Calcium Entry Research Articles

Knockdown of TcGluCl leads to the premature pupation of Tribolium castaneum larvae possibly by influencing the calcium-mediating hormone homeostasis

The glutamate-gated chloride channels (GluCls) are widely existed in the neural and nonneural tissues of invertebrate. In addition to play important roles in signal transduction, the GluCls also showed multiple physiological functions in insects such as participate in the juvenile hormone synthesis. In the present study, the potential roles of TcGluCl in growth and development of the red flour beetle Tribolium castaneum were explored. Knockdown of TcGluCl showed no effects on the survivability, weight growth, final pupation rate, eclosion and fecundity of T. castaneum, whereas resulted in the significant premature pupation of larvae. Inhibition of TcGluCl expression significantly changed the levels of juvenile hormone and ecdysone as well as the expressions of hormone biosynthetic genes. The increased ecdysone level and decreased juvenile hormone level were observed at the late stage of dsGluCl-treated larvae. Knockdown of TcGluCl significantly reduced the expressions of TcSTIM1 and TcOrai1, which were the primary proteins in store-operated calcium entry (SOCE) mediated Ca2+ influx mechanism. Whilst the L-glutamic acid treatment led to the increased TcOrai1 expression in T. castaneum. These findings suggested that knockdown of TcGluCl increased the ecdysone level and contributed to the premature pupation of larvae, which might be due to the reduced Ca2+ influx caused by the decreased expressions of TcSTIM1 and TcOrai1. These studies provide novel insights on the function of GluCls in insects.

TRPC3 suppression ameliorates synaptic dysfunctions and memory deficits in Alzheimer's disease.

Transient receptor potential canonical (TRPC) channels are widely expressed in the brain; however, their precise roles in neurodegeneration, such as Alzheimer's disease (AD) remain elusive. Bioinformatic analysis of the published single-cell RNA-seq data collected from AD patient cohorts indicates that the Trpc3 gene is uniquely upregulated in excitatory neurons. TRPC3 expression is also upregulated in post-mortem AD brains, and in both acute and chronic mouse models of AD. Functional screening of TRPC3 antagonists resulted in a lead inhibitor JW-65, which completely rescued Aβ-induced neurotoxicity, impaired synaptic plasticity (e.g., LTP), and learning memory in acute and chronic experimental AD models. In cultured rat hippocampal neurons, we found that treatment with soluble β-amyloid oligomers (AβOs) induces rapid and sustained upregulation of the TRPC3 expression selectively in excitatory neurons. This aberrantly upregulated TRPC3 contributes to AβOs-induced Ca 2+ overload through the calcium entry and store-release mechanisms. The neuroprotective action of JW-65 is primarily mediated via restoring AβOs-impaired Ca 2+ /calmodulin-mediated signaling pathways, including calmodulin kinases CaMKII/IV and calcineurin (CaN). The synaptic protective mechanism via TRPC3 inhibition was further supported by hippocampal RNA-seq data from the symptomatic 5xFAD mice after chronic treatment with JW-65. Overall, these findings not only validate TRPC3 as a novel therapeutic target for treating synaptic dysfunction of AD but most importantly, disclose a distinct role of upregulated TRPC3 in AD pathogenesis in mediating Ca 2+ dyshomeostasis.

Effects of zinc in podocytes and cortical collecting duct in vitro and Dahl salt-sensitive rats in vivo

Zinc is one of the essential divalent cations in the human body and a fundamental microelement involved in the regulation of many cellular and subcellular functions. Experimental studies reported that zinc deficiency is associated with renal damage and could increase blood pressure. It was proposed that zinc dietary supplementation plays a renoprotective role. Our study aimed to investigate the effects of zinc on intracellular signaling in renal cells and explore the correlation between dietary zinc and the progression of salt-induced hypertension. The impact of extracellular zinc concentrations on two different kidney epithelial cell types - podocytes and principal cells of the cortical collecting duct (CCD), was tested. In podocytes, a rise in extracellular zinc promotes TRPC6 channel-mediated calcium entry but not altered intracellular zinc levels. However, we observe the opposite effect in CCD cells with no alteration in calcium levels and steady-state elevation in intracellular zinc. Moreover, prolonged extracellular zinc exposure leads to cytotoxic insults in CCD cells but not in podocytes, characterized by increased cell death and disrupted cytoskeletal organization. Next, we tested if dietary zinc plays a role in the development of hypertension in Dahl salt-sensitive rats. Neither zinc-rich nor deficient diets impact the regular development of salt-sensitive hypertension. These results suggest specialized roles for zinc in renal function, implicating its involvement in proliferation and apoptosis in CCD cells and calcium signaling and cytoskeletal dynamics modulation in podocytes. Further research is required to elucidate the detailed mechanisms of zinc action and its implications in renal health and disease.

STIM Proteins: The Gas and Brake of Calcium Entry in Neurons.

Stromal interaction molecules (STIM)s are Ca2+ sensors in internal Ca2+ stores of the endoplasmic reticulum. They activate the store-operated Ca2+ channels, which are the main source of Ca2+ entry in non-excitable cells. Moreover, STIM proteins interact with other Ca2+ channel subunits and active transporters, making STIMs an important intermediate molecule in orchestrating a wide variety of Ca2+ influxes into excitable cells. Nevertheless, little is known about the role of STIM proteins in brain functioning. Being involved in many signaling pathways, STIMs replenish internal Ca2+ stores in neurons and mediate synaptic transmission and neuronal excitability. Ca2+ dyshomeostasis is a signature of many pathological conditions of the brain, including neurodegenerative diseases, injuries, stroke, and epilepsy. STIMs play a role in these disturbances not only by supporting abnormal store-operated Ca2+ entry but also by regulating Ca2+ influx through other channels. Here, we review the present knowledge of STIMs in neurons and their involvement in brain pathology.

Systematic mapping of mitochondrial calcium uniporter channel (MCUC)-mediated calcium signaling networks

The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases.

Impaired calcium influx underlies skewed T helper cell differentiation in children with IgE-mediated food allergies.

Reasons for Th2 skewing in IgE-mediated food allergies remains unclear. Clinical observations suggest impaired T cell activation may drive Th2 responses evidenced by increased atopic manifestations in liver transplant patients on tacrolimus (a calcineurin inhibitor). We aimed to assess differentiation potential, T cell activation and calcium influx of naïve CD4+ T cells in children with IgE-mediated food allergies. Peripheral blood mononuclear cells from infants in the Starting Time for Egg Protein (STEP) Trial were analyzed by flow cytometry to assess Th1/Th2/Treg development. Naïve CD4+ T cells from children with and without food allergies were stimulated for 7 days to assess Th1/Th2/Treg transcriptional factors and cytokines. Store operated calcium entry (SOCE) was measured in children with and without food allergies. The effect of tacrolimus on CD4+ T cell differentiation was assessed by treating stimulated naïve CD4+ T cells from healthy volunteers with tacrolimus for 7 days. Egg allergic infants had impaired development of IFNγ+ Th1 cells and FoxP3+ transitional CD4+ T cells compared with non-allergic infants. This parallels reduced T-bet, IFNγ and FoxP3 expression in naïve CD4+ T cells from food allergic children after invitro culture. SOCE of naïve CD4+ T cells was impaired in food allergic children. Naïve CD4+ T cells treated with tacrolimus had reduced IFNγ, T-bet, and FoxP3, but preserved IL-4 expression. In children with IgE-mediated food allergies, dysregulation of T helper cell development is associated with impaired SOCE, which underlies an intrinsic impairment in Th1 and Treg differentiation. Along with tacrolimus-induced Th2 skewing, this highlights an important role of SOCE/calcineurin pathway in T helper cell differentiation.

Identification of Noncoding Functional Regulatory Variants of STIM1 Gene in Idiopathic Pulmonary Arterial Hypertension.

STIM1 (stromal interaction molecule 1) regulates store-operated calcium entry and is involved in pulmonary artery vasoconstriction and pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial hypertension (PAH). Bioinformatics analysis and a 2-stage matched case-control study were conducted to screen for noncoding variants that may potentially affect STIM1 transcriptional regulation in 242 patients with idiopathic PAH and 414 healthy controls. Luciferase reporter assay, real-time quantitative polymerase chain reaction, western blot, 5-ethynyl-2'-deoxyuridine (EdU) assay, and intracellular Ca2+ measurement were performed to study the mechanistic roles of those STIM1 noncoding variants in PAH. Five noncoding variants (rs3794050, rs7934581, rs3750996, rs1561876, and rs3750994) were identified and genotyped using Sanger sequencing. Rs3794050, rs7934581, and rs1561876 were associated with idiopathic PAH (recessive model, all P<0.05). Bioinformatics analysis showed that these 3 noncoding variants possibly affect the enhancer function of STIM1 or the microRNA (miRNA) binding to STIM1. Functional validation performed in HEK293 and pulmonary artery smooth muscle cells demonstrated that the noncoding variant rs1561876-G (STIM1 mutant) had significantly stronger transcriptional activity than the wild-type counterpart, rs1561876-A, by affecting the transcriptional regulatory function of both hsa-miRNA-3140-5p and hsa-miRNA-4766-5p. rs1561876-G enhanced intracellular Ca2+ signaling in human pulmonary artery smooth muscle cells secondary to calcium-sensing receptor activation and promoted proliferation of pulmonary artery smooth muscle cells under both normoxia and hypoxia conditions, suggesting a possible contribution to PAH development. The potential clinical implications of the 3 noncoding variants of STIM1, rs3794050, rs7934581, and rs1561876, are 2-fold, as they may help predict the risk and prognosis of idiopathic PAH and guide investigations on novel therapeutic pathway(s).

Dysregulation of Store-Operated Calcium Entry in Myelofibrosis-Derived Monocytes

Dysregulation of Store-Operated Calcium Entry in Myelofibrosis-Derived Monocytes

MPN-614 Dysregulation of Store-Operated Calcium Entry in Myelofibrosis-Derived Monocytes

MPN-614 Dysregulation of Store-Operated Calcium Entry in Myelofibrosis-Derived Monocytes

Effect of calcium contamination on lignosulfonate mud, lime mud, and potassium chloride mud

A significant amount of the oil produced in Turkiye is extracted from the Southeastern Anatolia Region. In the drilling operations conducted around Batman and Diyarbakir, which are key areas for oil production in this region, formation-related problems are encountered, leading to increased drilling costs and time loss. The primary issues among these problems are the wellbore stability and the contamination of drilling mud with clay and calcium. In this study, experimental investigations were conducted in wells A-1, A-2, and A-3 located in region A, and well B-1 located in region B in the Batman and Diyarbakir areas. The investigations observed formation-related well stability issues and contamination of drilling mud with calcium and clay. Drilling mud experiments based on actual well data were conducted to address the changes in formation-related drilling mud. In wells A-1 and B-1, the drilling of formations containing gypsum and anhydrite was carried out using lignosulfonate mud, during which heavy calcium contamination occurred in the system mud, causing problems in the drilling operations. In well A-2, the drilling of gypsum and anhydrite in the Kayaköy formation, and shale and marl formations in the Kastel and Karadut formations, was done using KCl/Polymer mud. Experiments conducted on the drilling mud observed changes in the mud and examined the issues arising in the drilling operations. In well A-3, the drilling of rocks belonging to the same formations was performed, and lime mud was used in this well to counteract the possibility of gas influx, calcium contamination, and carbonate-bicarbonate contamination. The calcium concentration ranged from 120 to 800 mg/l in A-1 well, 80–800 mg/l in A-2 well, 440–680 mg/l in A-3 well, and 160–720 mg/l in B-1 well. Although calcium entrance to the drilling mud is inevitable during the drilling of calcium-bearing formation, this study shows that it is possible to drill these formations safely by the interventions at right times by the drilling fluid engineer.

Sodium butyrate prevents cytokine-induced β-cell dysfunction through restoration of stromal interaction molecule 1 expression and activation of store-operated calcium entry.

Sodium butyrate (NaB) improves β-cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been fully elucidated. In this study, we investigated the impact of NaB on β-cell function and calcium (Ca2+) signaling using exvivo and invitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 β cells. Consistently, NaB improved glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the β cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1β-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent β-cell death in response to IL-1β treatment. Mechanistic experiments revealed that NaB mediated these beneficial effects in the β-cell through histone deacetylase (HDAC) inhibition, iNOS suppression, and modulation of AKT-GSK-3 signaling. Taken together, these data support a model whereby NaB treatment promotes β-cell function and Ca2+ homeostasis under proinflammatory conditions through pleiotropic effects that are linked with maintenance of SOCE. These results also suggest a relationship between β-cell SOCE and gut microbiome-derived butyrate that may be relevant in the treatment and prevention of diabetes.

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.

PACS-1 Interacts with TRPC3 and ESyt1 to Mediate Protein Trafficking while Promoting SOCE and Cooperatively Regulating Hormone Secretion.

Corticotropic cells of the anterior pituitary gland release adrenocorticotropic hormone (ACTH) in a regulated manner to promote the production of cortisol and androgens. The process of ACTH secretion is partly mediated by the phosphofurin acidic cluster sorting protein 1 (PACS-1); however, the underlying mechanisms behind this regulation remain unclear. Herein, we demonstrated PACS-1 interactions with the short transient receptor potential channel 3 (TRPC3) calcium transporter and the extended synaptotagmin-1 (ESyt1) endoplasmic reticulum-plasma membrane tethering protein. Importantly, PACS-1 promoted interactions between TRPC3 and ESyt1 and regulated their plasma membrane localization. Lastly, we demonstrated that PACS-1 is required for a proper store-operated calcium entry (SOCE) response and that ESyt1 regulates ACTH secretion through an unknown mechanism regulated by PACS-1. Overall, our study provides new insights into the physiological role PACS-1 plays in modulating intracellular calcium levels and regulating ACTH secretion in corticotropic cells.

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.

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Abstract Mo029: Angiotensin Receptor Neprilysin Inhibitor improves cardiac hemodynamics and arrhythmogenesis in mitral regurgitation-induced heart failure—from bench to bedside approach

Background: An effective pharmacological therapy yet exists to mitigate mitral regurgitation (MR) progression before surgical interventions. Angiotensin receptor-neprilysin inhibitor (ARNI) has shown benefits in patients with heart failure (HF) with reduced ejection fraction but whether it can reduce degenerative MR induced HF remains unknown. Aim: To test the efficacy of ARNI in improving cardiac function in degenerative MR -induced HF. Methods and Results: Using a novel rat model of degenerative MR, we found that in degenerative MR rats treated with ARNI, left ventricular function and atrial fibrillation (AF) inducibility significantly improved compared with those treated with valsartan only (fractional shortening 61.78±7.26% v.s. 55.07±6.63%). Also, the AF inducibility dropped from 83% to 25% in MR rats treated with ARNI than to 58.3% in those treated with valsartan. ARNI treatment demonstrated significant attenuation of fibrosis, apoptosis and expressions of endoplasmic reticulum (ER) stress and inflammatory-associated proteins in left ventricular tissues compared to valsartan. In a shear stress model, the treatment of ARNI on cardiomyocytes mitigated the flow triggered activities of both ER released calcium signaling and store-operated calcium entry. Parallelly, we prospectively included 65 patients with degenerative MR receiving valsartan or ARNI and followed Holter electrocardiography and echocardiography. Notably, compared with degenerative MR patients receiving valsartan, those prescribed with ARNI demonstrated significant improvement of AF burden and left ventricular function. Conclusions: Degenerative MR-induced cardiovascular dysfunction including fibrosis and ER stress, were more effectively attenuated by ARNI treatment than by valsartan alone.

Mitochondria can substitute for parvalbumin to lower cytosolic calcium levels in the murine fast skeletal muscle.

Parvalbumin (PV) is a primary calcium buffer in mouse fast skeletal muscle fibers. Previous work showed that PV ablation has a limited impact on cytosolic Ca2+ ([Ca2+]cyto) transients and contractile response, while it enhances mitochondrial density and mitochondrial matrix-free calcium concentration ([Ca2+]mito). Here, we aimed to quantitatively test the hypothesis that mitochondria act to compensate for PV deficiency. We determined the free Ca2+ redistribution during a 2 s 60 Hz tetanic stimulation in the sarcoplasmic reticulum, cytosol, and mitochondria. Via a reaction-diffusion Ca2+ model, we quantitatively evaluated mitochondrial uptake and storage capacity requirements to compensate for PV lack and analyzed possible extracellular export. [Ca2+]mito during tetanic stimulation is greater in knock-out (KO) (1362 ± 392 nM) than in wild-type (WT) (855 ± 392 nM), p < 0.05. Under the assumption of a non-linear intramitochondrial buffering, the model predicts an accumulation of 725 μmoles/L fiber (buffering ratio 1:11 000) in KO, much higher than in WT (137 μmoles/L fiber, ratio 1:4500). The required transport rate via mitochondrial calcium uniporter (MCU) reaches 3 mM/s, compatible with available literature. TEM images of calcium entry units and Mn2+ quenching showed a greater capacity of store-operated calcium entry in KO compared to WT. However, levels of [Ca2+]cyto during tetanic stimulation were not modulated to variations of extracellular calcium. The model-based analysis of experimentally determined calcium distribution during tetanic stimulation showed that mitochondria can act as a buffer to compensate for the lack of PV. This result contributes to a better understanding of mitochondria's role in modulating [Ca2+]cyto in skeletal muscle fibers.

Are Aminoglycoside Antibiotics TRPing Your Metabolic Switches?

Transient receptor potential (TRP) channels are broadly implicated in the developmental programs of most tissues. Amongst these tissues, skeletal muscle and adipose are noteworthy for being essential in establishing systemic metabolic balance. TRP channels respond to environmental stimuli by supplying intracellular calcium that instigates enzymatic cascades of developmental consequence and often impinge on mitochondrial function and biogenesis. Critically, aminoglycoside antibiotics (AGAs) have been shown to block the capacity of TRP channels to conduct calcium entry into the cell in response to a wide range of developmental stimuli of a biophysical nature, including mechanical, electromagnetic, thermal, and chemical. Paradoxically, in vitro paradigms commonly used to understand organismal muscle and adipose development may have been led astray by the conventional use of streptomycin, an AGA, to help prevent bacterial contamination. Accordingly, streptomycin has been shown to disrupt both in vitro and in vivo myogenesis, as well as the phenotypic switch of white adipose into beige thermogenic status. In vivo, streptomycin has been shown to disrupt TRP-mediated calcium-dependent exercise adaptations of importance to systemic metabolism. Alternatively, streptomycin has also been used to curb detrimental levels of calcium leakage into dystrophic skeletal muscle through aberrantly gated TRPC1 channels that have been shown to be involved in the etiology of X-linked muscular dystrophies. TRP channels susceptible to AGA antagonism are critically involved in modulating the development of muscle and adipose tissues that, if administered to behaving animals, may translate to systemwide metabolic disruption. Regenerative medicine and clinical communities need to be made aware of this caveat of AGA usage and seek viable alternatives, to prevent contamination or infection in in vitro and in vivo paradigms, respectively.

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.

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.