The Ca[2+]-activated Cl[-] channel TMEM16A translates cytosolic Ca[2+] signals into transmembrane Cl[-] fluxes, supporting essential physiological functions across diverse tissues. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phosphoinositide enriched in the inner leaflet of the plasma membrane, has emerged as a key regulator of TMEM16A. However, the role of endogenous PI(4,5)P2 and the specific binding sites mediating its regulatory effects remain poorly understood. Here, using electrophysiology, voltage-sensitive 5-phosphatases, site-directed mutagenesis, and molecular dynamics simulations, we show that endogenous PI(4,5)P2 facilitates TMEM16A activation by lowering the apparent gating energy by ∼0.89 kcal/mol at physiological Ca[2+] concentrations. This energetic contribution remains consistent across Ca[2+]-dependent activation states, within experimental resolution. Cumulative disruption of previously identified PI(4,5)P2 binding sites 1 or 3 revealed that Site 1 promotes pore opening, whereas Site 3 functions in a manner consistent with a modulation of the apparent gating energy without detectable effects on pore function. Simultaneous disruption of both sites produced no additional change in apparent gating energy, suggesting that Sites 1 and 3 act as a functional unit that defines the energetic minimum required for channel opening. Residue R484 serves as a structural bridge coupling both Sites under endogenous PI(4,5)P2 conditions. Together, these findings support a two-site model in which endogenous PI(4,5)P2 shapes the energetic landscape, thereby facilitating TMEM16A pore opening. These findings contribute to understanding the role of PI(4,5)P2 as a central endogenous modulator of TMEM16A.

Microglia, the resident macrophages of the central nervous system, continuously survey the parenchyma with their highly motile processes. While calcium (Ca[2+]) signaling is a potential regulator of this process, the intrinsic mechanisms controlling process dynamics remain poorly defined. Here, we characterized microglial Ca[2+] signals evoked by purinergic receptor activation both in vitro and in situ. In cultured microglia, UDP/UTP-induced P2Y6 receptor activation evoked robust Ca[2+] signals with a sustained component mediated by store-operated Ca[2+] entry (SOCE). In contrast, Ca[2+] signals induced by P2Y6 and P2Y12/P2Y13 receptor activation in acutely isolated microglia or in situ microglia were dominated by intracellular store release, with minimal SOCE contribution. Functionally, Ca[2+] signaling induced by P2Y6 activation or store depletion suppressed dorsal ruffling-an F-actin-dependent process-in cultured microglia. In brain slices, these Ca[2+] signals triggered the retraction of fine processes and reduced process surveillance, while chemogenetic activation of Ca[2+] signaling in vivo similarly decreased process complexity. Crucially, this microglial structural remodeling was associated with exacerbated status epilepticus. Thus, our study demonstrates that microglial Ca[2+] signals act as a potent suppressor of process dynamics, thereby regulating neuronal excitability.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with disease progression and distant metastasis being the major determinants of mortality. The ion channel transient receptor potential melastatin-4 (TRPM4) has been associated with several types of cancer and has been shown to regulate key cellular functions. This study investigates the role of TRPM4 in two CRC cell lines of Dukes' stage C, DLD-1 and LoVo, using 2D and 3D models. CRISPR/Cas9-mediated knockout (KO) of TRPM4 led to a complete loss of Ca[2+]-activated non-selective cation channel current (ICAN) demonstrating that in Dukes' stage C CRC cells ICAN is exclusively mediated by TRPM4. TRPM4 deficiency in LoVo and in DLD-1 cells increased store-operated Ca[2+] signaling. Loss of TRPM4 resulted in unchanged proliferation, reduced metabolic activity, and migration in DLD-1 cells. LoVo TRPM4 KO cells presented decreased metabolic activity but showed increased proliferation and increased migration, that was associated with higher expression levels of fibronectin 1 (FN1). In 3D models, TRPM4 KO consistently reduced tumor spheroid formation and outgrowth in both cell lines. Gene expression analysis revealed differential regulation of genes related to WNT/β-catenin signaling, epithelial-to-mesenchymal transition (EMT), and cell adhesion upon TRPM4 KO. These findings suggest that TRPM4 supports tumor growth and dissemination in CRC. However, TRPM4 differentially affects expression levels of cancer-related genes, emphasizing the need for personalized approaches when considering TRPM4 as a therapeutic target in CRC.

Pigmentation is a critical protective mechanism that safeguards the skin against UV-induced damage, whereas dysregulated pigmentation predisposes to pigmentary disorders and skin malignancies. Although calcium signaling has emerged as an important regulator of melanogenesis, the identity of the calcium-handling proteins and the molecular mechanisms linking calcium dynamics to pigmentation remain poorly understood. Here, we identify the ER calcium pump SERCA2b as a negative regulator of pigmentation through modulation of ER stress and mitochondrial calcium uptake. We demonstrate that SERCA2b expression inversely correlates with pigmentation levels, and gain- and loss-of-function studies establish SERCA2b as a suppressor of melanogenesis. Mechanistically, SERCA2b depletion induces adaptive ER stress, enhances ER-mitochondrial proximity, and promotes mitochondrial calcium uptake. Notably, mutations in SERCA2b are associated with Darier disease, a condition characterized by hyperpigmented skin lesions, although the underlying mechanism remains unknown. To address this, we generated SERCA2b mutants corresponding to variants identified in Indian Darier's disease patients and examined their effects on pigmentation, ER stress, and mitochondrial calcium dynamics. The mutant phenotypes closely recapitulated SERCA2b loss-of-function effects, demonstrating that adaptive ER stress and enhanced mitochondrial calcium signaling underlie hyperpigmentation associated with Darier's disease. Importantly, treatment with 4-phenylbutyrate (4-PBA), an FDA-approved ER stress alleviator, rescued mutant-induced hyperpigmentation, reduced ER stress, and normalized mitochondrial calcium uptake. Collectively, our findings uncover a previously unrecognized role of SERCA2b in skin pigmentation, establish a mechanistic link between SERCA2b mutations and hyperpigmentation; and identify adaptive ER stress pathways as potential therapeutic target for pigmentary disorders.

Lysosomal Ca[2+] release can trigger endoplasmic reticulum (ER) Ca[2+] mobilisation, which would be expected to activate store-operated Ca[2+] entry (SOCE). Recent work by Lee et al. reveals that activation of the lysosomal two-pore channel TPC2 instead suppresses SOCE despite ER Ca[2+] depletion. TPC2-derived Ca[2+] signals recruit calmodulin to promote STIM1 inactivation, imparing STIM oligomerisation and coupling to Orai1. These findings identify a lysosomal feedback pathway that restrains Ca[2+] influx and expand the role of TPC2 from an initiator of intracellular Ca[2+] release to a brake on SOCE.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe ciliopathy caused by mutations in Polycystic kidney and hepatic disease 1 gene (PKHD1), resulting in progressive renal and hepatobiliary disease for which effective targeted therapies are lacking. Although reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression has been implicated in hepatic cystogenesis, whether pharmacological restoration of CFTR is associated with improvement of the underlying cellular abnormalities remains unclear. We investigated the effects of the CFTR correctors VX-809 and VX-445, and compared them with the CFTR inhibitor CFTRinh-172, in cholangiocytes isolated from Pkhd1[del3-4/del3-4] mice. FPC-deficient cholangiocytes exhibited enhanced cyst growth and proliferation together with marked reductions in CFTR, polycystin-1 (PC1), and polycystin-2 (PC2) protein expression. These changes were accompanied by profound remodeling of intracellular signaling, including elevated resting cytosolic Ca[2+], an increased thapsigargin-releasable endoplasmic reticulum Ca[2+] pool, increased STIM1 expression, reduced IP3 receptor expression, a marked increase in intracellular cAMP, and a switch in adenylyl cyclase isoform expression characterized by increased AC3 and reduced AC6. CFTR localization was also altered, with redistribution toward the apical membrane. Treatment with either VX-809 or VX-445 improved many of these abnormalities by increasing CFTR expression, partially restoring PC1 and PC2 expression, attenuating abnormal Ca[2+] and cAMP signaling, reducing cholangiocyte proliferation and cyst growth, and shifting CFTR membrane distribution toward the WT pattern. In contrast, CFTRinh-172 failed to reproduce many of the same effects, indicating that inhibition of chloride conductance alone does not account for the broader cellular effects of the CFTR correctors. These findings identify coordinated remodeling of Ca[2+]- and cAMP-dependent signaling as a central feature of ARPKD cholangiocytes and show that CFTR correctors are associated with improvement of multiple disease-associated pathways. Our results support further evaluation of clinically available CFTR correctors as a potential therapeutic strategy for hepatobiliary disease in ARPKD.

IL-33 is an alarmine released upon tissue injury. Mast cells (MCs) highly express the IL-33 receptor T1/ST2 (IL-33R) and are thus highly susceptible to released IL-33. Activation of the IL-33R on MCs induces the MyD88-dependent activation of the TAK1-IKK2-p65/RelA signaling pathway, ERK1/2 and p38 which together mediate the production of IL-6, IL-13 and IL-2. Whereas MC-derived IL-6 and IL-13 act as pro-inflammatory cytokines, MC-derived IL-2 acts as an anti-inflammatory cytokine by inducing Treg cell expansion in vitro and in vivo. The mechanism, which mediate this pleiotropic nature within IL-33-activated MCs is unknown. We hypothesized that precisely balanced signaling pathways define whether MCs act as pro-inflammatory cells by the production of IL-6 and IL-13, or as anti-inflammatory cells by the production of IL-2. Surprisingly, we identified Ca[2+] as the determinant factor, which balances the IL-33-induced production of IL-6, IL-13 and of IL-2. Thereby, whereas Ca[2+] activates the Calcineurine (CaN)-NFATc2-ERK1/2 signaling pathway, which selectively mediates the production of IL-2, Ca[2+] simultaneously limits the activation of p38 and the subsequent production of IL-6 and IL-13 in IL-33-activated MCs. Given that IL-2 producing MCs act as anti-inflammatory cells, our data show that the Ca[2+]-CaN-NFATc2-ERK1/2 signaling pathway determines the anti-inflammatory potential of IL-33-activated MCs.

Ferroptosis is executed by iron-dependent phospholipid peroxidation, yet the upstream signals that determine when oxidizable lipid pools become lethal remain incompletely resolved. Recent work identifies a calcium-dependent protein kinase C beta (PKCβ) pathway that phosphorylates acyl-CoA synthetase long-chain family member 4 (ACSL4) and arachidonate 15-lipoxygenase (ALOX15), and reports relocation of an ACSL4-PKCβ-ALOX15 complex to lipid droplets during ferroptotic stress. These findings connect calcium dynamics to lipid metabolism through a spatially organized phosphorylation program. We propose that lipid droplets should not be classified as constitutively protective or pro-ferroptotic organelles. Instead, they may undergo a calcium-gated licensing transition: under basal or transient stress, neutral-lipid storage buffers polyunsaturated fatty acids; under sustained, spatially restricted calcium signaling, PKCβ-dependent phosphorylation may convert the lipid-droplet surface into a catalytic staging platform that supplies and oxidizes ferroptosis-sensitive lipids. This model explains why lipid-droplet biogenesis can suppress ferroptosis in one context but support it in another, and it predicts a relay from lipid droplets to endoplasmic-reticulum and endoplasmic-reticulum-mitochondria contact sites where phospholipid peroxidation can propagate. Distinguishing initiating calcium microdomains from late calcium influx through damaged plasma membranes will be essential. Phosphosite-resolved imaging, organelle-targeted calcium sensors, contact-site lipidomics, and tissue-specific perturbation of PKCβ should test this framework and may enable selective ferroptosis induction in cancer while preserving calcium-overloaded tissues such as the exocrine pancreas.

Glioma is an aggressive primary tumor of the central nervous system. Calcium (Ca[2+]) signaling between the ER and mitochondria is essential for cell survival and death regulation. Calumenin (CALU), an ER-resident Ca[2+]-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 Ca[2+] responses, intracellular Ca[2+] store-associated fluorescence, and mitochondrial Ca[2+]-associated fluorescence signals by means of Fluo-4, Mag-Fluo-4, and Rhod-2 probes, respectively. Mitochondrial Ca[2+]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 Ca[2+] transport genes. CALU depletion reduced intracellular Ca[2+] store-associated fluorescence, enhanced store-operated Ca[2+] entry (SOCE), and induced sustained cytosolic Ca[2+]elevation accompanied by enhanced mitochondrial Ca[2+]-associated fluorescence signals and enhanced mitochondrial Ca[2+] 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 Ca[2+] homeostasis in glioma cells. Loss of CALU induces SOCE-associated Ca[2+]dysregulation, mitochondrial dysfunction, and ROS-mediated apoptosis, highlighting CALU as a potential therapeutic target for glioma.

Ca[2+]/calmodulin-dependent protein kinase I (CaMKI), a multifunctional CaM-activated protein kinase, is involved in various Ca[2+] 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 Ca[2+]. ATP induced CaMKIα phosphorylation within 5-10 min; phosphorylation was then blocked by CaMKK inhibitor TIM-063, or by the depletion of extracellular Ca[2+]. 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 Ca[2+] chelation using BAPTA-AM and thapsigargin; however, not by extracellular Ca[2+] 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 Mg[2+]/Mn[2+]-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 Ca[2+] 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 Ca[2+] 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 Ca[2+] 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 Ca[2+] 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 Ca[2+] entry and mitochondrial buffering, repair, and fate stability.

Palmitate-induced lipotoxicity contributes to pancreatic β-cell dysfunction, but the underlying molecular mechanisms remain incompletely understood. Because sarco/endoplasmic reticulum Ca[2+]-ATPase (SERCA) is essential for Ca[2+] 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 Ca[2+]-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.

The aim of this study was to analyse how fertilization-associated [Ca[2+]]ᵢ dynamics and metabolic responses evolve across IVF media, to identify relationships with developmental potential. Cytosolic Ca[2+] oscillations initiate egg activation and couple Ca[2+] signalling to mitochondrial metabolism, yet how IVF media shape this Ca[2+]-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 [Ca[2+]]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 [Mg[2+]]o/[Ca[2+]]o ratio. These recordings were analysed with dedicated algorithms to extract dynamic descriptors of Ca[2+] excitation, Ca[2+] release, inter-spike timing, and FAD redox state. The analysis revealed that IVF media modulate two interdependent functional layers of egg activation. The [Mg[2+]]o/[Ca[2+]]o ratio controlled PLCζ/InsP3-dependent Ca[2+] 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 Ca[2+] release, extended refractory periods, and lower oscillation frequency. These functional signatures provided a rationale for designing M16CORRECTED, combining adjustment of the [Mg[2+]]o/[Ca[2+]]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 Ca[2+] excitation with metabolic compensation to improve developmental competence.

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 Ca[2+] signaling at both intracellular and intercellular levels. The most direct form of intercellular Ca[2+] 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 Ca[2+] play crucial roles as mediators of intercellular communication through GJs. Intracellular Ca[2+] 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, Ca[2+] 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.