The effect of genetic polymorphisms in STIM1 and ORAI1 on erythropoietin resistance in Egyptian patients with end-stage renal disease.

Chronic renal failure (CRF) is an incurable disease with unique challenges. Anemia is a frequent complication affecting dialysis patients. Erythropoietin (EPO) is used to treat anemia, but a poor response may result. We investigated genetic polymorphisms of store-operated calcium channel (SOC) signaling, an important erythropoietin-activated pathway that may induce EPO resistance in patients with renal failure. A total of 108 end stage renal disease (ESRD) patients were selected for this study. Patients were divided into two groups according to their erythropoietin resistance index (ERI): 39 patients with an ERI>10 and 69 patients with an ERI<10. We selected four tagging single nucleotide polymorphisms (tSNPs) in STIM1 and five in ORAI1 in our study. A polymerase chain reaction was performed, and genotyping against EPO resistance was correlated. Patients with the AG genotype of rs1561876 in STIM1, the TC genotype of rs6486795 in ORAI1, and the TG or GG genotypes of rs12320939 in ORAI1 were associated with an increased risk of erythropoietin resistance. Overall, we reported a moderately significant relationship between genetic polymorphisms of STIM1 and EPO resistance. We also reported a highly significant relationship between genetic polymorphisms of ORAI1 and EPO resistance. The (A-A-G) haplotype of STIM1 and the (G-T-G-T-A, G-C-G-C-G, or G-T-T-C-G) haplotypes of ORAI1 were significantly associated with EPO resistance.

Feedback modulation of Orai1α and Orai1ß protein content mediated by STIM proteins.

Store-operated Ca2+ entry is a mechanism controlled by the filling state of the intracellular Ca2+ stores, predominantly the endoplasmic reticulum (ER), where ER-resident proteins STIM1 and STIM2 orchestrate the activation of Orai channels in the plasma membrane, and Orai1 playing a predominant role. Two forms of Orai1, Orai1α and Orai1ß, have been identified, which arises the question whether they are equally regulated by STIM proteins. We demonstrate that STIM1 preferentially activates Orai1α over STIM2, yet both STIM proteins similarly activate Orai1ß. Under resting conditions, there is a pronounced association between STIM2 and Orai1α. STIM1 and STIM2 are also shown to influence the protein levels of the Orai1 variants, independently of Ca2+ influx, via lysosomal degradation. Interestingly, Orai1α and Orai1ß appear to selectively regulate the protein level of STIM1, but not STIM2. These observations offer crucial insights into the regulatory dynamics between STIM proteins and Orai1 variants, enhancing our understanding of the intricate processes that fine-tune intracellular Ca2+ signaling.

Thermosensing ability of TRPC5: current knowledge and unsettled questions.

Our understanding of how the mammalian somatosensory system detects noxious cold is still limited. While the role of TRPM8 in signaling mild non-noxious coolness is reasonably understood, the molecular identity of channels transducing painful cold stimuli remains unresolved. TRPC5 was originally described to contribute to moderate cold responses of dorsal root ganglia neurons in vitro, but mice lacking TRPC5 exhibited no change in behavioral responses to cold temperature. The question of why a channel endowed with the ability to be activated by cooling contributes to the cold response only under certain conditions is currently being intensively studied. It seems increasingly likely that the physiological detection of cold temperatures involves multiple different channels and mechanisms that modulate the threshold and intensity of perception. In this review, we aim to outline how TRPC5 may contribute to these mechanisms and what molecular features are important for its role as a cold sensor.

An Orai1 gain-of-function tubular aggregate myopathy mouse model phenocopies key features of the human disease.

Tubular aggregate myopathy (TAM) is a heritable myopathy primarily characterized by progressive muscle weakness, elevated levels of creatine kinase (CK), hypocalcemia, exercise intolerance, and the presence of tubular aggregates (TAs). Here, we generated a knock-in mouse model based on a human gain-of-function mutation which results in a severe, early-onset form of TAM, by inducing a glycine-to-serine point mutation in the ORAI1 pore (Orai1G100S/+ or GS mice). By 8 months of age, GS mice exhibited significant muscle weakness, exercise intolerance, elevated CK levels, hypocalcemia, and robust TA presence. Unexpectedly, constitutive Ca2+ entry in mutant mice was observed in muscle only during early development and was abolished in adult skeletal muscle, partly due to reduced ORAI1 expression. Consistent with proteomic results, significant mitochondrial damage and dysfunction was observed in skeletal muscle of GS mice. Thus, GS mice represent a powerful model for investigation of the pathophysiological mechanisms that underlie key TAM symptoms, as well as those compensatory responses that limit the damaging effects of uncontrolled ORAI1-mediated Ca2+ influx.

Discovery of selective Orai channel blockers bearing an indazole or a pyrazole scaffold.

The calcium release activated calcium (CRAC) channel is highly expressed in T lymphocytes and plays a critical role in regulating T cell proliferation and functions including activation of the transcription factor nuclear factor of activated T cells (NFAT), cytokine production and cytotoxicity. The CRAC channel consists of the Orai pore subunit and STIM (stromal interacting molecule) endoplasmic reticulum calcium sensor. Loss of CRAC channel mediated calcium signaling has been identified as an underlying cause of severe combined immunodeficiency (SCID), leading to drastically weakened immunity against infections. Gain-of-function mutations in Orai and STIM have been associated with tubular aggregated myopathy (TAM), a skeletal muscle disease. While a number of small molecules have shown activity in inhibiting the CRAC signaling pathway, the usefulness of those tool compounds is limited by their off-target activity against TRPM4 and TRPM7 ion channels, high lipophilicity, and a lack of understanding of their mechanism of action. We report structure-activity relationship (SAR) studies that resulted in the characterization of compound 4k [1-(cyclopropylmethyl)-N-(3-fluoropyridin-4-yl)-1H-indazole-3-carboxamie] as a fast onset, reversible, and selective CRAC channel blocker. 4k fully blocked the CRAC current (IC50: 4.9 µM) and the nuclear translocation of NFAT at 30 and 10 µM, respectively, without affecting the electrophysiological function of TRPM4 and TRPM7 channels. Computational modeling appears to support its direction binding to Orai proteins that form the transmembrane CRACchannel.

Differential functional role of Orai1 variants in constitutive Ca2+ entry and calcification in luminal breast cancer cells.

Resting cytosolic Ca2+ concentration is tightly regulated to fine-tune Ca2+-dependent cellular functions. Luminal breast cancer cells exhibit constitutive Ca2+ entry mediated by Orai1 and the secretory pathway Ca2+-ATPase, SPCA2, which result in mammary microcalcifications that constitute a prognostic marker of mammary lesions. Two Orai1 isoforms have been identified, the full-length Orai1α, consisting of 301 amino acids, and the short variant, Orai1ß, lacking the 63 or 70 N-terminal amino acids comprising residues involved in channel inactivation and binding sites with Orai1 partners. We show that only the mammalian-specific Orai1α rescues SPCA2-dependent constitutive Ca2+ entry in Orai1-KO MCF7 cells, a widely used luminal breast cancer cell line. FRET analysis and immunoprecipitation revealed that Orai1α shows a greater ability to interact with SPCA2 than Orai1ß. Deletion of the first 38 amino acids in Orai1α reduced the interaction with SPCA2 to a similar extent as Orai1ß, thus suggesting that the N-terminal 38 amino acids play a relevant role in Orai1α-SPCA2 interaction. Finally, Orai1α, but not Orai1ß, rescue the ability of Orai1-deficient cells to form in vitro microcalcifications. These findings provide compelling evidence for a functional role of Orai1α in constitutive Ca2+ entry in MCF7 cells, which might be a target to prevent the development of mammary microcalcifications in luminal breast cancer.

Phenotypic Heterogeneity in ORAI-1-Associated Congenital Myopathy.

Introduction ORAI-1 is a plasma membrane calcium release-activated calcium channel that plays a crucial role in the excitation-contraction of skeletal muscles. Loss-of-function mutations of ORAI-1 cause severe combined immunodeficiency, nonprogressive muscle hypotonia, and anhidrotic ectodermal dysplasia. Autosomal dominant gain-of-function mutation causes Stormorken's syndrome, which includes tubular aggregate myopathy along with bleeding diathesis. Methods This is a description of a genetically confirmed case of ORAI-1-associated myopathy with clinical, histopathological, and imaging characteristics and a detailed literature review. Results We report an 18-year-old woman who presented with 2-and-a-half year history of slowly progressive proximal lower limb weakness and ophthalmoparesis. Her serum creatine kinase levels were normal. Magnetic resonance imaging of the muscle showed predominant fatty infiltration of the glutei and quadriceps femoris. Histopathological analysis of muscle biopsy was suggestive of congenital fiber-type disproportion (CFTD). Clinical exome sequencing showed novel homozygous nonsense pathogenic variant NC_000012.12 (NM_032790.3): c.205G > T (p.Glu69Ter) in ORAI-1 gene. Conclusion This report expands the phenotypic spectrum of ORAI-1-related myopathy to include congenital myopathy-CFTD with ophthalmoparesis, a novel manifestation.

Activated human Orai1 channel in lipid biolayer may exist as a pentamer.

The human Orai1 (hOrai1) channel plays a crucial role in extracellular Ca2+ influx and has emerged as an attractive drug target for various diseases. However, the activated structure of the hOrai1 channel assembly within a lipid bilayer remains unknown. In this study, we expressed and purified the hOrai1 channel covalently linked to two SOAR tandems (HOSS). Patch-clamp experiments in whole-cell configuration showed that HOSS is constitutively active. Biochemical characterization confirmed that the purified HOSS channels were successfully incorporated into MSP1E3D1 nanodiscs. Negative staining revealed that the HOSS channels resemble a mushroom, with the body representing the hOrai1 channel and the leg representing the SOAR domain. Surprisingly, 2D analysis of cryo-EM data demonstrated a pentameric assembly of HOSS in a lipid bilayer. Our findings suggest that the hOrai1 channel may assemble into different oligomeric states in response to varying membrane environments.

ELD607 specifically traffics Orai1 to the lysosome leading to inhibition of store operated calcium entry.

Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1's (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1's α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607's mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.

Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer.

ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.

Orai1 and Orai3 act through distinct signalling axes to promote stemness and tumorigenicity of breast cancer stem cells.

BACKGROUND: One of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood. METHODS: A highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo. RESULTS: Orai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis. CONCLUSION: We found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.

Targeting STIM1 Contributes to Alleviating Remodeling of Vascular Smooth Muscle Cells in Atherosclerosis.

BACKGROUND: Remodeling of vascular smooth muscle cells (VSMCs), as a pathological hallmark of cardiovascular diseases, is related to the molecular rewiring of Calcium signaling, which induces upregulation of stromal interaction molecule (STIM) proteins. This study analyzed the influence of STIM1 proteins on the remodeling of VSMCs in atherosclerosis (AS). METHODS: After oxidized low-density lipoprotein (ox-LDL) treatment and transfection, VSMC viability, migration, and invasion were separately measured using Cell Counting Kit-8, Scratch assay, and Transwell assay. An animal AS model was constructed, and histological analysis via hematoxylin-eosin staining was conducted on the aorta. RESULTS: Ox-LDL promoted expression of STIM1 and Orai calcium release-activated calcium modulator 1 (Orai1). STIM1 or Orai1 downregulation suppressed viability, migration, invasion, and phenotypic switching of ox-LDL-treated VSMCs, whereas STIM1 or Orai1 upregulation had opposite effects. Orai1 level was upregulated by STIM1 overexpression. Orai1 silencing reversed the effects of STIM1 overexpression in VSMCs. STIM1 deficiency alleviated AS and regulated expression of Orai1 and phenotypic switch-related factors in vivo. CONCLUSION: STIM1 deficiency suppresses viability, migration, invasion, and phenotypic switching of ox-LDL-induced VSMCs and alleviates AS by inhibiting Orai1.

Combination of the CRAC Channel Inhibitor CM4620 and Galactose as a Potential Therapy for Acute Pancreatitis.

Acute pancreatitis (AP) is a life-threatening inflammatory disease with no specific therapy. Excessive cytoplasmic Ca2+ elevation and intracellular ATP depletion are responsible for the initiation of AP. Inhibition of Ca2+ release-activated Ca2+ (CRAC) channels has been proposed as a potential treatment, and currently, a novel selective CRAC channel inhibitor CM4620 (Auxora, CalciMedica) is in Phase 2b human trials. While CM4620 is on track to become the first effective treatment for AP, it does not produce complete protection in animal models. Recently, an alternative approach has suggested reducing ATP depletion with a natural carbohydrate galactose. Here, we have investigated the possibility of using the smallest effective concentration of CM4620 in combination with galactose. Protective effects of CM4620, in the range of 1-100 n m, have been studied against necrosis induced by bile acids, palmitoleic acid, or l-asparaginase. CM4620 markedly protected against necrosis induced by bile acids or asparaginase starting from 50 n m and palmitoleic acid starting from 1 n m. Combining CM4620 and galactose (1 m m) significantly reduced the extent of necrosis to near-control levels. In the palmitoleic acid-alcohol-induced experimental mouse model of AP, CM4620 at a concentration of 0.1 mg/kg alone significantly reduced edema, necrosis, inflammation, and the total histopathological score. A combination of 0.1 mg/kg CM4620 with galactose (100 m m) significantly reduced further necrosis, inflammation, and histopathological score. Our data show that CM4620 can be used at much lower concentrations than reported previously, reducing potential side effects. The novel combination of CM4620 with galactose synergistically targets complementary pathological mechanisms of AP.

Exploring potential ion channel targets for rheumatoid arthritis: combination of network analysis and gene expression analysis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial membrane that leads to the destruction of cartilage and bone. Currently, pharmacological targeting of ion channels is being increasingly recognized as an attractive and feasible strategy for the treatment of RA. The present work employs a network analysis approach to predict the most promising ion channel target for potential RA-treating drugs. A protein-protein interaction map was generated for 343 genes associated with inflammation in RA and ion channel genes using Search Tool for the Retrieval of Interacting Genes and visualized using Cytoscape. Based on the betweenness centrality and traffic values as key topological parameters, 17 hub nodes were identified, including FOS (9800.85), tumor necrosis factor (3654.60), TGFB1 (3305.75), and VEGFA (3052.88). The backbone network constructed with these 17 hub genes was intensely analyzed to identify the most promising ion channel target using network analyzer. Calcium permeating ion channels, especially store-operated calcium entry channels, and their associated regulatory proteins were found to highly interact with RA inflammatory hub genes. This significant ion channel target for RA identified by theoretical and statistical studies was further validated by a pilot case-control gene expression study. Experimental verification of the above findings in 75 RA cases and 25 controls showed increased ORAI1 expression. Thus, with a combination of network analysis approach and gene expression studies, we have explored potential targets for RA treatment.

Septin regulation of Orai-mediated Ca2+ entry - a novel target for neurodegeneration.

Aberrant Ca2+ signaling is an early hallmark of multiple neurodegenerative syndromes including Alzheimer's and Parkinson's disease (AD and PD) as well as classes of rare genetic disorders such as Spinocebellar Ataxias. Therapeutic strategies that target aberrant Ca2+ signals whilst allowing normal neuronal Ca2+ signals have been a challenge. In a recent study Princen et al., performed a screen in the tauP301L cell model of AD for drugs that could specifically ameliorate the excess Ca2+ entry observed. They identified a class of compounds referred to as ReS19-T that interact with Septins, previously identified as regulators of the Store-operated Ca2+ entry channel Orai. Drug treatment of the cellular model, a mouse model and human iPSC derived neurons alleviate cellular and systemic deficits associated with tauP301L. Comparison of Septin filament architecture in disease conditions with and without the drug treatment indicate that excess Ca2+ entry is a consequence of abnormal Septin filament architecture resulting in aberrant ER-PM contacts. The importance of membrane contacts for maintaining precise cellular signaling has been recognized previously. However, the molecular mechanism by which Septin filaments organize the ER-PM junctions to regulate Ca2+ entry through Orai remains to be fully understood.

STIM1: A new player in nuclear dynamics? Lessons learnt from tubular aggregate myopathy.

Two recent papers have highlighted that STIM1, a key component of Store-operated Ca2+-entry, is able to translocate to the nucleus and participate in nuclear Ca2+-handling and in DNA repair. These finding opens new avenues on the role that this Ca2+-sensing protein may have in health and disease.

Targeting calciumopathy for neuroprotection: focus on calcium channels Cav1, Orai1 and P2X7.

As the uncontrolled entry of calcium ions (Ca2+) through plasmalemmal calcium channels is a cell death trigger, the conjecture is here raised that mitigating such an excess of Ca2+ entry should rescue from death the vulnerable neurons in neurodegenerative diseases (NDDs). However, this supposition has failed in some clinical trials (CTs). Thus, a recent CT tested whether isradipine, a blocker of the Cav1 subtype of voltage-operated calcium channels (VOCCs), exerted a benefit in patients with Parkinson's disease (PD); however, outcomes were negative. This is one more of the hundreds of CTs done under the principle of one-drug-one-target, that have failed in Alzheimer's disease (AD) and other NDDs during the last three decades. As there are myriad calcium channels to let Ca2+ ions gain the cell cytosol, it seems reasonable to predict that blockade of Ca2+ entry through a single channel may not be capable of preventing the Ca2+ flood of cells by the uncontrolled Ca2+ entry. Furthermore, as Ca2+ signaling is involved in the regulation of myriad functions in different cell types, it seems also reasonable to guess that a therapy should be more efficient by targeting different cells with various drugs. Here, we propose to mitigate Ca2+ entry by the simultaneous partial blockade of three quite different subtypes of plasmalemmal calcium channels that is, the Cav1 subtype of VOCCs, the Orai1 store-operated calcium channel (SOCC), and the purinergic P2X7 calcium channel. All three channels are expressed in both microglia and neurons. Thus, by targeting the three channels with a combination of three drug blockers we expect favorable changes in some of the pathogenic features of NDDs, namely (i) to mitigate Ca2+ entry into microglia; (ii) to decrease the Ca2+-dependent microglia activation; (iii) to decrease the sustained neuroinflammation; (iv) to decrease the uncontrolled Ca2+ entry into neurons; (v) to rescue vulnerable neurons from death; and (vi) to delay disease progression. In this review we discuss the arguments underlying our triad hypothesis in the sense that the combination of three repositioned medicines targeting Cav1, Orai1, and P2X7 calcium channels could boost neuroprotection and delay the progression of AD and other NDDs.

Interstitial cell of Cajal-like cells (ICC-LC) exhibit dynamic spontaneous activity but are not functionally innervated in mouse urethra.

Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit+ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca2+ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca2+ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca2+ signalling. ICC-LC generated Ca2+ waves spontaneously that propagated on average 40.1 ± 0.7 µm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca2+ signalling between ICC-LC was obtained. Ca2+ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca2+ channels (GSK-7975A). Phenylephrine increased Ca2+ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca2+ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.

ER and SOCE Ca2+ signals are not required for directed cell migration in human iPSC-derived microglia.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

Loss of Calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy R1/2A (LGMD R1/2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wild-type (WT) mice, we determined whether loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD R1/2A pathology.