TMEM16A-mediated Cl[-] fluxes promote contraction and set vascular tone in smooth muscle cells. In myocytes, CaMKII-mediated phosphorylation downregulates Cl[-] fluxes, while Ca[2+]-dependent phosphatases such as calcineurin (CaN) upregulate them. In HEK293 cells, we showed that increased intracellular Ca[2+] promotes CaN association with FKBP12 and TMEM16A, and that the resulting complex upregulates TMEM16A activity. However, the specific binding site mediating the interaction between TMEM16A, CaN, and FKBP12 remained unknown. Here, using a stable CaN-knockdown cell line, we show that endogenous FKBP12 still co-immunoprecipitated with TMEM16A in adherent HEK293 (HEK-AD293) cells. In addition, we observed an association between TMEM16A and recombinant FKBP12 on the cytoplasmic face of plasma membranes from HEK-AD293 cells, indicating that FKBP12 can associate with TMEM16A-containing plasma membranes without an intact cytoplasmic environment. Co-localization of TMEM16A-EGFP and mRFP-FKBP12 in HEK-AD293 cells stimulated with the Ca[2+] ionophore ionomycin, along with live-cell fluorescence complementation, provided fluorescence-based indirect evidence for this interaction. Finally, using GST pull-down and fluorescent complementation, we found that the TMEM16A C-terminus is necessary for FKBP12 binding. Consistent with this, C-terminally truncated TMEM16A currents were insensitive to the CaN inhibitor FK506; cyclosporin and FK506 had no effect on TMEM16A activity in CaN-depleted cells. In summary, we provide evidence that FKBP12 directly binds to the C-terminus of TMEM16A, potentially serving as a molecular anchor that recruits and localizes CaN to the channel.

Calcium-calmodulin signaling represents a central mechanism through which intracellular calcium fluctuations coordinate diverse cellular processes. Despite the identification of numerous calmodulin binding proteins, a systems level understanding of how these interactions integrate into global signaling networks and influence disease relevant pathways remains incomplete. Here, we constructed a comprehensive human calmodulin interactome by integrating experimentally validated protein-protein interactions from the IntAct and BioGRID databases, identifying 919 calmodulin associated proteins. Network analysis revealed highly connected hub proteins, including SRC and UBC, and delineated three major functional clusters enriched in translational machinery, calcium dependent kinase signaling, and actin cytoskeleton organization. Functional enrichment analyses further highlighted strong associations with phosphosignaling pathways, cytoskeletal dynamics, and hormone mediated signaling processes. Further, classification of interactome into functional categories of interest identified 102 kinases, 16 phosphatases, 24 metabolic enzymes, 29 ion channel proteins, and 56 transcription factors, underscoring the broad regulatory scope of calmodulin signaling. Integration and evaluation of publicly available calcium perturbation proteomics datasets to assess potential Ca[2+]-dependent and Ca[2+]-independent calmodulin interactions identified 43 presumed calmodulin-interacting proteins exhibiting consistent calcium-responsive regulation. Importantly, kinase substrate network mapping revealed that 46 presumed calmodulin interacting kinases phosphorylate 155 human transcription factors, suggesting formation of an extensive calmodulin linked phospho-regulatory transcriptional network. Collectively, these findings establish a systems level framework of the calmodulin signaling axis and highlight its potential relevance in transcriptional regulation, providing a resource for future mechanistic and therapeutic investigations.

Astrocytes and microglia maintain cortical homeostasis via dynamic intracellular calcium (Ca[2+]) signaling, but their state-dependent activity and neuronal dependencies in the auditory cortex remain poorly understood. Here, using in vivo two-photon imaging, combined with cell-type-specific viral expression of GCaMP sensors, we monitored somatic Ca[2+] transients in awake and anesthetized mice. Wakefulness significantly enhanced the active cell fraction, frequency, and amplitude of somatic Ca[2+] events in both glial populations compared to the anesthetized state. Local application of tetrodotoxin (TTX) completely abolished microglial Ca[2+] activity, revealing an obligatory dependence on neuronal firing. In contrast, astrocytic Ca[2+] events persisted at similar frequencies but exhibited significantly reduced peak amplitudes, indicating partial autonomy coupled with neuronal modulation. Together, our findings demonstrate that wakefulness coordinately boosts glial Ca[2+] activity, while microglia and astrocytes rely on fundamentally distinct relationships with local neuronal networks.

TRPV3 is a heat- and ligand-gated calcium-permeable ion channel, which is mostly expressed in the skin and plays important roles in skin physiology. Targeting TRPV3 with selective ligands represents a promising therapeutic strategy for the correction of cutaneous pathological processes. For the rational drug design and prognosis of clinical outcomes, it is essential to understand the mechanisms of ligand action. Here, we study the mechanism of TRPV3 activation by cannabidiol (CBD) and inhibition by 14-deoxy-11,12-didehydroandrographolide (DDA). Pharmacological data were obtained via calcium imaging in TRPV3-expressing CHO cells and two-electrode voltage-clamp recordings in Xenopus oocytes. We show that CBD activates human TRPV3 with the EC50 of 12.8 µM, while DDA potently inhibits CBD-evoked activation with the IC50 of 7 nM. DDA acts as a modality-specific inhibitor: a competitive reversible antagonist for CBD activation, a negative allosteric modulator for 2-aminoethoxydiphenyl borate (2-APB) activation, and is neutral to heat activation. Using molecular docking and site-directed mutagenesis, we show that both CBD and DDA bind to the vanilloid site, an important regulatory pocket in the transmembrane region of TRPV3. Thus, single-residue substitutions in the vanilloid site disrupt activation by CBD (A560L) or increase its activation potency (L664S), and abrogate TRPV3 inhibition by DDA (L557A, L664S). Our results expand the pharmacological profiles of CBD and DDA and highlight their potential as drug candidates for TRPV3-targeting therapeutics.

Erythrocyte-derived microvesicles have been reported to be elevated in patients exposed to supraphysiological shear stress in the context of blood-contacting medical devices, and they have also been implicated in thrombosis and inflammation. Intracellular calcium has been proposed as a mediator of erythrocyte vesiculation. However, whether intracellular calcium accumulation is necessary for microvesicle formation under the short millisecond duration, high-intensity shear conditions present clinically remains unclear. In this study, we investigated whether calcium accumulation is necessary for shear-induced microvesicle formation using two distinct experimental setups that contain supraphysiological shear stresses. Human RBCs were exposed to transient (0 ms to 10 ms), supraphysiological shear rates (100,000 s[-1]) in microfluidic devices that have been previously shown to generate microvesicles. The proportion of post-shear, calcium-positive RBCs was assessed using Fluo-4, AM via flow cytometry. A second experiment included bovine erythrocytes circulating through a CentriMag blood pump for six hours in calcium-containing and calcium-free media. The pump imposed multiple exposures to supraphysiological shear stress of extremely short durations. Generated microvesicle release was quantified using carboxyfluorescein succinimidyl ester labeling. Despite exposure to extreme shear, microfluidic experiments revealed minimal calcium-positive RBCs following shear exposure. No detectable sustained calcium in the erythrocytes was observed. In the mock loop, CFSE-positive MV concentrations increased in both calcium media and calcium-free media with six hours of circulation time, independent of calcium presence. These findings suggest that, under the conditions of these experiments, erythrocyte vesiculation can occur in the absence of detectable calcium accumulation and independently of extracellular calcium availability.

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.