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Stromal interaction molecule 1 (STIM1) is critical for store-operated Ca 2+ entry (SOCE) and T cell activation. T helper 1 (T H 1) cells, which express T-bet (encoded by TBX21), mediate immunity to intracellular pathogens. Although SOCE is known to regulate other T H lineages, its role in Th1 differentiation remains unclear. Here, we report a patient with an intronic loss-of-function mutation in STIM1, which abolishes SOCE and causes immunodeficiency. We demonstrate that SOCE promotes nuclear factor of activated T cells (NFAT) binding to conserved noncoding sequence (CNS)-12 in the TBX21 enhancer and enables NFAT to synergize with STAT1 to mediate TBX21 expression. While SOCE-deficient CD4 + T cells have reduced expression of TBX21 in the absence of interleukin-12 (IL-12), their expression of IL-12 receptors β1 and β2 is increased, sensitizing them to IL-12 signaling and allowing IL-12 to rescue T-bet expression. Our study reveals that the STIM1-SOCE-NFAT signaling axis is essential for the differentiation of Th1 cells depending on the cytokine milieu., Competing Interests: Competing interests: S.F. is a scientific cofounder and consultant of Calcimedica, and an inventor on a patent Regulators of NFAT (WO/2007/081804) related to this paper. The other authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Membrane contact sites (MCS) provide specialized conduits for inter-organelle communications to maintain cellular homeostasis. Most organelles are interconnected, which supports their coordination and function. M-phase (mitosis or meiosis) is associated with dramatic cellular remodeling to support cell division, including the equal distribution of organelles to the two daughter cells. However, the fate of MCS in M-phase is poorly understood. Here we review recent advances arguing for differential remodeling of endoplasmic reticulum (ER) MCS with the plasma membrane (PM, ERPMCS) and the mitochondria (MERCS) during cell division., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2025.)

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, the details of its signal transduction cascade remain elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/β hydrolase domain-containing protein 2) as an essential mPRβ co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires mPRβ. This PLA2 activity bifurcates P4 signaling by inducing clathrin-dependent endocytosis of mPRβ, resulting in the production of lipid messengers that are G-protein coupled receptor agonists. Therefore, P4 drives meiosis by inducing an ABHD2 PLA2 activity that requires both mPRβ and ABHD2 as obligate co-receptors., Competing Interests: NN, LA, LZ, AH, SY, MD, NA, QC, KS, SG, KM No competing interests declared, (© 2024, Nader et al.)

Ca2+ tunneling requires both store-operated Ca2+ entry (SOCE) and Ca2+ release from the endoplasmic reticulum (ER). Tunneling expands the SOCE microdomain through Ca2+ uptake by SERCA into the ER lumen where it diffuses and is released via IP3 receptors. In this study, using high-resolution imaging, we outline the spatial remodeling of the tunneling machinery (IP3R1; SERCA; PMCA; and Ano1 as an effector) relative to STIM1 in response to store depletion. We show that these modulators redistribute to distinct subdomains laterally at the plasma membrane (PM) and axially within the cortical ER. To functionally define the role of Ca2+ tunneling, we engineered a Ca2+ tunneling attenuator (CaTAr) that blocks tunneling without affecting Ca2+ release or SOCE. CaTAr inhibits Cl- secretion in sweat gland cells and reduces sweating in vivo in mice, showing that Ca2+ tunneling is important physiologically. Collectively our findings argue that Ca2+ tunneling is a fundamental Ca2+ signaling modality., (© 2024 Courjaret et al.)

Store-operated calcium entry (SOCE) is essential for cellular signaling. Earlier studies of the pyrazole derivative BTP2, an efficient inhibitor SOCE, identified that SOCE blockade suppresses proinflammatory gene expression. The impact of SOCE blockade on gene expression at the whole transcriptome level, however, is unknown. To fill this gap, we performed RNA sequencing (RNA-seq) and investigated at the whole transcriptome level the effect of BTP2 on gene expression in human peripheral blood mononuclear cells signaled with phytohemagglutinin. Our global gene expression analysis identified that SOCE blockade spares activation-induced expression of anti-inflammatory genes (e.g., IL10, TGFB1, FOXP3, and CTLA4) whereas the induced expression of proinflammatory genes such as IFNG and cytopathic genes such as GZMB are inhibited. We validated the differential expression of immunoregulatory genes identified by RNA-seq using preamplification-enhanced RT-qPCR assays. Because IL-2/IL2RA interaction is essential for T cell clonal expansion, we investigated and confirmed that BTP2 inhibits IL2RA expression at the protein level using multiparameter flow cytometry. Our elucidation that SOCE blockade spares activation-induced expression of anti-inflammatory genes while blocking pro-inflammatory gene expression suggests that SOCE blockers may represent a novel class of immunoregulatory drugs of value for treating autoimmune disease states and organ transplantation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, the details of its signal transduction cascade remain elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/β hydrolase domain-containing protein 2) as an essential mPRβ co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires mPRβ. This PLA2 activity bifurcates P4 signaling by inducing clathrin-dependent endocytosis of mPRβ, resulting in the production of lipid messengers that are G-protein coupled receptors agonists. Therefore, P4 drives meiosis by inducing an ABHD2 PLA2 activity that requires both mPRβ and ABHD2 as obligate co-receptors.

Background: TRPM4 is a broadly expressed, calcium-activated, monovalent cation channel that regulates immune cell function in mice and cell lines. Clinically, however, partial loss- or gain-of-function mutations in TRPM4 lead to arrhythmia and heart disease, with no documentation of immunologic disorders., Objective: To characterize functional cellular mechanisms underlying the immune dysregulation phenotype in a proband with a mutated TRPM4 gene., Methods: We employed a combination of biochemical, cell biological, imaging, omics analyses, flow cytometry, and gene editing approaches., Results: We report the first human cases to our knowledge with complete loss of the TRPM4 channel, leading to immune dysregulation with frequent bacterial and fungal infections. Single-cell and bulk RNA sequencing point to altered expression of genes affecting cell migration, specifically in monocytes. Inhibition of TRPM4 in T cells and the THP-1 monocyte cell line reduces migration. More importantly, primary T cells and monocytes from TRPM4 patients migrate poorly. Finally, CRISPR knockout of TRPM4 in THP-1 cells greatly reduces their migration potential., Conclusion: Our results demonstrate that TRPM4 plays a critical role in regulating immune cell migration, leading to increased susceptibility to infections., Competing Interests: Disclosure statement The Weill Cornell Medicine Qatar Cores are supported by the Biomedical Research Program at Weill Cornell Medicine Qatar, a program funded by Qatar Foundation. This publication was made possible by Path Towards Precision Medicine fourth Cycle grant PPM 04-0128-200015 from the Qatar National Research Fund (a member of Qatar Foundation). The findings herein reflect the work and are solely the responsibility of the authors. This work was also supported by Sidra Medicine and the Biomedical Research Program at Weill Cornell Medicine–Qatar, a program funded by Qatar Foundation. Disclosure of potential conflicts of interest: The authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Store operated Ca 2+ entry (SOCE) is a ubiquitous signalling module with established roles in the immune system, secretion and muscle development. Recent evidence supports a complex role for SOCE in the nervous system. In this review we present an update of the current knowledge on SOCE function in the brain with a focus on its role as a regulator of brain activity and excitability., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Mitochondria-ER contact sites (MERCS) are involved in energy homeostasis, redox and Ca 2+ signaling, and inflammation. MERCS are heavily studied; however, little is known about their regulation during mitosis. Here, we show that MERCS expand during mitosis in three cell types using various approaches, including transmission electron microscopy, serial EM coupled to 3D reconstruction, and a split GFP MERCS marker. We further show enhanced Ca 2+ transfer between the ER and mitochondria using either direct Ca 2+ measurements or by quantifying the activity of Ca 2+ -dependent mitochondrial dehydrogenases. Collectively, our results support a lengthening of MERCS in mitosis that is associated with improved Ca 2+ coupling between the two organelles. This augmented Ca 2+ coupling could be important to support the increased energy needs of the cell during mitosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Authors.)

Competing Interests: None.

Store-operated Ca 2+ entry (SOCE) is a ubiquitous Ca2+ signaling modality mediated by Orai Ca 2+ channels at the plasma membrane (PM) and the endoplasmic reticulum (ER) Ca 2+ sensors STIM1/2. At steady state, Orai1 constitutively cycles between an intracellular compartment and the PM. Orai1 PM residency is modulated by its endocytosis and exocytosis rates. Therefore, Orai1 trafficking represents an important regulatory mechanism to define the levels of Ca 2+ influx. Here, we present a protocol using the dually tagged YFP-HA-Orai1 with a cytosolic YFP and extracellular hemagglutinin (HA) tag to quantify Orai1 cycling rates. For measuring Orai1 endocytosis, cells expressing YFP-HA-Orai1 are incubated with mouse anti-HA antibody for various periods of time before being fixed and stained for surface Orai1 with Cy5-labeled anti-mouse IgG. The cells are fixed again, permeabilized, and stained with Cy3-labeled anti-mouse IgG to reveal anti-HA that has been internalized. To quantify Orai1 exocytosis rate, cells are incubated with anti-HA antibody for various incubation periods before being fixed, permeabilized, and then stained with Cy5-labeled anti-mouse IgG. The Cy5/YFP ratio is plotted over time and fitted with a mono-exponential growth curve to determine exocytosis rate. Although the described assays were developed to measure Orai1 trafficking, they are readily adaptable to other PM channels. Key features Detailed protocols to quantify endocytosis and exocytosis rates of Orai1 at the plasma membrane that can be used in various cell lines. The endocytosis and exocytosis assays are readily adaptable to study the trafficking of other plasma membrane channels., Competing Interests: Competing interestsThe authors declare no conflict of interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Store-operated calcium entry (SOCE) contributes to several physiological and pathological conditions including transcription, secretion, immunodeficiencies, and cancer. SOCE has been shown to be important for breast cancer cell migration where knockdown of SOCE components (STIM1 or Orai1) decreases cancer metastasis. Here we show unexpectedly that complete knockout of STIM1 (STIM1-KO) using gene editing in metastatic MDA-MB-231 breast cancer cells results in faster migration and enhanced invasion capacity. In contrast, Orai1-KO cells, which have similar levels of SOCE inhibition as STIM1-KO, migrate slower than the parental cell line. This shows that the enhanced migration phenotype of STIM1-KO cells is not due to the loss of Ca 2+ entry through SOCE, rather it involves transcriptional remodeling as elucidated by RNA-seq analyses. Interestingly, NFAT1 is significantly downregulated in STIM1-KO cells and overexpression of NFAT1 reversed the enhanced migration of STIM1-KO cells. STIM1 knockout in other breast cancer cells, independent of their metastatic potential, also enhanced cell migration while reducing NFAT1 expression. These data argue that in breast cancer cells STIM1 modulates NFAT1 expression and cell migration independently of its role in SOCE., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Calcium is a critical signaling molecule in many cell types including immune cells. The calcium-release activated calcium channels (CRAC) responsible for store-operated calcium entry (SOCE) in immune cells are gated by STIM family members functioning as sensors of Ca 2+ store content in the endoplasmic reticulum. We investigated the effect of SOCE blocker BTP2 on human peripheral blood mononuclear cells (PBMC) stimulated with the mitogen phytohemagglutinin (PHA). We performed RNA sequencing (RNA-seq) to query gene expression at the whole transcriptome level and identified genes differentially expressed between PBMC activated with PHA and PBMC activated with PHA in the presence of BTP2. Among the differentially expressed genes, we prioritized genes encoding immunoregulatory proteins for validation using preamplification enhanced real time quantitative PCR assays. We performed multiparameter flow cytometry and validated by single cell analysis that BTP2 inhibits cell surface expression CD25 at the protein level. BTP2 reduced significantly PHA-induced increase in the abundance of mRNAs encoding proinflammatory proteins. Surprisingly, BTP2 did not reduce significantly PHA-induced increase in the abundance of mRNAs encoding anti-inflammatory proteins. Collectively, the molecular signature elicited by BTP2 in activated normal human PBMC appears to be tipped towards tolerance and away from inflammation.

Background and Purpose: Senescent preadipocytes promote adipose tissue dysfunction by secreting pro-inflammatory factors, although little is known about the mechanisms regulating their production. We investigated if up-regulated purinoceptor function sensitizes senescent preadipocytes to cognate agonists and how such sensitization regulates inflammation., Experimental Approach: Etoposide was used to trigger senescence in 3T3-L1 preadipocytes. CRISPR/Cas9 technology or pharmacology allowed studies of transcription factor function. Fura-2 imaging was used for calcium measurements. Interleukin-6 levels were quantified using quantitative PCR and ELISA. Specific agonists and antagonists supported studies of purinoceptor coupling to interleukin-6 production. Experiments in MS1 VEGF angiosarcoma cells and adipose tissue samples from obese mice complemented preadipocyte experiments., Key Results: DNA damage-induced senescence up-regulated purinoceptor expression levels in preadipocytes and MS1 VEGF angiosarcoma cells. ATP-evoked Ca 2+ release was potentiated in senescent preadipocytes. ATP enhanced interleukin-6 production, an effect mimicked by ADP but not UTP, in a calcium-independent manner. Senescence-associated up-regulation and activation of the adenosine A 3 receptor also enhanced interleukin-6 production. However, nucleotide hydrolysis was not essential because exposure to ATPγS also enhanced interleukin-6 secretion. Pharmacological experiments suggested coupling of P2X ion channels and P2Y 12 -P2Y 13 receptors to downstream interleukin-6 production. Interleukin-6 signalling exacerbated inflammation during senescence and compromised adipogenesis., Conclusions and Implications: We report a previously uncharacterized link between cellular senescence and purinergic signalling in preadipocytes and endothelial cancer cells, raising the possibility that up-regulated purinoceptors play key modulatory roles in senescence-associated conditions like obesity and cancer. There is potential for exploitation of specific purinoceptor antagonists as therapeutics in inflammatory disorders., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Loss of function mutations in store-operated Ca 2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1) hypomorph mouse model was generated with significant reduction in Ca 2+ influx through store-operated Ca 2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Cell lipids are differentially distributed in distinct organelles and within the leaflets of the bilayer. They can further form laterally defined sub-domains within membranes with important signaling functions. This molecular and spatial complexity offers optimal platforms for signaling with the associated challenge of dissecting these pathways especially that lipid metabolism tends to be highly interconnected. Lipid signaling has historically been implicated in gamete function, however the detailed signaling pathways involved remain obscure. In this review we focus on oocyte and sperm maturation in an effort to consolidate current knowledge of the role of lipid signaling and set the stage for future directions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mostafa, Nader and Machaca.)

Store-operated Ca 2+ entry (SOCE) is a Ca 2+ influx pathway present in practically every cell type in metazoans and mediates a variety of physiological functions. Defects in SOCE are associated with immunodeficiencies and defects in skeletal muscle development and function. The molecular machinery underpinning SOCE can be complex and cell type specific, however the minimal functional SOCE unit consists of the endoplasmic reticulum (ER) Ca 2+ sensor STIM1 and the plasma membrane (PM) Ca 2+ -selective channel Orai1. STIM1 localizes to ER-PM contact sites (CS) following store depletion, where it recruits and gates Orai1. STIM1 is a phosphoprotein that is hyper-phosphorylated during cell division. STIM1 phosphorylation has been implicated in several functions, including modulation of cellular metabolism, SOCE inactivation during M-phase, ER segregation during mitosis, modulation of SOCE levels, and cell migration. However, the role of STIM1 phosphorylation in the majority of these processes is controversial bringing into question the physiological function of STIM1 phosphorylation, if any. Here we review the role and modulation of STIM1 phosphorylation under various conditions and argue that except for the modulation of energy metabolism, the physiological function of STIM1 phosphorylation remains unclear., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca 2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and I CRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Risk genes for Mendelian (single-gene) disorders (SGDs) are consistent across populations, but pathogenic risk variants that cause SGDs are typically population-private. The goal was to develop "QChip1," an inexpensive genotyping microarray to comprehensively screen newborns, couples, and patients for SGD risk variants in Qatar, a small nation on the Arabian Peninsula with a high degree of consanguinity. Over 10 8 variants in 8445 Qatari were identified for inclusion in a genotyping array containing 165,695 probes for 83,542 known and potentially pathogenic variants in 3438 SGDs. QChip1 had a concordance with whole-genome sequencing of 99.1%. Testing of QChip1 with 2707 Qatari genomes identified 32,674 risk variants, an average of 134 pathogenic alleles per Qatari genome. The most common pathogenic variants were those causing homocystinuria (1.12% risk allele frequency), and Stargardt disease (2.07%). The majority (85%) of Qatari SGD pathogenic variants were not present in Western populations such as European American, South Asian American, and African American in New York City and European and Afro-Caribbean in Puerto Rico; and only 50% were observed in a broad collection of data across the Greater Middle East including Kuwait, Iran, and United Arab Emirates. This study demonstrates the feasibility of developing accurate screening tools to identify SGD risk variants in understudied populations, and the need for ancestry-specific SGD screening tools., (© 2022. The Author(s).)

Store-operated Ca 2+ entry (SOCE) is a ubiquitous Ca 2+ influx pathway required for multiple physiological functions including cell motility. SOCE is triggered in response to depletion of intracellular Ca 2+ stores following the activation of the endoplasmic reticulum (ER) Ca 2+ sensor STIM1, which recruits the plasma membrane (PM) Ca 2+ channel Orai1 at ER-PM junctions. STIM1 is phosphorylated dynamically, and this phosphorylation has been implicated in several processes including SOCE inactivation during M-phase, maximal SOCE activation, ER segregation during mitosis, and cell migration. Human STIM1 has 10 Ser/Thr residues in its cytosolic domain that match the ERK/CDK consensus phosphorylation. We recently generated a mouse knock-in line where wild-type STIM1 was replaced by a non-phosphorylatable STIM1 with all ten S/Ts mutated to Ala (STIM1-10A). Here, we generate mouse embryonic fibroblasts (MEF) from the STIM1-10A mouse line and a control MEF line (WT) that express wild-type STIM1 from a congenic mouse strain. These lines offer a unique model to address the role of STIM1 phosphorylation at endogenous expression levels in contrast to previous studies that relied mostly on overexpression. We show that STIM1 phosphorylation at ERK/CDK sites is not required for SOCE activation, cell migration, or ER partitioning during mitosis. These results rule out STIM1 phosphorylation as a regulator of SOCE, migration, and ER distribution in mitosis., (Copyright © 2021. Published by Elsevier Ltd.)

Store-operated Ca 2+ entry (SOCE) represents a predominant Ca 2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca 2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient's lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID.

Ca 2+ signaling is ubiquitous in eukaryotic cells and modulates many cellular events including cell migration. Directional cell migration requires the polarization of both signaling and structural elements. This polarization is reflected in various Ca 2+ signaling pathways that impinge on cell movement. In particular, store-operated Ca 2+ entry (SOCE) plays important roles in regulating cell movement at both the front and rear of migrating cells. SOCE represents a predominant Ca 2+ influx pathway in non-excitable cells, which are the primary migrating cells in multicellular organisms. In this review, we summarize the role of Ca 2+ signaling in cell migration with a focus on SOCE and its diverse functions in migrating cells and cancer metastasis. SOCE has been implicated in regulating focal adhesion turnover in a polarized fashion and the mechanisms involved are beginning to be elucidated. However, SOCE is also involved is other aspects of cell migration with a less well-defined mechanistic understanding. Therefore, much remains to be learned regarding the role and regulation of SOCE in migrating cells.

Our current understanding of the molecular mechanisms underlying activation of store-operated Ca 2+ entry (SOCE) relies in large part on studies that modulate the expression of STIM1 and Orai1. Shen et al. present the first detailed study to address the dynamics and stoichiometry of endogenous STIM1 and Orai1. They argue for an active SOCE cluster centered around a single Orai1 channel per punctum linked to 12 STIM1 dimers, which could have significant implications on SOCE-dependent Ca 2+ signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Regulation of Ca 2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca 2+ influx pathway in oocytes is Store-Operated Ca 2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260-275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

There a few reports of rhodamine-based fluorescent sensors for selective detection of only Al 3+ , due to the challenge of identifying a suitable ligand for binding Al 3+ ion. The use of fluorophore moieties appended to a polymer backbone for sensing applications is far from mature. Here, we report a new fluorescent probe/monomer 4 and its ROMP derived polymer P for specific detection of Al 3+ ions. Both monomer 4 and its polymer P exhibit high selectivity toward only Al 3+ with no interference from other metal ions, having a limit detection of 0.5 and 2.1 µM, respectively. The reversible recognition of monomer 4 and P for Al 3+ was also proved in presence of Na 2 EDTA by both UV-Vis and fluorometric titration. The experimental data indicates the behavior of 4 and P toward Al 3+ is pH independent in medium conditions. In addition, the switch-on luminescence response of 4 at acidic pH (0 < 5.0), allowed us to specifically stain lysosomes (pH ~ 4.5-5.0) in live cells.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRβ is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRβ signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRβ into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: KM is a co-founder of Valdia Health.

Voltage-gated L-type Ca 2+ channel (Ca v 1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca 2+ signaling machinery, including down-regulation of Ca v 1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca 2+ sensor proteins and the plasma membrane ORAI Ca 2+ channels. STIM/ORAI proteins mediate store-operated Ca 2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca 2+ , causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca 2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Ca v 1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca 2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated., Competing Interests: The authors declare no competing interest.

Contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM) regulate both non-vesicular lipid transfer as well as Ca 2+ signaling with multiple interactions between the two pathways. Here I discuss recent findings that offer exciting insights into the role of store-operated Ca 2+ entry (SOCE), Oxysterol-binding protein (OSBP)-related proteins ORP3, Arf5 and the Arf GEF IQSec1 in this crosstalk and how they regulate cell migration and focal adhesion disassembly., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Store-operated Ca 2+ entry (SOCE) has been shown to be important for breast cancer metastasis in xenograft mouse models. The ER Ca 2+ sensor STIM1 and Orai plasma membrane Ca 2+ channels molecularly mediate SOCE. Here we investigate the role of the microRNA machinery in regulating STIM1 expression. We show that STIM1 expression is regulated post-transcriptionally by the miRNA machinery and identify miR-223 and miR-150 as regulators of STIM1 expression in the luminal non-aggressive MCF7 breast cancer cell line. In contrast, STIM1 expression in the more aggressive basal triple-negative MDA-MB-231 cell line is not significantly modulated by a single miRNA species but is rather upregulated due to inhibition of the miRNA machinery through downregulation of Ago2. Consistently, overexpression of Ago2 results in decreased STIM1 protein levels in MDA-MB-231 cells. Clinically, STIM1 and Ago2 expression levels do not correlate with breast cancer progression, however in the basal subtype high STIM1 expression is associated with poorer survival. Our findings show that STIM1 expression is differentially regulated by the miRNA machinery in different cell types and argue for a role for this regulation in breast cancer.

TPEN is an amino chelator of transition metals that is effective at the cellular and whole organism levels. Although TPEN of often used as a selective zinc chelators, it has affinity for copper and iron and has been shown to chelate both biologically. We have previously shown that TPEN selectively kills colon cancer cells based on its ability to chelate copper, which is highly enriched in colon cancer cells. The TPEN-copper complex is redox active thus allowing for increased ROS production in cancer cells and as such cellular toxicity. Here we generate TPEN derivatives with the goal of increasing its selectivity for copper while minimizing zinc chelation to reduce potential side effects. We show that one of these derivatives, TPEEN despite the fact that it exhibits reduced affinity for transition metals, is effective at inducing cell death in breast cancer cells, and exhibits less toxicity to normal breast cells. The toxicity effect of the both chelators coupled to the metal content of the different cell types reveals that they exhibit their toxicity through chelating redox active metals (iron and copper). As such TPEEN is an important novel chelators that can be exploited in anti-cancer therapies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Store-operated Ca 2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca 2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca 2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca 2+ influx that is required for cell cycle progression., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Store-operated Ca 2+ entry (SOCE) is a ubiquitous pathway for Ca 2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca 2+ -sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca 2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca 2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca 2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1 -/- , STIM2 -/- , and STIM1/2 -/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca 2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are widely expressed intracellular Ca 2+ channels that evoke large local increases in cytosolic Ca 2+ concentration. By depleting the ER of Ca 2+ , IP 3 Rs also activate store-operated Ca 2+ entry (SOCE). Immobile IP 3 Rs close to the plasma membrane (PM) are the only IP 3 Rs that respond to physiological stimuli. The association of these 'licensed' IP 3 Rs with the ER-PM junctions where STIM interacts with Orai PM Ca 2+ channels may define the autonomous functional unit for SOCE. Ca 2+ entering cells through SOCE can be delivered directly to specific effectors, or it may reach them only after the Ca 2+ has been sequestered by the ER and then released through IP 3 Rs. This 'tunnelling' of Ca 2+ through the ER to IP 3 Rs selectively delivers Ca 2+ to different effectors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Store-operated Ca 2+ entry (SOCE) encodes a range of cellular responses downstream of Ca 2+ influx through the SOCE channel Orai1. Orai1 recycles at the plasma membrane (PM), with ~40% of the total Orai1 pool residing at the PM at steady state. The mechanisms regulating Orai1 recycling remain poorly understood. We map the domains in Orai1 that are required for its trafficking to and recycling at the PM. We further identify, using biochemical and proteomic approaches, the CCT [chaperonin-containing TCP-1 (T-complex protein 1)] chaperonin complex as a novel regulator of Orai1 recycling by primarily regulating Orai1 endocytosis. We show that Orai1 interacts with CCT through its intracellular loop and that inhibition of CCT-Orai1 interaction increases Orai1 PM residence. This increased residence is functionally significant as it results in prolonged Ca 2+ signaling, early formation of STIM1-Orai1 puncta, and more rapid activation of NFAT (nuclear factor of activated T cells) downstream of SOCE. Therefore, the CCT chaperonin is a novel regulator of Orai1 trafficking and, as such, a modulator of Ca 2+ signaling and effector activation kinetics.

Agonist-dependent Ca 2+ mobilization results in Ca 2+ store depletion and Store-Operated Calcium Entry (SOCE), which is spatially restricted to microdomains defined by cortical ER - plasma membrane contact sites (MCS). However, some Ca 2+ -dependent effectors that localize away from SOCE microdomains, are activated downstream of SOCE by mechanisms that remain obscure. One mechanism proposed initially in acinar cells and termed Ca 2+ tunneling, mediates the uptake of Ca 2+ flowing through SOCE into the ER followed by release at distal sites through IP 3 receptors. Here we show that Ca 2+ tunneling encodes exquisite specificity downstream of SOCE signal by dissecting the sensitivity and dependence of multiple effectors in HeLa cells. While mitochondria readily perceive Ca 2+ release when stores are full, SOCE shows little effect in raising mitochondrial Ca 2+ , and Ca 2+ -tunneling is completely inefficient. In contrast, gK Ca displays a similar sensitivity to Ca 2+ release and tunneling, while the activation of NFAT1 is selectively responsive to SOCE and not to Ca 2+ release. These results show that in contrast to the previously described long-range Ca 2+ tunneling, in non-specialized HeLa cells this mechanism mediates spatially restricted Ca 2+ rise within the cortical region of the cell to activate a specific subset of effectors.

The proper maturation of both male and female gametes is essential for supporting fertilization and the early embryonic divisions. In the ovary, immature fully-grown oocytes that are arrested in prophase I of meiosis I are not able to support fertilization. Acquiring fertilization competence requires resumption of meiosis which encompasses the remodeling of multiple signaling pathways and the reorganization of cellular organelles. Collectively, this differentiation endows the egg with the ability to activate at fertilization and to promote the egg-to-embryo transition. Oocyte maturation is associated with changes in the electrical properties of the plasma membrane and alterations in the function and distribution of ion channels. Therefore, variations on the pattern of expression, distribution, and function of ion channels and transporters during oocyte maturation are fundamental to reproductive success. Ion channels and transporters are important in regulating membrane potential, but also in the case of calcium (Ca 2+ ), they play a critical role in modulating intracellular signaling pathways. In the context of fertilization, Ca 2+ has been shown to be the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. These early events include the block of polyspermy, the completion of meiosis and the transition to the embryonic mitotic divisions. In this review, we discuss the role of ion channels during oocyte maturation, fertilization and early embryonic development. We will describe how ion channel studies in Xenopus oocytes, an extensively studied model of oocyte maturation, translate into a greater understanding of the role of ion channels in mammalian oocyte physiology.

Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRβ (PAQR8) partner that is required for mPRβ plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The G2-M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C-terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub-cellular location. We further demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo-like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure-function relationship and the role of transition metals in regulating meiosis., (© 2017 Wiley Periodicals, Inc.)

Background: Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes., Methods: We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis., Results: After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges., Conclusions: The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony., (Copyright © 2017. Published by Elsevier Inc.)

Ca 2+ signalling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium-binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca 2+ concentration. In electrically non-excitable cells, for example epithelial cells, this is achieved by primary release of Ca 2+ from the endoplasmic reticulum via Ca 2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca 2+ concentration activates Ca 2+ extrusion, and in order for cells not to run out of Ca 2+ , there is a need for compensatory Ca 2+ uptake from the extracellular fluid. This Ca 2+ uptake occurs through a process known as store-operated Ca 2+ entry. Ideally Ca 2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca 2+ tunnelling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca 2+ entering via store-operated Ca 2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Ca 2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca 2+ entry (SOCE) in regulating the temporal dynamics of Ca 2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca 2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca 2+ release leads to Ca 2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca 2+ signal. SOCE has also been implicated in fueling Ca 2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP 3 to gate IP 3 receptors (IP 3 R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca 2+ oscillations and produces a tonic Ca 2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca 2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca 2+ uptake into the ER, followed by release through open IP 3 Rs at distant sites, a process we refer to as "Ca 2+ teleporting." Therefore, sustained SOCE activation in the presence of an IP 3 -dependent "leak" pathway at the ER membrane results in a switch from oscillatory to tonic Ca 2+ signaling. J. Cell. Physiol. 232: 1095-1103, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca 2+ signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca 2+ signaling, including most prominently an inhibition of the passive ER Ca 2+ leak and the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca 2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca 2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca 2+ signaling machinery are different.