experimental design, data analysis, data interpretation, and manuscript preparation.GF-M, TR-G, and TB-L contributed to the generation and characterization of the experimental model of MetS in rats, isolation of cardiomyocytes, confocal imaging of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


They mediate their physiological effects through releasing Ca2+ from acidic organelles in response to cues such as the second messenger, NAADP. Here, we review emerging evidence linking TPCs to disease. We discuss how perturbing both local and global Ca2+ changes mediated by TPCs through chemical and/or molecular manipulations can induce or reverse disease phenotypes. We cover evidence from models of Parkinson's disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes. A need for more drugs targeting TPCs is identified.Two-pore channels (TPCs) are Ca Structuhannels) . TPCs arhannels) ,8 or thehannels) . This ishannels) ,11.2+ [2+ mobilizing messenger NAADP [2+ from so called acidic Ca2+ stores [2) [2+ permeability and NAADP sensitivity of TPCs [2+, albeit less direct than electrophysiology, affirm the role of TPCs in NAADP-mediated Ca2+ signalling [TPCs are thought to be non-selective cation channels permeable to Ca2+ . They weer NAADP ,8,12,13.er NAADP , has an + stores ,16. NAAD+ stores ,18 throu+ stores  and neur+ stores  in mammaores [2) ,22. Howe of TPCs ,23. This of TPCs . Certaingnalling ,29.2+ in lysosomes is on par with that of the better characterised ER Ca2+ stores; ~500 μM [2+ signals. During signalling, local Ca2+ signals evoked by NAADP are thought to be amplified by Ca2+ release channels on the ER resulting in global Ca2+ signals that pervade the cell [2+-induced Ca2+ release and facilitated by membrane contact sites between the organelles [2+ signals act in their own – in ‘constitutive mode’ - to regulate cellular processes. Key here is the TPC interactome which reveals association with a number of proteins involved in membrane fusion, trafficking and organisation [The luminal concentration of Ca ~500 μM . Howeverthe cell ,32. Suchganelles ,34. But nisation ,35. Suchnisation , endo-lynisation ,37, retrnisation  and membnisation .2+ channel modulators as inhibitors of NAADP-mediated Ca2+ release [2+ channel blockers [+ channel blockers) which like Ca2+ channel blockers demonstrably inhibit endogenous NAADP-evoked Ca2+ signals and Ca2+ signals evoked by recombinant TPCs [2+ and Na+ channels. This suggests that core structural determinants underlying drug block were probably in place in a primordial two-domain channel early in the evolution of the voltage-gate ion channel superfamily. This is prior to the duplication events that led to extant four domain Ca2+ and Na+ channels and their subsequent divergence allowing them to distinguish Ca2+ and Na+ channel blockers [The pharmacology of TPCs is in its infancy. Indirect blockers include Ned-19 which is an NAADP antagonist discovered through ligand-based virtual screening . Early w release . These b release . Consistblockers . This siant TPCs . TPCs thblockers .In this contribution, we review how chemical and molecular strategies are converging to uncover roles for TPCs in disease. These diseases affect a number of organs such as the brain, liver, heart and the pancreas. They include cancer and Ebola infection and possibly others .Fig. 1TP2LRRK2 gene which represents the most common cause of familial disease.Parkinson's disease is a common and disabling neurodegenerative disorder, characterised by a progressive decline in the control of voluntary movement. The neuropathological hallmark of Parkinson's is the selective death of dopaminergic neurons in the substantia nigra pars compacta and, in the surviving neurons, an accumulation of α-synuclein aggregates . For manLRRK2 encodes a large multi-domain enzyme and the prevalent G2019S mutation in Parkinson's affects its kinase domain enhancing catalytic activity [activity . It is uactivity . Indeed,activity  and seveactivity  with whiactivity .2+ chelation, NAADP antagonism (with Ned-19) or by overexpressing a non-conducting TPC2 pore mutant [2+ channel CaV 2.1 and increases channel activity [2+ channels.Hilfiker and colleagues identified autophagic defects in cells overexpressing wild-type and mutant (G2019S) LRRK2 . These ae mutant ,48. Thisactivity , hints t2 and the trafficking GTPase, Rab7) and molecular silencing of TPC2 (but not TPC1) [2+ signals evoked by NAADP were enhanced in LRRK2- in Parkinson's. Thus, these results further support the hypothesis that pathological LRRK2 enhances TPC2 activity to cause dysfunction.LRRK2/TPC action was also studied in fibroblasts derived from Parkinson's patients with the G2019S LRRK2 mutation . Using vot TPC1) . AdditioTPCN2 was one of the top 20 genes with altered expression [2+ levels were reduced in fibroblasts from in Parkinson's patients with a mutation in GBA1 [2+ levels were depleted in GBA1-Parkinson's due to excessive TPC2 activity, however this has yet to be established. Intriguingly, a recent study demonstrated that Parkinson's is associated with multiple genes underlying other lysosomal storage diseases whereby almost half of the cohort analysed had 1 or more putative damaging mutations [2+, including NAADP-evoked signals [Although patients carrying mutations in LRRK2 present with a phenotype clinically and pathologically indistinguishable to idiopathic Parkinson's, it is possible that LRRK2 studies might not apply to the whole Parkinson's population. Interestingly, a recent transcriptomic meta-analysis of blood samples from sporadic Parkinson's patients found that pression . Additio in GBA1 . This ge in GBA1 . It is putations . That ly signals , is disrIn summary, pathogenic LRRK2 increases TPC2 functionality likely through a direct interaction, to disrupt autophagy and endo-lysosomal morphology. Furthermore, it is possible that TPC2 dysfunction features in other forms of Parkinson's.3Non-alcoholic fatty liver disease (NAFLD) is the most common chronic disorder of the liver characterised by fat accumulation and fibrosis. NAADP is detectable in hepatocytes , liver hIn mice fed with a Western style high cholesterol diet, the livers from the transgenic animals were discoloured and weighed more than those from wild type mice . Liver cvia receptor-mediated endocytosis and delivered to the lysosome [2+-dependent block in fusion between late endosomes and lysosomes was the underlying cause. Consistent with this, the effects of TPC2 knockout on EGF distribution could be pheno-copied by buffering local Ca2+ changes with BAPTA and rescued by a wild-type TPC2 but not a non-conducting pore mutant [Mechanistic studies in mouse embryonic fibroblasts suggest that TPC2 knockout disturbs trafficking of LDL receptors . LDL reclysosome . Pulse clysosome . Howevere mutant ,48. Whete mutant . Again, e mutant . Furthere mutant .2+ signalling provides a likely mechanism for liver dysfunction in NAFLD.In summary, de-regulated trafficking of receptors upon TPC2 knockout probably through disrupted local Ca4The Ebola virus causes an often fatal haemorrhagic fever in humans exemplified by the 2014 outbreak in West Africa. Work by Davey and colleagues identified TPCs as potential drug targets for combatting Ebola infection .in vitro [2+ channel blockers and Ned-19. The former included tetrandrine a plant alkaloid derived from a Chinese herb that is often used in traditional medicine [in vivo in a mouse model of Ebola infection [Many viruses highjack the endocytic system to enter cells . For Eboin vitro . Over-exin vitro ,48 also medicine . Tetrandnfection .2-stimulated currents through TPC1 and TPC2, and NAADP-evoked Ca2+ signals live cells [Mechanistically, tetrandrine blocked NAADP- and PIPve cells . It alsove cells . Howeverve cells . One potve cells ,65 is thve cells . Unexplave cells ,66. The 2+-dependent fusion events.In summary, molecular and chemical targeting of TPCs provides a novel strategy for combatting Ebola uncovering a role for TPCs in viral entry probably through regulation of Ca5Cancer needs little introduction. It is a pervasive problem. Many cellular processes are subverted during carcinogenesis including the process of cell migration (underlying metastasis) and angiogenesis (underlying tumour vascularisation). TPC expression in cancer was originally characterised in SKBR3 (human breast cancer) and PC12 (rat pheochromocytoma) cells where TPC1 transcript levels were reported to be ~3–8 fold higher than TPC2 .TPCN1, NCBI Gene ID: 53373) and 11 , respectively. The gene encoding TPC3, which is surprisingly absent from select mammals including the mouse, is a pseudogene in humans but likely functional in closely related primates [TPCN2  [Inhibiting TPCs was also associated with reduced adhesion and reduced formation of the leading edge in cancer cell lines . These fst cells . Neural st cells . Migrati in vivo . In vitrachinery . Indeed,+ fluxes . This is+ fluxes . Althoug+ fluxes  vs lysosceptors) .2+ signals deriving from acidic Ca2+ stores might also contribute to cancer. NAADP signalling has previously been implicated in agonist-evoked Ca2+ signalling in endothelial cells in response to cues such acetylcholine [in vitro [in vivo Matrigel assays showed that vascularisation by VEGF was inhibited by Ned-19 and in TPC2 but not in TPC1 knockout mice. TPC2 knockout mice however appeared to develop normally suggesting that compensatory changes maintain angiogenesis during development of these animals. Nevertheless, these studies highlight a role for TPCs in a process crucial for cancer progression.Global Calcholine  and histlcholine . Couplinlcholine  is relevin vitro . ImportaXenopus oocytes in a manner that requires interactivity with members of the Rab family of trafficking proteins [2+ store) to potentially regulate melanin production through changes in luminal pH [Melanoma is a cancer that derives from pigmented melanocytes. A link between pigmentation and TPCs has been long appreciated through GWAS which correlated two non-synonymous variants in TPC2 with hair colour . Overexpproteins . And, a proteins . Collectminal pH ,85. Howeminal pH .2+ signalling. TPCs seem to be required for cellular processes that underpin cancer cell survival. And thus present themselves as viable therapeutic targets.In summary, cancer likely features changes in TPC expression and defects in both local and global TPC-mediated Ca62+ signals in individual live cells [The actions of NAADP in the heart have been long appreciated from early radioligand binding and flux assays using cardiac microsomes  through ve cells . TPCs arve cells . TPCs ha2+ has long been considered an upstream culprit in ischemia-reperfusion injury but the underlying mechanisms are not well understood.Interrupting blood supply to the heart causes ischemia and tissue injury. This can occur during myocardial infarction. Reperfusion is essential to restore oxygen and nutrients. But this process in itself exacerbates damage through opening of the mitochondrial transition pore. Cain vivo model of ischemia-reperfusion injury [In vitro, Ca2+ oscillations, and subsequent cell death of cardiomyocytes upon re-oxygenation following ischemia were inhibited by Ned-K and the lysosomotropic agent, GPN. The principle Ca2+ release channel in cardiomyocytes is the type 2 ryanodine receptor and previous studies have implicated this protein in reperfusion-induced Ca2+ oscillations [2+ release from the sarcoplasmic reticulum which is ultimately received by mitochondria resulting in MTP opening [TPC1 and TPC2 levels are reportedly higher in left ventricular samples from ischemic as well as dilated hearts from patients suffering from heart failure . Davidson injury . This efllations . This le opening . Knockdo opening .per se are unlikely to trigger Ca2+ release through TPCs. One possibility is that TPCs become sensitised to NAADP during re-perfusion perhaps in response to oxidative stress, a known determinant of ischemia-reperfusion injury. Indeed, plant TPCs are reportedly responsive to oxidants [How TPCs are activated during reperfusion remains to be established. NAADP levels decrease upon ischemia and increase upon reperfusion . Howeveroxidants .Interestingly, extracellular NAADP appears to paradoxically protect hearts against ischemic injury ,95. SuchAdrenaline plays a key role in the pathophysiology of the heart. Low level stimulation of β-adrenergic receptors acutely increases contractility  whereas more intense stimulation induces arrhythmias. Chronic stimulation induces hypertrophy. Accumulating evidence points to NAADP as a second messenger for adrenaline in the heart .2+ transients in electrically-paced cardiomyocytes and arrhythmias in vivo [2+ transients were also prevented by bafilomycin indicating a requirement for acidic organelles and thus pointing to TPCs. Terrar and colleagues provided direct evidence for TPC2 in the actions of adrenaline [in vivo was also modestly reduced upon TPC2 knock-out.Work by Guse and colleagues provided evidence that the NAADP antagonist BZ194  could pr in vivo . The arrrenaline . In thei2+ transients, effects that could be mimicked by Ned-19 and bafilomycin [2+ transients in previous work [2+ handling proteins such as voltage-gated Ca2+ channels, and more controversially ryanodine receptors, may be more complex. It is tempting to speculate that cyclic AMP-dependent protein kinase phosphorylates TPCs to augment their action. Indeed, evidence for positive regulation of TPC2 by PKA has recently been provided [2+ may enhance Ca2+ release from the SR possibly through promoting Ca2+ uptake through a CamKinase II-dependent mechanism [Mechanistically, these blunted responses were attributed to reduced action of adrenaline on the amplitude of evoked Cailomycin . Bafilomous work . Neverthprovided . Howeverechanism .2+ signalling. Chemical targeting of NAADP in vivo has proven beneficial.In sum, TPCs have emerged as common mediators of cardiac dysfunction in several scenarios of heart disease through disrupting global Ca7Diabetes is a metabolic disorder characterised by hyper-glycemia resulting from either deficient secretion of insulin or insulin action. Functional NAADP effects in the insulin secreting beta cells of the endocrine pancreas has been long known  where it2+-dependent manner in response to glucose. The prevailing view is that this is due to inhibition of ATP-sensitive K+ channels, membrane depolarisation and activation of voltage-gated Ca2+ channels. However, it is increasingly appreciated that other mechanisms likely contribute to stimulus-secretion coupling [2+ signals can be inhibited by interfering with NAADP [2+ signals [Insulin is a key anabolic hormone that controls blood sugar by promoting glucose uptake into peripheral tissues. It is secreted by beta cells in a Cacoupling . Indeed,th NAADP ,103. Con signals . Double  signals  although signals .in vivo studies of these animals showed that blood glucose levels were modestly increased in glucose tolerance tests. Reduced insulin secretion and elevated blood glucose are both key diabetic traits. Mice lacking TPC2 also show decreased insulin secretion in response to glucose challenge in vivo [ex vivo studies using whole pancreata were less clear [in vivo were paradoxically either slightly reduced [2+ signalling, insulin secretion or glucose tolerance [in vivo, a perennial concern for transgenic mice studies. Here, a recent study focusing on alpha cells of the endocrine pancreas is potentially relevant. Alpha cells secrete glucagon which has the opposite effect to insulin on blood sugar levels. Knock-out of TPC2 but less so TPC1 was shown to reduce glucagon secretion [in vivo. Clearly further studies are required to relate loss of TPCs in single cells through organs to whole animal phenotypes particularly for TPC2.Importantly, whole pancreata derived from TPC1 knock-out mice secrete less insulin in response to elevated glucose . Moreove in vivo  althoughss clear . Glucose reduced  or uncha reduced . Beta-ceolerance . One posecretion . Consequ2+ but in mammalian TPCs, lack of Ca2+ coordinating residues likely renders this region Ca2+-insensitive. In human TPC2, the corresponding residue is again leucine like the rat TPC2 variant  has also been implicated in diabetes. Fan et al. identified a variant within an intron toward the 3′ of the gene  underlie familial forms of Alzheimer's disease . In cell reduced  and this reduced . In PS1-enhanced . Intriguenhanced . This waFurther phenotypic analyses of TPC knockout mice have implicated TPC1 in infertility through a requirement for the acrosome reaction in sperm . The MenInterestingly, a recent GWAS linked TPC2 to systemic lupus erythematosus , a chronIn sum, TPCs might be involved in a number of additional disorders affecting the brain, reproductive system, skeletal muscle, adipose tissue and the immune system. But further supporting evidence is required.9As discussed, NAADP antagonists have proven beneficial in correcting disease phenotypes in cellular and animal models of Parkinson's (Ned-K), cancer (Ned-19) and heart arrhythmia (BZ194), often mimicking the effects of TPC knockdown/knockout. They, thus present themselves as lead drugs for combatting TPC dysfunction. But because the effects of NAADP on TPCs is likely indirect, a clear mechanism of action is difficult to ascertain as NAADP-binding proteins continue to evade molecular identification.2+ channels is not without its challenges. Nevertheless, successful drugging of voltage-gated Ca2+ channels (with for example dihydropyridines) and ryanodine receptors (with dantrolene) in hypertension and malignant hyperthermia offer hope. In this context, emerging structures of TPCs [An alternative (and complementary) approach to manipulate this pathway is to directly target TPCs. Success with tetrandrine in combatting Ebola infection is most encouraging in this respect although its pleiotropic actions should not be ignored . Indeed, of TPCs ,126 shou2+ dependent output, lead the way with the availability of both activators and inhibitors [Finally, with growing evidence linking disorders of the pancreas and liver with loss of TPC function should focus attention on strategies to boost TPC activity. It is intriguing that tolbutamide, a diabetic drug which targets kATP channels to stimulate insulin secretion appears to require TPC2 for its activity . Adminishibitors .No doubt future advances will offer us new approaches to target TPCs in disease.Transparency document.Image 1


The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss- and gain-of-function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five ‘disease-switch’ residues  that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease-switch residue mutations to commonly display signalling bias. For example, two FHH-associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH-associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease-switch residues together with four reported disease-switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease-switch residues are located at sites critical for CaSR activation and play a role in mediating signalling biasThe calcium-sensing receptor (CaSR) is a homodimeric G-protein-coupled receptor that signals via intracellular calcium (Ca Stimulation with Ca2+e induced a concentration-dependent increase in pERK responses in WT, Arg27 and Glu27 CaSR-expressing cells , but reduced responses at high [Ca2+]e (5-10 mM) compared to WT , and therefore these disease-switch residues are not involved in regulating the basal activity of the CaSR. However, mutations affecting the Gln27, Ser657 and Thr828 disease-switch residues altered the maximal responses of the CaSR without influencing EC50 values, which may indicate that these mutations altered the intrinsic signalling efficacy of the CaSR without affecting agonist affinity  or MAPK pathway activation . In particular, the FHH1-associated p.Asn178Asp and p.Ser820Ala mutations led to reductions in Ca2+i mobilisation without any significant impairment of pERK responses. Thus, these mutations may result in the CaSR adopting conformational states that favour signalling away from Gq/11-mediated PLC activation and towards Gi/o and/or β-arrestin-mediated MAPK signalling, as has been recently demonstrated for a CaSR mutation that disrupts a salt-bridge within the TMD  is biased towards the Ca2+i mobilisation pathway   and the 5FBK, 5FBH, 5K5S, 5K5T] (4OR2)  . CrystalS, 5K5T] ,4. The HS, 5K5T] ,48. The ] (4OR2)  template] (4OR2) . Figures2. Mutations were introduced into the reported WT pEGFP-N1-CaSR construct by site-directed mutagenesis using the Quikchange Lightning Kit (Agilent Technologies) and gene-specific primers (SigmaAldrich), as described. WT and mutant pEGFP-N1-CaSR constructs, and luciferase reporter constructs  were transiently transfected into HEK293 cells using Lipofectamine 2000 (LifeTechnologies) 48 h before experiments, as described (Studies were performed in HEK293 cells maintained in DMEM-Glutamax media (ThermoFisher) with 10% fetal bovine serum (Gibco) at 37°C, 5% COescribed .Expression of WT and mutant proteins by the pEGFP-N1-CaSR constructs was confirmed by Western blot analysis, with the calnexin housekeeping protein being used as a loading control . For cel2+e-induced Ca2+i responses were measured by Fluo-4 calcium assays as previously described  at 37°C with an excitation filter of 485 nm and an emission filter of 520 nm. The peak mean fluorescence ratio of the transient response following each individual stimulus was measured using MARS data analysis software (BMG Labtech). Responses were normalized to the maximal response of WT expressing cells and plotted using GraphPad Prism  on a Veritas Luminometer (Promega), as previously described (2 concentrations (0.1 mM). Assays were performed in 8–12 biological replicates  for each of the expression constructs. Statistical analysis was performed using two-way ANOVA. To construct the bias plots the responses at each [Ca2+]e in Ca2+i (x-axis) and pERK (y-axis) assays, were plotted against each other, as previously described  value was calculated using the following equation for each mutant and WT CaSR:2+i) and ‘.2’ denotes response 2 (pERK). The bias factor was calculated relative to WT as follows:Bias factor calculations were based on previous studies ,54. To nP-value < 0.05 was considered significant for all analyses. Comparison of EC50 values was performed using the F test  were used for all statistical comparisons. All data was analysed by two-way ANOVA with Tukey’s multiple-comparisons test. Statistical analyses were undertaken using GraphPad Prism (GraphPad), and a value of e F test .Supplementary DataClick here for additional data file.