Glioma is an aggressive primary tumor of the central nervous system. Calcium (Ca2+) signaling between the ER and mitochondria is essential for cell survival and death regulation. Calumenin (CALU), an ER-resident Ca2+-binding protein, has been implicated in several cancers, but its role in glioma remains unclear. Public datasets (TCGA, CGGA) were analyzed to assess CALU expression and prognosis. We manipulated CALU through transfection technology and tested its role in Ca2+ responses, intracellular Ca2+ store-associated fluorescence, and mitochondrial Ca2+-associated fluorescence signals by means of Fluo-4, Mag-Fluo-4, and Rhod-2 probes, respectively. Mitochondrial Ca2+uptake was further assessed using an isolated mitochondrial Calcium Green-5 N assay. Mitochondrial function was assessed through the detection of MMP and mPTP utilizing JC-1 staining and calcein-AM/cobalt assay. Mitochondrial ROS and total ROS were estimated via mitoSox red staining and DCFH-DA assay. The cell apoptosis was appraised utilizing flow cytometry and TUNEL staining. A xenograft model using U87 glioma cells was established to further explore the role of CALU in vivo. CALU expression was highly expressed in gliomas and correlated with poor survival and mitochondrial Ca2+ transport genes. CALU depletion reduced intracellular Ca2+ store-associated fluorescence, enhanced store-operated Ca2+ entry (SOCE), and induced sustained cytosolic Ca2+elevation accompanied by enhanced mitochondrial Ca2+-associated fluorescence signals and enhanced mitochondrial Ca2+ uptake. These changes were associated with decreased MMP, increased mitochondrial calcein fluorescence, elevated ROS generation, and facilitated apoptosis. In vivo, CALU knockdown reduced tumor growth, increased 4-HNE and cleaved caspase-3 expression and reduced Ki-67 expression, which were partially reversed by NAC treatment. These findings suggest that CALU may contribute to the maintenance of intracellular Ca2+ homeostasis in glioma cells. Loss of CALU induces SOCE-associated Ca2+dysregulation, mitochondrial dysfunction, and ROS-mediated apoptosis, highlighting CALU as a potential therapeutic target for glioma.
Ca²⁺/calmodulin-dependent protein kinase I (CaMKI), a multifunctional CaM-activated protein kinase, is involved in various Ca²⁺ signaling pathways including neuronal development. Here, we characterize the phosphorylation at Thr177 (an activation Thr residue) and subsequent dephosphorylation dynamics of CaMKIα in HeLa cells upon physiological stimulation that elevates intracellular Ca²⁺. ATP induced CaMKIα phosphorylation within 5-10 min; phosphorylation was then blocked by CaMKK inhibitor TIM-063, or by the depletion of extracellular Ca²⁺. This was followed by gradual dephosphorylation to basal levels within 30-60 min. Histamine induced CaMKIα phosphorylation, peaking within 3-4 min; this process was abolished by treatment with either TIM-063 or intracellular Ca²⁺ chelation using BAPTA-AM and thapsigargin; however, not by extracellular Ca²⁺ depletion. CaMKIα was then rapidly dephosphorylated to basal levels within 10 min. Consistently, histamine-induced (but not ATP-induced) CaMKIα phosphorylation was absent in triple IP₃ receptor-knockout HeLa cells. Dephosphorylation of CaMKIα after ATP-induced phosphorylation was unaffected by okadaic acid or CaMK phosphatase (CaMKP, known as PPM1F) inhibitors (ANS and ANDS). We found that HeLa cell extracts contained Mg2+/Mn2+-dependent CaMKIα dephosphorylation activity that was insensitive to ANS and ANDS. Furthermore, co-expression of PP2Cα fully abolished ATP-, histamine-, or ionomycin-stimulated CaMKIα phosphorylation, which is consistent with in vitro dephosphorylation of CaMKIα at Thr177 by recombinant PP2Cα. Taken together, these results reveal that agonist-induced Ca²⁺ influx from the extracellular space or release from intracellular stores transiently activates CaMKK-CaMKIα signaling in HeLa cells, which is shut off by dephosphorylation catalyzed by PP2Cα as a promising candidate for CaMKIα phosphatase.
Piezo1-derived Ca²⁺ signals provide a mechanosensitive route through which mechanical inputs are decoded by mitochondria. Piezo1 converts membrane tension, fluid shear stress, and matrix stiffening into ionic signals, but mitochondrial outcomes depend on how Ca²⁺ is spatially routed, buffered, and amplified. This review integrates plasma membrane-initiated entry, endoplasmic reticulum (ER)-mitochondria communication, voltage-dependent anion channel (VDAC)/mitochondrial calcium uniporter (MCU)-related transfer, cytoskeletal organization, and selected organelle-associated Piezo1 signals. We examine how mechanical dose, pathological microenvironments, and cell state shift mitochondrial decoding from adaptive bioenergetic, redox, and quality-control responses toward Ca²⁺ overload and organelle failure. A shared mitochondrial stress state can then bias apoptosis, ferroptosis, inflammatory death, or senescence, producing tissue-specific outcomes and therapeutic opportunities. The translational goal is not indiscriminate Piezo1 blockade, but restoration of a safe coupling range between Piezo1-derived Ca²⁺ entry and mitochondrial buffering, repair, and fate stability.
In T cells, Dual Oxidase 2 (DUOX2) is responsible for reducing NAADPH to NAADP, a potent second messenger that evokes Ca2+ signaling. However, specific DUOX2 activation mechanisms, particularly upon T-cell receptor (TCR) activation, remained unclear. New findings from Winterberg K. et al. identify two key kinases, PKCθ and PKA Cβ2, that directly phosphorylate and activate DUOX2, thereby accounting for basal TCR-independent and TCR-evoked NAADP/Ca2+ signaling and dictating cytokine production.
Palmitate-induced lipotoxicity contributes to pancreatic β-cell dysfunction, but the underlying molecular mechanisms remain incompletely understood. Because sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is essential for Ca2+ homeostasis and β-cell function, we investigated whether palmitate affects SERCA activity through oxidative and structural perturbations. To model lipotoxic stress and explore a potential mechanism of β-cell injury, SERCA1a-enriched sarcoplasmic reticulum vesicles were exposed to palmitate to examine its effects on pump activity and oxidative/structural changes. We found that palmitate caused a concentration-dependent inhibition of enzyme activity accompanied by changes in thiol accessibility, protein carbonylation, lipid peroxidation, and altered fluorescence signals consistent with perturbation of the transmembrane region of SERCA1a. These effects closely paralleled the loss of Ca2+-ATPase function. In INS-1E β-cells, palmitate reduced viability, induced apoptosis, impaired glucose-stimulated insulin secretion, and decreased SERCA2b expression. Together, these findings identify SERCA as a target of palmitate-induced lipotoxic injury and support a model in which SERCA dysfunction may contribute to β-cell failure under lipotoxic conditions.
Maintaining epithelial cell polarity is a crucial defense against neoplastic progression, ensuring precise spatial and temporal organization of signals. Central to this structural and functional integrity is the compartmentalization of Ca2+ signaling at both intracellular and intercellular levels. The most direct form of intercellular Ca2+ propagation is mediated by gap junction channels (GJs). Their precursors, unopposed hemichannels (HCs), have been recognized for connecting the intracellular and extracellular environments, allowing the passage of a diverse range of small molecules. Although the precise molecular signals that govern specific cellular outcomes in intercellular communication have yet to be fully elucidated, inositol 1,4,5-trisphosphate (InsP3) and Ca2+ play crucial roles as mediators of intercellular communication through GJs. Intracellular Ca2+ release is primarily mediated by specialized release channels, specifically inositol 1,4,5-trisphosphate receptors (ITPRs), which are the predominant release pathways in polarized liver epithelial cells. In the liver, ITPRs isoforms 2 and 3 are specifically targeted to the apical membranes of hepatocytes and cholangiocytes, respectively. Under physiological conditions, Ca2+ signaling is restricted to sub-apical trigger zones to regulate critical cellular functions, most notably, secretion. However, the transition to a pathological state is characterized by the collapse of apical signaling microdomains. Loss of ITPRs apical polarity leads to a shift from compartmentalized signaling to generalized dysregulation and GJs instability. This review synthesizes the current evidence on how the spatial organization of the ITPRs and GJs axis maintains homeostasis and how its breakdown facilitates the progression of liver malignancies.
Bacillus amyloliquefaciens amy-1 is recognized as a probiotic for animals, and its exopolysaccharides (EPS) can lower blood sugar by activating specific human bitter-taste receptor type 2 (TAS2R) subtypes, particularly TAS2R14 and TAS2R38. Humans have 26 TAS2R subtypes, with some exhibiting different activities depending on genotype. However, differences in activity among TAS2R14 genotypes remain poorly studied. This research used THP-1 monocytes to identify TAS2Rs with high expression levels and analyzed how different TAS2R14 genotypes and other highly expressed subtypes respond to EPS. Accordingly, 8 genotypes of TAS2R14 (TIL, AIL, TVL, TIF, AVL, TVF, AIF, and AVF) that were created using site-directed mutagenesis, and TAS2R subtypes 5, 7, 16, 19, 20, 31, 41, and 42 were transfected into Gα-gustducin/HEK293T cells. Ca2+ fluorescence analysis with Fluo-4 showed that EPS activates all tested TAS2Rs, but with varying sensitivity and Ca2+ release levels. TAS2R14 (TIL) and TAS2R7 had the strongest activation. These results may inform further research on the activation of specific TAS2Rs by EPS and its potential as a therapeutic agent for human health.
Transient receptor potential vanilloid 2 (TRPV2) is highly expressed in immune cells, including mast cells, yet its functional role in mast cell physiology remains incompletely understood. Given the increasing global prevalence of immune disorders, elucidating TRPV2-mediated mechanisms may uncover novel therapeutic approaches. Using fluorometric Ca2+ influx assays and electrophysiological patch-clamp recordings, this study analyzes synergistic effects of the TRPV2 activators AV2-1, cannabidiol and probenecid. Superadditive TRPV2 activation was observed upon co-application of probenecid and cannabidiol, as well as by the combination of AV2-1 and cannabidiol. The effects of the activators were validated in primary mouse bone marrow-derived mast cells (mBMMCs) and the human mast cell-like cell line HMC-1.2. Activation of TRPV2 by the cannabidiol/probenecid combination or by AV2-1 alone induced mBMMC degranulation, as assessed by β-hexosaminidase release, histamine secretion, and increased CD63 surface expression measured by flow cytometry, without evidence of long-term cytotoxicity. TRPV2-dependent degranulation was additive to FcεRI-mediated responses induced by anti-DNP-IgE/DNP-HSA stimulation, indicating mechanistically distinct signaling pathways. In addition, TRPV2 activation enhanced mBMMC migration. All observed effects were abolished by the TRPV2 inhibitor IV2-1 or genetic deletion of TRPV2 in transwell migration assays. These findings demonstrate that pharmacological activation of TRPV2 modulates mast cell effector functions and migration, supporting a role for TRPV2 as an immunomodulatory ion channel and a potential therapeutic target in immune system-related pathologies.
Neural crest cells (NCCs) are multipotent stem cells that migrate extensively during embryogenesis and give rise to a broad spectrum of derivatives, including melanocytes, Schwann cells, chromaffin cells, and neurons of the enteric nervous system. NCC proliferation, migration, or differentiation defects lead to a group of disorders collectively known as neurocristopathies. Understanding the signaling pathways that govern NCC behavior is therefore crucial for elucidating the origins of these conditions. Store-Operated Calcium Entry (SOCE) is recognized as the principal calcium influx mechanism in non-excitable cells and regulates key cellular processes such as proliferation, migration, and differentiation. Accumulating evidence indicates a significant role for Ca2+ signaling and SOCE in multiple NCC-derived cell types. This review examines current knowledge on Ca2+ signaling mediated by SOCE in NCCs and their derivatives, and discusses how dysregulated SOCE signaling may influence the pathogenesis of neurocristopathies.
Platelet activation arises from the integration of multiple biochemical cues, but how platelets respond to spatially heterogeneous combinations of agonists and inhibitors remains poorly understood. Here we applied spatially multiplexed stimulation using two independently controlled light-sensitive compounds to generate localized regions enriched in ADP, epinephrine, or nitric oxide, as well as their combinations. Calcium imaging of platelet populations revealed that different spatial contexts produced qualitatively distinct activation dynamics. In regions with high ADP, responses were robust and were selectively suppressed by nitric oxide, whereas epinephrine had little additional effect. In regions with low ADP, epinephrine enhanced calcium signals, indicating increased sensitivity to weak stimulation. A distinct activation mode emerged when low ADP was combined with both epinephrine and nitric oxide: platelets exhibited delayed yet sustained calcium elevations, suggesting nonlinear integration of sensitizing and inhibitory inputs. These results show that spatial variation in multiple stimuli can give rise to diverse platelet activation patterns, highlighting the importance of local agonist composition in shaping platelet signaling dynamics.
The aim of this study was to analyse how fertilization-associated [Ca²⁺]ᵢ dynamics and metabolic responses evolve across IVF media, to identify relationships with developmental potential. Cytosolic Ca²⁺ oscillations initiate egg activation and couple Ca²⁺ signalling to mitochondrial metabolism, yet how IVF media shape this Ca²⁺-metabolic response remains poorly understood. In the standard media tested here, full-term development remained close to 20%, highlighting the need to understand how fertilization media constrain developmental competence.Simultaneous microfluidic recordings of [Ca²⁺]ᵢ using Fura-2 and FAD autofluorescence were obtained from 319 oocytes across 19 conditions, including standard mouse media, commercial IVF media, and experimental formulations differing in energy substrates and extracellular [Mg²⁺]ₒ/[Ca²⁺]ₒ ratio. These recordings were analysed with dedicated algorithms to extract dynamic descriptors of Ca²⁺ excitation, Ca²⁺ release, inter-spike timing, and FAD redox state.The analysis revealed that IVF media modulate two interdependent functional layers of egg activation. The [Mg²⁺]ₒ/[Ca²⁺]ₒ ratio controlled PLCζ/InsP₃-dependent Ca²⁺ excitability, shaping oscillation number, mean frequency, and train duration. Energetic substrates provided a second level of control: glucose stabilized redox dynamics, whereas pyruvate or lactate were associated with redox imbalance, prolonged Ca²⁺ release, extended refractory periods, and lower oscillation frequency.These functional signatures provided a rationale for designing M16CORRECTED, combining adjustment of the [Mg²⁺]ₒ/[Ca²⁺]ₒ ratio with reduced pyruvate and lactate. This formulation stabilized FAD redox dynamics, prolonged the oscillatory regime, and increased full-term survival to 52%. These findings indicate that IVF media can be rationally tuned by balancing Ca²⁺ excitation with metabolic compensation to improve developmental competence.
Cardiomyopathy and arrhythmia development significantly contribute to mortality in patients with Duchenne muscular dystrophy (DMD), a fatal muscle disorder caused by dystrophin deficiency. Previous studies suggested that abnormal Ca handling is an important causative factor for cardiac involvement in DMD. Here, we aimed to provide an extensive analysis of Ca handling properties and arrhythmia vulnerability of cardiomyocytes from the dystrophin-deficient mdx mouse, the most common DMD animal model. Whole cell patch clamp experiments showed that currents through L-type Ca channels are similar in wild-type and mdx cardiac Purkinje myocytes. Intracellular Ca measurements revealed that the decay of electrically evoked Ca transients is significantly prolonged in mdx compared to wild-type ventricular cardiomyocytes, thereby suggesting slowed Ca removal from the cytosol by the sarcoplasmic/endoplasmic reticulum Ca ATPase. The diastolic sarcoplasmic reticulum Ca leak and the resting concentration of free Ca in the cytosol were not significantly increased in mdx compared to wildtype ventricular cardiomyocytes. The occurrence of arrhythmogenic delayed afterdepolarizations (DADs), which are provoked by dysregulated Ca handling, was also not enhanced in mdx compared to wild-type ventricular cardiomyocytes. In conclusion, this work provides evidence that the only significant Ca handling abnormality in mdx cardiomyocytes is slowed removal of cytosolic Ca after release from the sarcoplasmic reticulum. Since the susceptibility of mdx ventricular cardiomyocytes to DADs was normal, cardiac arrhythmias in mdx mice might primarily be triggered by other arrhythmogenic mechanisms than DADs.
Endothelial-to-mesenchymal transition (EndMT) is a cell trans-differentiation process which occurs during embryonic development as well as in a variety of pathological contexts. During this process, cells lose their endothelial lineage markers and gain mesenchymal ones. It is stimulated by several factors, such as TGF-β, high glucose, or hypoxia, which trigger intracellular pathways, mainly the Smad and ERK pathways, leading to modulation of transcription factors. While Ca2+ signaling is known to participate in a wide variety of cellular processes, its role in the context of EndMT is only partially understood, although several Ca2+-carrying and Ca2+-regulated proteins have been shown to influence EndMT. These include Ca2+-permeable channels from the mechanosensitive Piezo family and members of the TRP family; the actors involved in store-operated Ca2+ entry, including IP3-receptors located on the endoplasmic reticulum membrane, the endoplasmic reticulum Ca2+ sensor STIM1, and the Ca2+-permeable plasma membrane channel Orai1; the mitochondrial Ca2+uniporter complex; Ca2+-activated channels such as the non-selective monovalent cation channel TRPM4 and the big-conductance K+ channel BKCa; Ca2+-regulated signaling pathways such as the Ca2+-calmodulin-calcineurin-NFAT pathway and the calpain-ERK pathway.After a brief description of Ca2+ signaling in endothelial cells, this review will specifically detail the available data on the Ca2+ signaling in EndMT. Links between Ca2+ signaling and other signaling pathways leading to EndMT will be discussed. Finally, molecular targets that may be valuable to explore in order to prevent EndMT will be highlighted.
Many intracellular pathogens manipulate host cell calcium to facilitate their survival and replication. Live-cell microscopy using fluorescent calcium indicators has become an indispensable tool for characterizing the mechanisms underlying both homeostatic and pathogen-induced cellular calcium dynamics, but such imaging must be coupled with robust quantitative analysis. Further, calcium imaging is most powerful when paired with reductive studies targeting calcium-modulating proteins. The lack of specific inhibitors or agonists to directly target most pathogen-induced calcium signals precludes many of the approaches that have allowed for robust characterization of major eukaryotic cell calcium signaling mechanisms, such as ER Ca2+ release by inositol triphosphate receptors. Given this, we sought to develop quantitative imaging pipelines tailored for the characterization of pathogen-induced calcium signals. Using rotavirus as a prototypical calcium-modulating pathogen, we developed and optimized a suite of computational tools for automated quantitation of both intra- and inter-cellular calcium signals detected via live-cell imaging of infected epithelial monolayers expressing genetically encoded calcium indicators. Using recombinant strains of rotavirus that express fluorescent markers, we developed a system that allows for automated detection of rotavirus-infected cells and normalization of signals to infectivity. All tools were built in ImageJ, making them freely available and adaptable across operating systems and microscope setups. These tools required minimal active time from the user and allowed for the extraction of signal parameters previously unquantifiable, increasing the speed and breadth of characterization.
During the past decade, advancements in CryoEM have afforded significant insight into transmembrane regions of ion channels. Portions of cytosolic amino acids have been structurally and biophysically characterized; however, understanding structural relationships and how components integrate into a functional ion channel required clarification. Here, we have calculated an all-atom model for the complete sequence of the cardiac voltage-gated calcium ion channel (CaV1.2 alpha-1C and beta-3 subunits) in a lipid bilayer with explicit salt and water. Four one-microsecond molecular dynamics (MD) simulations of the isolated alpha subunit improved backbone torsion angles for non-transmembrane residues relative to the AlphaFold model. Two 500 ns MD simulations of CaV1.2 (alpha and beta subunits) provided insight into the binding interface and structural features of the complex. The time component of our MD simulation provided unique insight into side chain dynamics, solvation of voltage sensors, and features of the channel pore. Repeating the alpha subunit simulations with a highly penetrant Timothy Syndrome point mutation (G406R) predicted changes to side chain dynamics that may contribute to a destabilization mechanism of an inactivated channel configuration. Lastly, a comparison of our WT post-MD model with empirical data obtained in the presence of small molecules yielded mechanistic insights into several small molecule CaV1.2 interactions.
Stromal Interaction Molecule 1 (STIM1) is a ubiquitous protein that triggers extracellular Ca2+ entry after endoplasmic reticulum store depletion, a mechanism called store-operated Ca2+ entry (SOCE). In skeletal muscle, a longer splicing variant of STIM1 accounts for half of the SOCE. In this study, we deciphered the impact of the canonical STIM1, or the long muscle variant (STIM1L) on human muscle regeneration. We showed that both protein knockdowns led to defects in excitation-contraction coupling. Furthermore, STIM1 but not STIM1L functionally interacts with PMCA1, enhancing Ca2+ extrusion. Additionally, STIM1L-dependent SOCE did not trigger NFATc1 translocation, while the SOCE induced by STIM1 led to NFATc1 translocation both at rest and under stimulated conditions. In parallel, the downregulation of PMCA1 decreased basal Ca2+ entry as well as NFATc1 translocation. Overall, we propose that STIM1 but not STIM1L increases PMCA1-dependent Ca2+ extrusion, facilitating NFATc1 activation, possibly through the attenuation of Ca2+-dependent inactivation of Orai1. This mechanism, in turn, favors skeletal muscle maturation by increasing myotube growth.
A healthy pregnancy depends on proper placental development, which requires tightly regulated calcium (Ca2+) signaling. In non-excitable cells, store-operated Ca2+ entry (SOCE) is mediated by Ca2+ release-activated Ca2+ (CRAC) channels, composed of Orai1, Orai2 and Orai3 subunits and activated by stromal interaction molecules (STIM1 and STIM2). In this study, we investigated the potential role of CRAC channels in extravillous trophoblast (EVT) function. Using the human placenta-derived EVT cell line HTR8/SVneo, we combined CRISPR/Cas9, immunocytochemistry, confocal microscopy, whole-cell patch-clamp electrophysiology, Ca2+ imaging, pharmacological CRAC channel inhibitors, and functional assays of cell proliferation, migration, and invasion to determine the contribution of Orai1 to the regulation of key trophoblast functions. In human primary EVTs, STIM1-2 and Orai1-3 mRNA expression was analyzed from single-cell RNAsequencing data (Cambridge Trophoblast Atlas). Similar to human primary EVT, HTR8/SVneo cells expressed all STIM and Orai isoforms, and Orai1 is the predominant pore-forming ion channel subunit. Targeted deletion of Orai1 or its pharmacological inhibition with BTP2 or GSK-7975A significantly reduced ICRAC, SOCE, and impaired EVT cell viability, migration, and invasion. In conclusion, our findings demonstrate the role of Orai1 in mediating ICRAC, SOCE, and SOCE-dependent EVT functions and suggest its potential involvement in EVT (patho)physiology.
Normal microvascular function is critical for tissue perfusion, and intestinal vascular dysfunction is a major pathological feature of colitis and sepsis. Although dimethyl sulfoxide (DMSO) as a versatile solvent exerts antiinflammatory and vasoactive properties in conduit vessels, its pharmacological effects on arterioles have not been explored. This study repurposed it as a potential therapeutic agent for these two gut-derived inflammatory diseases, aiming to elucidate its direct effects and underlying mechanisms on intestinal resistance arterioles under both physiological and pathological conditions. Mulvany-style wire myography was used to assess DMSOinduced vasorelaxation in murine mesenteric arterioles. Intracellular Ca2+ dynamics in human umbilical vein endothelial cells (HUVECs) were assessed using Ca2+ imaging, and the membrane potential changes were monitored by patch clamp. Mouse models of dextran sulfate sodium (DSS)-induced colitis and cecal ligation puncture (CLP)-induced sepsis were used. To this end, we showed that DMSO elicited concentration-dependent vasorelaxation of mesenteric arterioles, predominantly mediated by endothelium-dependent hyperpolarization (EDH) mechanism in healthy mice. DMSO activated PLC/IP3/IP3R signaling pathway and store-operated Ca2+ entry (SOCE) in endothelial cells to trigger EDH-mediated vasorelaxation. In colitic and septic mice, DMSO significantly alleviated the severity of the diseases, such as body weight, stool score, colon length, and survival rate. Moreover, DMSO/EDH-mediated vasorelaxation was largely preserved in colitic and septic mice. Importantly, DMSO pretreatment restored the ACh/EDH-mediated vasorelaxation impaired in colitic and septic mice. This study reveals a previously overlooked pharmacological effect of DMSO as a common solvent, strongly suggesting that it could be repositioned as a safe therapeutic agent for improving microcirculation under inflammatory conditions, such as colitis and sepsis.
