Calcium Entry Research Articles

Trimetazidine Affects Mitochondrial Calcium Uniporter Expression to Restore Ischemic Heart Function via Reactive Oxygen Species/NFκB Pathway Inhibition.

Studies have demonstrated the roles of trimetazidine beyond being an antianginal agent in ischemic heart disease (IHD) treatment associated with mechanisms of calcium regulation. Our recent studies revealed that mitochondrial calcium uniporter (MCU, the pore-forming unit responsible for mitochondrial calcium entrance) inhibition provided cardioprotective effects for failing hearts. Because trimetazidine and MCU are associated with calcium homeostasis, we hypothesized that trimetazidine may affect MCU to restore the failing heart function. In the present study, we tested this hypothesis in the context of cardiac ischemia in vivo and in vitro. The IHD model was established in male C57BL/6 mice followed by trimetazidine administration intraperitoneally at 20 mg/kg q.o.d for 8 weeks. In vitro studies were performed in a hypoxia model using primary rat neonate cardiomyocytes. The mice survival outcomes and heart function, pathohistologic, and biological changes were analyzed. The results demonstrated that trimetazidine treatment resulted in longer life spans and heart function improvement accompanied by restoration of mitochondrial calcium levels and increase in ATP production via MCU down-regulation. Studies in vitro further showed that trimetazidine treatment and MCU inhibition decreased reactive oxygen species (ROS) production, inhibited the NFκB pathway, and protected the cardiomyocytes from hypoxic injury, and vice versa. Thus, the present study unveils a unique mechanism in which trimetazidine is involved in ameliorating the ischemic failing heart via MCU down-regulation and the following mitochondrial calcium homeostasis restoration, ROS reduction, and cardiomyocyte protection through NFκB pathway inhibition. This mechanism provides a novel explanation for the treatment effects of trimetazidine on IHD.

Orai, RyR, and IP3R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca2+ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.

Store-operated calcium entry inhibition ameliorates Ang II induced podocyte apoptosis and mitochondria respiratory dysfunction

Purpose and hypothesis: Podocyte injury induced by Angiotensin II (Ang II) is the key factor contributing to proteinuria and glomerulopathy in many kidney diseases including diabetic nephropathy and hypertensive kidney disease. Recent studies suggest that mitochondria dysfunction mediates Ang II-induced podocyte injury. However, the mechanism for Ang II-induced podocyte mitochondria damage is poorly understood. Store-operated Ca2+ entry (SOCE) has multiple functions in both excitable and non-excitable cells. Ang II can activate SOCE by releasing endoplasmic reticulum Ca2+ through generation of IP3. In addition, Ang II also activates Transient Receptor Potential Conventional Member 6 (TRPC6) channel through diacylglycerol (DAG). Our previous study demonstrated that enhanced SOCE mediated high glucose-induced podocyte cytoskeleton remodeling. However, the role of SOCE in podocyte injury by Ang II is not clear. The present study was carried out to test the hypothesis that enhanced SOCE contributed to Ang II-induced podocyte apoptosis and mitochondria respiration dysfunction. Methods: All experiments were carried out using cultured immortalized human podocytes. BTP2 (4 μM) was used to inhibit SOCE. SAR 7334 (100 nM) was used to block TRPC6 channel. Podocyte apoptosis was determined by flow cytometry using Annexin V/Propidium iodide (PI) staining. The conventional whole-cell patch-clamp was performed to measure store-operated Ca2+ channel (SOC) currents. Mitochondrial respiration function and mitochondria ATP production were evaluated by oxygen consumption rate (OCR) using seahorse analysis. Results: Ang II (1 μM) evoked inward currents, which were significantly blunted by BTP2 (10 μM), an SOC channel blocker. Ang II (1 μM) treatments for 24 hours significantly increased podocyte apoptosis and reduced podocyte basal OCR, maximal OCR, spare capacity and mitochondria ATP production. All these responses were significantly blunted by BTP2. However, Ang II treatment for 30 min did not induce a significant response of OCR, but significantly reduced maximal respiration and spare capacity. These responses were also significantly inhibited by BTP2 (10 μM), but not by SAR 7334 (100 nM). Conclusion: SOCE contributed to Ang II-induced podocyte apoptosis and mitochondria respiratory dysfunction. Translational Project Award from American Heart Association (20TPA35500045, to RM); National Institute of Diabetes and Digestive and Kidney Disease (DK115424-01, to RM); Predoctoral Fellowship from American Heart Association (903925, to YT). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging impairs the clustering and reduces the activity of the L-type calcium channels in the heart's pacemaker

Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. In mammals, including humans, aging leads to the slowdown of the pacemaker rate, which can lead to pathological dysfunction and death. However, the mechanisms behind the age-associated slowdown of the pacemaker are not well understood. The automaticity of the pacemaker relies on a finely tuned balance of ionic currents, where L-type calcium channels play a central role. CaV1.2 and CaV1.3 channels are key players involved in the diastolic depolarization phase and action potential upstroke. In this project, we studied the effects of aging on CaV1.2 and CaV1.3 calcium channel function and regulation. Here we recorded the pacemaker activity and calcium currents on isolated spindle pacemaker cells with patch clamp. The clustering of LTCC has been highlighted with Super-resolution microscopy (GSD). We found that in mouse pacemaker cells, aging causes a reduction of calcium entry through L-type calcium channels, which is not accompanied by a reduction of protein expression. In turn, we found that aging disrupts calcium channel function at different levels. First, aging reduces the expression of L-type calcium channels at the plasma membrane. Second, aging reduces the clustering and nano-organization of L-type calcium channels. And finally, aging reduces the open channel probability, a sign of decreased functional coupling. Moreover, aging also alters the association between L-type calcium channels and beta-adrenergic receptors, altering the adrenergic modulation of the pacemaker cells. Next, we tested the hypothesis that reduced open probability of L-type calcium channels was responsible for a slower firing frequency. Interestingly, we found that the application of BayK was enough to restore intrinsic pacemaker rates in old cells to those levels observed in young cells. Overall, our findings provide evidence that the proper organization and function of L-type calcium channels is affected by aging and that this dysfunction contributes to the slowdown of pacemaker cells in old animals.

Lithium sensitizes P2Y2-dependent intracellular Ca2+ signaling in the collecting duct cells

INTRODUCTION. Lithium salts are used as mood stabilizers to treat bipolar disorder and depression. A common renal side effect of lithium therapy is nephrogenic diabetes insipidus (NDI), manifesting in polyuria. Lithium decreases the numbers of aquaporin 2 (AQP2) water channels in collecting duct cells reducing water reabsorption in response to vasopressin (AVP). Stimulation of purinergic P2Y2 receptors (P2Y2R) can impair AVP signaling and deletion of P2Y2R in mice slows down the progression of lithium-induced NDI. Interestingly, stimulation of P2Y2R triggers Ca2+ release from the endoplasmic reticulum (ER) in collecting duct cells. The interplay between P2Y2R and intracellular Ca2+ ([Ca2+]i) signaling in lithium-induced NDI has not been tested. We hypothesize that lithium facilitates P2Y2-mediated Ca2+ signaling in collecting duct cells, attenuating AVP-dependent water transport and disrupting [Ca2+]i balance. METHODS. This project combines water permeability tests in cultured cell monolayers, immunoblotting and ratiometric Fura-2 Ca2+-imaging to study the effects of lithium on P2Y2-dependent [Ca2+]i mobilization in a culture of mouse renal cortical collecting duct cells, mpkCCDcl4. RESULTS. Upon stimulation with 1 nM AVP, mpkCCDcl4 cells incubated in the medium containing 10 mM LiCl for 4 days exhibited a 2-fold lower abundance of membrane resident AQP2, phosphorylated at serine 269, when compared to control cells. Transepithelial water transport was also significantly slower in LiCl-treated than in control cells, further validating the detrimental effects of Li+ in our cell model. Saturating (10-100 uM) concentrations of a P2Y2R-selective ligand, uridine-5'-triphosphate (UTP), induced comparable [Ca2+]i responses in in control and LiCl-treated cells. When we assessed the P2Y2-mediated [Ca2+]i responses to a range of UTP concentrations (0.1-100 uM), the dose response curve was shifted to the left in LiCl-treated cells when compared to controls. The half-maximal effective UTP concentration, EC50, was 0.4±0.03 uM and 2.0±0.07 uM in Li-treated and control cells, respectively, indicating higher sensitivity to UTP in LiCl-treated cells. Next, we evaluated how P2Y2-dependent depletion of the ER Ca2+ stores, affects store-operated calcium entry (SOCE) in mpkCCDcl4 cells. The magnitude of SOCE-reporting Ca2+ signal was significantly (~25%) higher in LiCl-treated cells than in controls. CONCLUSIONS. Lithium facilitates P2Y2-mediated [Ca2+]i mobilization, reduces AVP-dependent transepithelial water transport and AQP2 trafficking in collecting duct cells. Sensitization of P2Y2-dependent Ca2+ signaling causes excessive depletion of the ER stores and requires higher activity of SOCE to compensate for Ca2+ loss and restore the balance. Our findings reveal previously unrecognized signaling determinants underlying the pathophysiological effects of lithium, pertaining to Ca2+ homeostasis in collecting duct cells. This work was supported by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) R01DK125464. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

STIM1: The lord of the rings?

STIM1 and Orai1 are the central core of the Store Operated Calcium Entry (SOCE). This calcium influx mechanism is triggered after the activation of Gq protein-coupled receptors at the plasma membrane (PM) that activate phospholipase C. The phospholipase C produces Inositol triphosphate (IP3) which rapidly diffuses throughout the cytosol, resulting in the binding and activation of IP3 receptors (IP3R) and the rapid efflux of calcium from the endoplasmic reticulum (ER) to the cytosol. The calcium depletion in the ER is sensed by the stromal interaction molecule 1 (STIM1) a single-pass transmembrane protein at the ER that binds intraluminal calcium through an EF-hand domain in its amino terminal region (Fig. 1A). The cytosolic portion of STIM1 contains multiple domains. The region that interacts and activates Orai channels is known as SOAR (the STIM1 Orai activating region) [1]. For SOAR be accessible to Orai1, STIM1 must get an extended conformation that unlocks SOAR from its coiled-coil 1 (CC1) region [2]. The extended conformation is triggered by calcium depletion at the ER that oligomerizes STIM1. The oligomers of STIM1 then translocate to a close distance between two opposing membranes, forming what is known as ER-PM junctions. STIM1 accumulates at ER-PM junctions conforming the denominated STIM1 puncta.

An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

Reorganization and Suppression of Store-Operated Calcium Entry in Podocytes of Type 2 Diabetic Rats.

Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca2+ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca2+ imaging indicated the involvement of ORAI and sodium-calcium exchanger in Ca2+ entry induced upon store depletion. In male rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of a store-operated Ca2+ entry (SOCE) in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca2+ influx such that TRPC6 channels lost their sensitivity to Ca2+ store depletion and ORAI-mediated Ca2+ entry was suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account when developing pharmacological treatment of the early stages of diabetic nephropathy.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SummaryAlterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. Subject areaCalcium, ROS, Cellular signaling, lung disease.

Angiotensin-(1−7) mediated calcium signalling by MAS

The G protein-coupled receptor, MAS, is the receptor of the endogenous ligand, Angiotensin (Ang)-(1−7). It is a promising drug target since the Ang-(1−7)/MAS axis is protective in the cardiovascular system. Therefore, a characterization of MAS signalling is important for developing novel therapeutics for cardiovascular diseases. In this paper, we show that Ang-(1−7) increases intracellular calcium in transiently MAS-transfected HEK293 cells. The calcium influx induced by the activation of MAS is dependent on plasma membrane Ca2+ channels, phospholipase C, and protein kinase C. Specifically, we could demonstrate that MAS employs non-selective, transient receptor potential channels (TRPs) for calcium entry.

Role of Orai3-Mediated Store-Operated Calcium Entry in Radiation-Induced Brain Microvascular Endothelial Cell Injury

Radiation-induced brain injury is a serious complication with complex pathogenesis that may accompany radiotherapy of head and neck tumors. Although studies have shown that calcium (Ca2+) signaling may be involved in the occurrence and development of radiation-induced brain injury, the underlying molecular mechanisms are not well understood. In this study, we used real-time quantitative polymerase chain reaction and Western blotting assays to verify our previous finding using next-generation sequencing that the mRNA and protein expression levels of Orai3 in rat brain microvascular endothelial cells (rBMECs) increased after X-ray irradiation. We next explored the role of Orai3 and Orai3-mediated store-operated Ca2+ entry (SOCE) in radiation-induced brain injury. Primary cultured rBMECs derived from wild-type and Orai3 knockout (Orai3(-/-)) Sprague-Dawley rats were used for in vitro experiments. Orai3-mediated SOCE was significantly increased in rBMECs after X-ray irradiation. However, X-ray irradiation-induced SOCE increase was markedly reduced in Orai3 knockout rBMECs, and the percentage of BTP2 (a nonselective inhibitor of Orai channels)-inhibited SOCE was significantly decreased in Orai3 knockout rBMECs. Functional studies indicated that X-ray irradiation decreased rBMEC proliferation, migration, and tube formation (a model for assessing angiogenesis) but increased rBMEC apoptosis, all of which were ameliorated by BTP2. In addition, occurrences of all four functional deficits were suppressed in X-ray irradiation-exposed rBMECs derived from Orai3(-/-) rats. Cerebrovascular damage caused by whole-brain X-ray irradiation was much less in Orai3(-/-) rats than in wild-type rats. These findings provide evidence that Orai3-mediated SOCE plays an important role in radiation-induced rBMEC damage and brain injury and suggest that Orai3 may warrant development as a potential therapeutic target for reducing or preventing radiation-induced brain injury.

Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM invitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing To Peripheral Sensitization

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing To Peripheral Sensitization

The Mechanism of the Anti-Cardiac Hypertrophy Effect of Glycyrrhizic Acid Is Related to Reducing STIM1-Dependent Store-Operated Calcium Entry

We explored the anti-cardiac hypertrophy mechanism of glycyrrhizic acid from the perspective of calcium regulation under pathological conditions. For this purpose, we used a rat model of myocardial hypertrophy induced by pressure overload. The effect of glycyrrhizic acid on BP was measured non-invasively with a sphygmomanometer and recorded in PC. In rats with modeled cardiac hypertrophy, the effect of GA on expression of type 1 matrix interaction molecules was determined in horizontal tissues and cultured cardiomyocytes of the left ventricle. The laser confocal microscopy and calcium ion probe Fluo-4 AM were used to assess the effect of glycyrrhizic acid on stromal interaction molecule 1 (STIM1)-dependent store-operated calcium entry in cultured cardiomyocytes derived from the hypertrophic myocardium. Glycyrrhizic acid exerted the anti-hypertrophic effect in rats with hypertrophic myocardium by down-regulating STIM1 protein expression and reducing the intensity of STIM1-dependent store-operated calcium entry.

Store-operated calcium entry enhances the polarization and chemotaxis of neutrophils in the peripheral venous blood of patients with bronchial asthma by upregulating ERM protein.

Neutrophils can be rapidly recruited and are largely abundant in the airways of patients with asthma. However, whether the polarization and chemotaxis of neutrophils in patients with asthma are abnormal, and the underlying mechanisms, have not been clarified. Pseudopods formation is the initial step of neutrophils' polarization, ezrin, radixin and moesin (ERM) play an important role in the polarization of neutrophils. As an important signaling molecule in cell physiological processes, Ca2+ has been shown to be involved in the polarity changes of neutrophils. This study thus aimed to explore polarization and chemotaxis of neutrophils in patients with asthma and the underlying mechanism. Fresh neutrophils were isolated using standard separation protocols. The polarization and chemotactic activity of neutrophils were observed using Zigmond chamber and Transwell migration assay under linear concentration gradients of N-formyl-methionine-leucine-phenylalanine (fMLP) or interleukin (IL)-8. The distribution of calcium, ERMs and F-actin in neutrophils were observed by confocal laser scanning microscope. The expression of the main components of ERMs (moesin and ezrin) was detected with reverse transcription-polymerase chain reaction (RT-PCR). Compared with those in the healthy control group, the polarization and chemotaxis of neutrophils in the venous blood of patients with asthma were significantly increased, and the expression and distribution of cytoskeletal proteins F-actin and ezrin were abnormal. The expression and function of key components of store-operated calcium entry (SOCE), stromal interaction molecule 1 (STIM1), STIM2, and Orai1 of neutrophils in patients with asthma were significantly increased. The polarization and chemotaxis of neutrophils in the venous blood of patients with asthma are increased. This may be due to the abnormal expression and distribution of ERM and F-actin as a result of abnormal SOCE function.

Large-scale interrogation of retinal cell functions by 1-photon light-sheet microscopy

Visual processing in the retina depends on the collective activity of large ensembles of neurons organized in different layers. Current techniques for measuring activity of layer-specific neural ensembles rely on expensive pulsed infrared lasers to drive 2-photon activation of calcium-dependent fluorescent reporters. We present a 1-photon light-sheet imaging system that can measure the activity in hundreds of neurons in the exvivo retina over a large field of view while presenting visual stimuli. This allows for a reliable functional classification of different retinal cell types. We also demonstrate that the system has sufficient resolution to image calcium entry at individual synaptic release sites across the axon terminals of dozens of simultaneously imaged bipolar cells. The simple design, large field of view, and fast image acquisition make this a powerful system for high-throughput and high-resolution measurements of retinal processing at a fraction of the cost of alternative approaches.

Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication.

Calcium (Ca2+), a ubiquitous second messenger, plays a crucial role in many cellular functions. Viruses often hijack Ca2+ signaling to facilitate viral processes such as entry, replication, assembly, and egress. Here, we report that infection by the swine arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV), induces dysregulated Ca2+ homeostasis, subsequently activating calmodulin-dependent protein kinase-II (CaMKII) mediated autophagy, and thus fueling viral replication. Mechanically, PRRSV infection induces endoplasmic reticulum (ER) stress and forms a closed ER-plasma membrane (PM) contacts, resulting the opening of store operated calcium entry (SOCE) channel and causing the ER to take up extracellular Ca2+, which is then released into the cytoplasm by inositol trisphosphate receptor (IP3R) channel. Importantly, pharmacological inhibition of ER stress or CaMKII mediated autophagy blocks PRRSV replication. Notably, we show that PRRSV protein Nsp2 plays a dominant role in the PRRSV induced ER stress and autophagy, interacting with stromal interaction molecule 1 (STIM1) and the 78 kDa glucose-regulated protein 78 (GRP78). The interplay between PRRSV and cellular calcium signaling provides a novel potential approach to develop antivirals and therapeutics for the disease outbreaks.

Chronic suppression of STIM1-mediated calcium signaling in Purkinje cells rescues the cerebellar pathology in spinocerebellar ataxia type 2

Distorted neuronal calcium signaling has been reported in many neurodegenerative disorders, including different types of spinocerebellar ataxias (SCAs). Cerebellar Purkinje cells (PCs) are primarily affected in SCAs and the disturbances in the calcium homeostasis were observed in SCA PCs. Our previous results have revealed that 3,5-dihydroxyphenylglycine (DHPG) induced greater calcium responses in SCA2-58Q PC cultures than in wild type (WT) PC cultures. Here we observed that glutamate-induced calcium release in PCs cells bodies is significantly higher in SCA2-58Q PCs from acute cerebellar slices compared to WT PCs of the same age. Recent studies have demonstrated that the stromal interaction molecule 1 (STIM1) plays an important role in the regulation of the neuronal calcium signaling in cerebellar PCs in mice. The main function of STIM1 is to regulate store-operated calcium entry through the TRPC/Orai channels formation to refill the calcium stores in the ER when it is empty. Here we demonstrated that the chronic viral-mediated expression of the small interfering RNA (siRNA) targeting STIM1 specifically in cerebellar PCs alleviates the deranged calcium signaling in SCA2-58Q PCs, rescues the spine loss in these cerebellar neurons, and also improves the motor decline in SCA2-58Q mice. Thus, our preliminary results support the important role of the altered neuronal calcium signaling in SCA2 pathology and also suggest the STIM1-mediated signaling pathway as a potential therapeutic target for treatment of SCA2 patients.

TRPV6 Calcium Channel Targeting by Antibodies Raised against Extracellular Epitopes Induces Prostate Cancer Cell Apoptosis

The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against extracellular epitopes corresponding to the extracellular loop between S1 and S2 (rb79) and the pore region (rb82). These antibodies generated a complex biphasic response with the transient activation of the TRPV6 channel. Store-operated calcium entry was consequently potentiated in the prostate cancer cell line LNCaP upon the treatment. Both rb79 and rb82 antibodies significantly decreased cell survival rate in a dose-dependent manner as compared to the control antibodies of the same isotype. This decrease was due to the enhanced cell death via apoptosis revealed using a sub-G1 peak in a cell cycle assay, TUNEL assay, and a Hoechst staining, having no effects in the PC3Mtrpv6-/- cell line. Moreover, all TUNEL-positive cells had TRPV6 membrane staining as compared to the control antibody treatment where TRPV6-positive cells were all TUNEL negative. These data clearly demonstrate that TRPV6 channel targeting using rb79 and rb82 antibodies is fatal and may be successfully used in the anticancer therapies.

CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets.

Altered intracellular Ca2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. Altered thapsigargin-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor [TMEM66]) were found in neonatal platelet as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca2+-dependent inactivation, so reproducing the Ca2+-homeostasis pattern observed in neonatal platelets. CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. S-STIM1 may avoid slow Ca2+-dependent inactivation and, subsequently, result in an enhanced thapsigargin-evoked SOCE as observed in neonatal platelets.