STIM1 Research Articles

Blockade of mGluR5 in nucleus accumbens modulates calcium sensor proteins, facilitates extinction, and attenuates reinstated morphine place preference in rats

Numerous findings confirm that the metabotropic glutamate receptors (mGluRs) are involved in the conditioned place preference (CPP) induced by morphine. Here we focused on the role of mGluR5 in the nucleus accumbens (NAc) as a main site of glutamate action on the rewarding effects of morphine. Firstly, we investigated the effects of intra-NAc administrating mGluR5 antagonist 3-((2-Methyl-1,3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP; 1, 3, and 10 μg/μl saline) on the extinction and the reinstatement phase of morphine CPP. Moreover, to determine the downstream signaling cascades of mGluR5 in morphine CPP, the protein levels of stromal interaction molecules (STIM1 and 2) in the NAc and hippocampus (HPC) were measured by western blotting. The behavioral data indicated that the mGluR5 blockade by MTEP at the high doses of 3 and 10 μg facilitated the extinction of morphine-induced CPP and attenuated the reinstatement to morphine in extinguished rats. Molecular results showed that the morphine led to increased levels of STIM proteins in the HPC and increased the level of STIM1 without affecting STIM2 in the NAc. Furthermore, intra-NAc microinjection of MTEP (10 μg) in the reinstatement phase decreased STIM1 in the NAc and HPC and reduced the STIM2 in the HPC. Collectively, our data show that morphine could facilitate brain reward function in part by increasing glutamate-mediated transmission through activation of mGluR5 and modulation of STIM proteins. Therefore, these results highlight the therapeutic potential of mGluR5 antagonists in morphine use disorder.

STIM1-dependent store-operated calcium entry mediates sex differences in macrophage chemotaxis and monocyte recruitment

Infiltration of monocyte-derived cells to sites of infection and injury is greater in males than in females, due in part, to increased chemotaxis, the process of directed cell movement toward a chemical signal. The mechanisms governing sexual dimorphism in chemotaxis are not known. We hypothesized a role for the store-operated calcium entry (SOCE) pathway in regulating chemotaxis by modulating leading and trailing edge membrane dynamics. We measured the chemotactic response of bone marrow derived macrophages (BMDMs) migrating towards complement component 5a (C5a). Chemotactic ability was dependent on sex and inflammatory phenotype (M0, M1, M2), and correlated with SOCE. Notably, females exhibited a significantly lower magnitude of SOCE than males. When we knocked out the SOCE gene, stromal interaction molecule 1 (STIM1), it eliminated SOCE and equalized chemotaxis across both sexes. Analysis of membrane dynamics at the leading and trailing edges showed that STIM1 influences chemotaxis by facilitating retraction of the trailing edge. Using BTP2 to pharmacologically inhibit SOCE mirrored the effects of STIM1 knockout, demonstrating a central role of STIM/Orai-mediated calcium signaling. Importantly, by monitoring the recruitment of adoptively transferred monocytes in an in vivo model of peritonitis, we show that increased infiltration of male monocytes during infection is dependent on STIM1. These data support a model in which STIM1-dependent SOCE is necessary and sufficient for mediating the sex difference in monocyte recruitment and macrophage chemotactic ability by regulating trailing edge dynamics.

Role of STIM1 in stretch-induced signaling in human airway smooth muscle.

Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1, and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways, including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.NEW & NOTEWORTHY Mechanical forces on the airway can contribute to altered contractility and remodeling in airway diseases, but the mechanisms are not clearly understood. Using human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic breathing and static pressure, we found that the effects of stretch are mediated through STIM1, resulting in the activation of multiple pathways, including Piezo channels and the inflammasome, with potential downstream influences on contractility and remodeling.

Role of KDM2B epigenetic factor in regulating calcium signaling in prostate cancer cells

KDM2B, a histone lysine demethylase, is expressed in a plethora of cancers. Earlier studies from our group, have showcased that overexpression of KDM2B in the human prostate cancer cell line DU-145 is associated with cell adhesion, actin reorganization, and improved cancer cell migration. In addition, we have previously examined changes of cytosolic Ca2+, regulated by the pore-forming proteins ORAI and the Ca2+ sensing stromal interaction molecules (STIM), via store-operated Ca2+ entry (SOCE) in wild-type DU-145. This study sought to evaluate the impact of KDM2B overexpression on the expression of key molecules (SGK1, Nhe1, Orai1, Stim1) and SOCE. Furthermore, this is the first study to evaluate KDM2B expression in circulating tumor cells (CTCs) from patients with prostate cancer. mRNA levels for SGK1, Nhe1, Orai1, and Stim1 were quantified by RT-PCR. Calcium signals were measured in KDM2B-overexpressing DU-145 cells, loaded with Fura-2. Blood samples from 22 prostate cancer cases were scrutinized for KDM2B expression using immunofluorescence staining and the VyCAP system. KDM2B overexpression in DU-145 cells increased Orai1, Stim1, and Nhe1 mRNA levels and significantly decreased Ca2+ release. KDM2B expression was examined in 22 prostate cancer patients. CTCs were identified in 45 % of these patients. 80 % of the cytokeratin (CK)-positive patients and 63 % of the total examined CTCs exhibited the (CK + KDM2B + CD45−) phenotype. To conclude, this study is the first to report increased expression of KDM2B in CTCs from patients with prostate cancer, bridging in vitro and preclinical assessments on the potentially crucial role of KDM2B on migration, invasiveness, and ultimately metastasis in prostate cancer.

Essential role of N-terminal SAM regions in STIM1 multimerization and function

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Ca2+-dependent contractile signaling induced by dsDNA-cGAS ligation mediates type 1 interferon generation and vascular injury

Acute Lung injury (ALI) is an uncontrolled systemic inflammatory response from bacterial or viral infection. The cyclic GMP-AMP synthase (cGAS) detects cytosolic double stranded DNA (dsDNA) release from bacteria, viruses, or apoptotic cells and induces innate immunity by generating the second messenger cGAMP and activation of stimulator of interferon genes (STING). However, the mechanisms regulating cGAS activity upon binding dsDNA remain elusive. Here, we show for the first in-depth analysis of mechanisms inducing cGAS activation in using lung endothelial cells (EC) and establish its translational relevance in causing ALI using a mouse model. We show that within 15 min, dsDNA ligation with cGAS mediates its interaction with a motor protein, myosin light chain 6 (MYL6), leading to phosphorylation of MLC and actin stress fiber formation. cGAS-MYL6-mediated MLC phosphorylation required calcium release and organization of an ER-Ca2+ sensor, Stromal Interaction Molecule 1 (STIM1) as puncta. Activated STIM1 also induced cGAS tyrosine phosphorylation at Y215/242 residues via cSrc. Interfering with MYL6 or cSrc expression impaired cGAS condensation and cGAMP generation. Liposome delivery of phosphodeficient cGAS (Y215/Y242àF215/F242) in EC of cGAS null mice blocked type1-IFN production during bacterial pneumonia. Our finding suggests that STIM1 is crucial in inducing cGAS activity and type1-IFN production. cGAS thus represents a novel target for inhibiting STING activation, thereby suppressing tissue injury and inflammation. NIH. This is the full abstract presented at the American Physiology Summit 2024 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.

Molecular Characterization of the Ca2+ Release Activated Ca2+ Current in Native Brain Capillary Endothelial Cells

Brain capillary endothelial cells (cECs) form the blood-brain barrier orchestrate on-demand increases in blood flow to serve the brain’s unique metabolic needs. An in vivo imaging study showed that brain capillaries generate an array of dynamic Ca2+ signals primarily dependent on the release of Ca2+ from the endoplasmic reticulum (ER). Little is known about the physiological functions and origins of these signals. Store-operated Ca2+ entry (SOCE) is a fundamental Ca2+ influx mechanism activated by emptying ER Ca2+ stores. Following store depletion, interactions between the ER Ca2+-sensor stromal interaction molecule 1 (STIM1) and Orai1, 2, and/or 3 ion channel subunits on the plasma membrane initiate the Ca2+-release activated Ca2+ current (ICRAC) and SOCE. ICRAC and SOCE in brain cECs are uncharacterized. Therefore, we investigated STIM and Orai expression and the biophysical properties of ICRAC in primary brain cECs. Using droplet digital PCR, we found that native brain cECs express Stim1, Stim2, Orai1, and Orai3 but not Orai2. Conventional whole-cell patch-clamp electrophysiology was used to record ICRAC from freshly isolated brain cECs. Currents were induced by including the slow Ca2+ buffer EGTA in the intracellular solution. In contrast to the strong inward rectification of canonical ICRAC, currents recorded from native brain cECs were dually rectifying. The inward and outward components were blocked by La3+, enhanced with 2-APB, and inhibited with the selective Orai1 channel antagonist Synta66. Both ICRAC components were increased when a divalent-free solution (Ca2+ replaced with Cs+) was used. To gain insight into the molecular composition of CRAC channels in brain cECs, we generated EC-specific knockout (ecKO) Stim1, Orai1, and Orai3 mice. ICRAC was absent in cECs obtained from Stim1-ecKO mice. In contrast, ICRAC recorded from Orai1-ecKO mice displayed strong inward rectification with no outward current and was insensitive to Synta66. ICRAC recorded from Orai3-ecKO mice were also inwardly rectifying but were highly sensitive to Synta66. ICRAC was undetectable in cells from Orai1-Orai3-ecKO mice. These data suggest that native ICRAC in brain cECs requires the expression of Stim1, Orai1, and Orai3. The outward component of native ICRAC in these cells requires the expression of Orai1 and Orai3, but the expression of either Orai1 or Orai3 alone is suffcient to maintain the inward component. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 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.

Store-operated calcium entry is necessary for sustained K+-induced capillary-to-arteriole dilation in the brain

Neurovascular coupling (NVC) is a physiological process that directs blood flow to the most active regions of the brain. The extensive network of capillaries in the brain acts as a sensory web that detects local neuronal activity and orchestrates the dilation of upstream arterioles to meet the brain’s metabolic demands. Activation of inwardly rectifying K+ (KIR) channels on brain capillary endothelial cells (cECs) by modest concentrations of extracellular K+ initiates retrograde propagating hyperpolarizing signals that induce vasodilation and increase blood flow. Brain capillaries exhibit diverse and dynamic patterns of Ca2+ signaling in vivo, but little is known about their origins or physiological function. Store-operated Ca2+ entry (SOCE) is fundamental to Ca2+homeostasis in non-excitable cells, but the contribution of SOCE to capillary-to-arteriole dilation has not been explored. Here, we report that brain cECs express the endoplasmic reticulum-resident Ca2+ sensing protein stromal interaction molecule 1 ( Stim1) and the Ca2+-selective Orai1 and Orai3 channels. Ca2+ imaging experiments using brain cECs from transgenic mice expressing GCaMP8 in the endothelium or loaded with Fluo-4 showed that these cells exhibited robust SOCE that was blocked by the Orai1 inhibitor Synta66. Using EC-specific knockout (ecKO) mice, we found that Stim1 and Orai1 but not Orai3 are required for SOCE. Ex vivo brain microvascular preparations consisting of cannulated and pressured parenchymal arterioles with intact capillary beds were used to examine the role of SOCE in capillary-to-arteriole dilation. Arteriole dilation in response to a train of K+ pulses applied to capillaries exhibited dramatic run-down when preparations were treated with Snyta66 or when tissues from Stim1-ecKO mice were used. Preparations from Orai3-ecKO mice did not demonstrate run-down. Moreover, inhibition of Ca2+-activated K+ (SK and IK) channels also induced run-down, suggesting that SOCE activates SK and IK channels to boost KIR-induced hyperpolarizing signals to dilate upstream arterioles during multiple stimulations. We conclude that brain cECs exhibit SOCE that requires Stim1 and Orai1. Our data further show that brain cEC SOCE is necessary for capillary-to-arteriole dilation during repeated stimulations, suggesting an essential role for SOCE in NVC. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 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.

Mechanisms of vibration-induced structural myocardial remodeling

The review analyzes literature data on structural changes in the heart of patients with vibration disease, as detected by echocardiographic methods. Particularly, it highlights concentric remodeling of the left ventricle chambers and disturbances in diastolic function. The review also discusses a 1.2-fold decrease in heart structure intensity compared to healthy individuals (p 0.05). Furthermore, it examines changes in morphometric and bioenergetic parameters of cardiomyocytes under different experimental vibration modes (7 and 56 sessions at a frequency of 8 Hz), confirming the disruptions in the relationship between the spatial configuration of the heart cavities, contractile ability, and energy supply potential. Loss of cardiac myofibrils represents the transition from myocardial hypertrophy to decompensation, accompanied by an increase in degenerative (dystrophic) signs such as the loss of sarcomeres in cardiomyocytes. Understanding these pathological (morphological) processes requires consideration of various mediators that regulate cell metabolism, proliferation, growth, and survival, including stromal interaction molecule, calcium ATPase of the endo(sarco)plasmic reticulum, inositol-1,4,5-triphosphate receptor, protein that forms CRAC channels, and transient receptor potential canonical. The degradation system of the extracellular matrix, including matrix metalloproteinases and tissue inhibitors, plays a crucial role in structural cardiac remodeling. This system regulates the rate of mRNA synthesis on the DNA matrix by binding to specific DNA regions that control cardiac nutrition and plasticity. The review suggests that these findings can help explain some patterns of cardiac remodeling development in patients with vibration disease and determine the direction of pathogenetically based therapy. This therapy should consider not only the vibration-protective effect of drugs but also their ability to inhibit and regress myocardial remodeling.

Rapamycin Controls Lymphoproliferation and Reverses T-Cell Responses in a Patient with a Novel STIM1 Loss-of-Function Deletion

PurposeDeficiency of stromal interaction molecule 1 (STIM1) results in combined immunodeficiency accompanied by extra-immunological findings like enamel defects and myopathy. We here studied a patient with a STIM1 loss-of-function mutation who presented with severe lymphoproliferation. We sought to explore the efficacy of the mTOR inhibitor rapamycin in controlling disease manifestations and reversing aberrant T-cell subsets and functions, which has never been used previously in this disorder.MethodsClinical findings of the patient were collected over time. We performed immunological evaluations before and after initiation of rapamycin treatment, including detailed lymphocyte subset analyses, alterations in frequencies of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes as well as T cell activation and proliferation capacities.ResultsA novel homozygous exon 2 deletion in STIM1 was detected in a 3-year-old girl with severe lymphoproliferation, recurrent infections, myopathy, iris hypoplasia, and enamel hypoplasia. Lymphoproliferation was associated with severe T-cell infiltrates. The deletion resulted in a complete loss of protein expression, associated with a lack of store-operated calcium entry response, defective T-cell activation, proliferation, and cytokine production. Interestingly, patient blood contained fewer cTFH and increased circulating follicular regulatory (cTFR) cells. Abnormal skewing towards TH2-like responses in certain T-cell subpopulations like cTFH, non-cTFH memory T-helper, and Treg cells was associated with increased eosinophil numbers and serum IgE levels. Treatment with rapamycin controlled lymphoproliferation, improved T-cell activation and proliferation capacities, reversed T-cell responses, and repressed high IgE levels and eosinophilia.ConclusionsThis study enhances our understanding of STIM1 deficiency by uncovering additional abnormal T-cell responses, and reveals for the first time the potential therapeutic utility of rapamycin for this disorder.

STIM1 promotes acquired resistance to sorafenib by attenuating ferroptosis in hepatocellular carcinoma

Dysregulated calcium (Ca2+) signaling pathways are associated with tumor cell death and drug resistance. In non-excitable cells, such as hepatocellular carcinoma (HCC) cells, the primary pathway for Ca2+ influx is through stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE). Previous studies have demonstrated the involvement of STIM1-mediated SOCE in processes such as genesis, metastasis, and stem cell self-renewal of HCC. However, it remains unclear whether STIM1-mediated SOCE plays a role in developing acquired resistance to sorafenib in HCC patients. In this study, we established acquired sorafenib-resistant (SR) HCC cell lines by intermittently exposing them to increasing concentrations of sorafenib. Our results showed higher levels of STIM1 and stronger SOCE in SR cells compared with parental cells. Deleting STIM1 significantly enhanced sensitivity to sorafenib in SR cells, while overexpressing STIM1 promoted SR by activating SOCE. Mechanistically, STIM1 increased the transcription of SLC7A11 through the SOCE-CaN-NFAT pathway. Subsequently, up-regulated SLC7A11 increased glutathione synthesis, resulting in ferroptosis insensitivity and SR. Furthermore, combining the SOCE inhibitor SKF96365 with sorafenib significantly improved the sensitivity of SR cells to sorafenib both in vitro and in vivo. These findings suggest a potential strategy to overcome acquired resistance to sorafenib in HCC cells.

Insights into the dynamics of the Ca2+ release-activated Ca2+ channel pore-forming complex Orai1.

An important calcium (Ca2+) entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel, which controls a series of downstream signaling events such as gene transcription, secretion and proliferation. It is composed of a Ca2+ sensor in the endoplasmic reticulum (ER), the stromal interaction molecule (STIM), and the Ca2+ ion channel Orai in the plasma membrane (PM). Their activation is initiated by receptor-ligand binding at the PM, which triggers a signaling cascade within the cell that ultimately causes store depletion. The decrease in ER-luminal Ca2+ is sensed by STIM1, which undergoes structural rearrangements that lead to coupling with Orai1 and its activation. In this review, we highlight the current understanding of the Orai1 pore opening mechanism. In this context, we also point out the questions that remain unanswered and how these can be addressed by the currently emerging genetic code expansion (GCE) technology. GCE enables the incorporation of non-canonical amino acids with novel properties, such as light-sensitivity, and has the potential to provide novel insights into the structure/function relationship of CRAC channels at a single amino acid level in the living cell.

Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+-release-activated Ca2+ channels constituted by Orai family members, with predominance of calcium release-activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT-29 and Caco-2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT-29 and Caco-2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta-66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Synthetic Biology Meets Ca2+ Release-Activated Ca2+ Channel-Dependent Immunomodulation.

Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor-ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis.

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

Vanillic acid restores homeostasis of intestinal epithelium in colitis through inhibiting CA9/STIM1-mediated ferroptosis

The damage of integrated epithelial epithelium is a key pathogenic factor and closely associated with the recurrence of ulcerative colitis (UC). Here, we reported that vanillic acid (VA) exerted potent therapeutic effects on DSS-induced colitis by restoring intestinal epithelium homeostasis via the inhibition of ferroptosis. By the CETSA assay and DARTS assay, we identified carbonic anhydrase IX (CAIX, CA9) as the direct target of VA. The binding of VA to CA9 causes insulin-induced gene-2 (INSIG2) to interact with stromal interaction molecule 1 (STIM1), rather than SREBP cleavage-activating protein (SCAP), leading to the translocation of SCAP-SREBP1 from the endoplasmic reticulum (ER) to the Golgi apparatus for cleavage into mature SREBP1. The activation of SREBP1 induced by VA then significantly facilitated the transcription of stearoyl-CoA desaturase 1 (SCD1) to exert an inhibitory effect on ferroptosis. By inhibiting the excessive death of intestinal epithelial cells caused by ferroptosis, VA effectively preserved the integrity of intestinal barrier and prevented the progression of unresolved inflammation. In conclusion, our study demonstrated that VA could alleviate colitis by restoring intestinal epithelium homeostasis through CA9/STIM1-mediated inhibition of ferroptosis, providing a promising therapeutic candidate for UC.

Calcium signaling in endothelial and vascular smooth muscle cells: sex differences and the influence of estrogens and androgens.

Calcium signaling in vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) is essential for the regulation of vascular tone. However, the changes to intracellular Ca2+ concentrations are often influenced by sex differences. Furthermore, a large body of evidence shows that sex hormone imbalance leads to dysregulation of Ca2+ signaling and this is a key factor in the pathogenesis of cardiovascular diseases. In this review, the effects of estrogens and androgens on vascular calcium-handling proteins are discussed, with emphasis on the associated genomic or nongenomic molecular mechanisms. The experimental models from which data were collected were also considered. The review highlights 1) in female ECs, transient receptor potential vanilloid 4 (TRPV4) and mitochondrial Ca2+ uniporter (MCU) enhance Ca2+-dependent nitric oxide (NO) generation. In males, only transient receptor potential canonical 3 (TRPC3) plays a fundamental role in this effect. 2) Female VSMCs have lower cytosolic Ca2+ levels than males due to differences in the activity and expression of stromal interaction molecule 1 (STIM1), calcium release-activated calcium modulator 1 (Orai1), calcium voltage-gated channel subunit-α1C (CaV1.2), Na+-K+-2Cl- symporter (NKCC1), and the Na+/K+-ATPase. 3) When compared with androgens, the influence of estrogens on Ca2+ homeostasis, vascular tone, and incidence of vascular disease is better documented. 4) Many studies use supraphysiological concentrations of sex hormones, which may limit the physiological relevance of outcomes. 5) Sex-dependent differences in Ca2+ signaling mean both sexes ought to be included in experimental design.

The D84G mutation in STIM1 causes nuclear envelope dysfunction and myopathy in mice.

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor located in the sarcoplasmic reticulum (SR) of skeletal muscle, where it is best known for its role in store-operated Ca2+ entry (SOCE). Genetic syndromes resulting from STIM1 mutations are recognized as a cause of muscle weakness and atrophy. Here, we focused on a gain-of-function mutation that occurs in humans and mice (STIM1+/D84G mice), in which muscles exhibited constitutive SOCE. Unexpectedly, this constitutive SOCE did not affect global Ca2+ transients, SR Ca2+ content, or excitation-contraction coupling (ECC) and was therefore unlikely to underlie the reduced muscle mass and weakness observed in these mice. Instead, we demonstrate that the presence of D84G STIM1 in the nuclear envelope of STIM1+/D84G muscle disrupted nuclear-cytosolic coupling, causing severe derangement in nuclear architecture, DNA damage, and altered lamina A-associated gene expression. Functionally, we found that D84G STIM1 reduced the transfer of Ca2+ from the cytosol to the nucleus in myoblasts, resulting in a reduction of [Ca2+]N. Taken together, we propose a novel role for STIM1 in the nuclear envelope that links Ca2+ signaling to nuclear stability in skeletal muscle.