Molecular Determinants of the Coupling between STIM1 and Orai Channels: DIFFERENTIAL ACTIVATION OF Orai1�C3 CHANNELS BY A STIM1 COILED-COIL MUTANT*

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca²⁺ release-activated Ca²⁺ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by ∼50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca²⁺ entry is a key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel (2–6). However, its molecular components have remained elusive until 4 years ago; the STIM1 (stromal interacting molecule 1) (7, 8) and later on Orai1 (9–11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca²⁺ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12, 13). Its N terminus is located in the ER lumen and contains an EF-hand Ca²⁺-binding motif, which senses the ER Ca²⁺ level, and a sterile α-motif, which is suggested to mediate homomeric STIM1 aggregation (14–16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14, 17, 18). Three recent studies have independently identified the ezrin-radixin-moesin domain as the essential Orai activating domain, named SOAR (STIM1 Orai-activating region) (20) which represents so far the shortest active fragment, OASF (Orai-activating small fragment) (21) or CAD (CRAC-activating domain) (22), which includes the second, more C terminally located coiled-coil domain and the following ∼55 amino acids. The latter amino acids are suggested to contain an additional cytosolic homomerization domain indispensable for OASF homomerization and Orai activation (21).The Orai family includes three highly Ca²⁺-selective ion channels (Orai1–3) that locate to the plasma membrane, and each protein contains four predicted transmembrane segments with cytosolic N and C termini (10). All three Orai proteins possess a conserved putative coiled-coil domain in the C terminus (23, 24), whereas only the N terminus of Orai1 consists of a proline/arginine-rich region (25). Orai1 has been assumed to act in concert with STIM1 (10, 27)-activating inward Ca²⁺ currents after store depletion. The two other members of the Orai family, Orai2 and Orai3, display similar but smaller store-operated inward Ca²⁺ currents when co-expressed with STIM1 with distinct inactivation profiles, permeability properties, and 2-aminoethoxydiphenyl borate sensitivity (28–32). Recently, we have provided evidence for a store depletion-induced, dynamic coupling of STIM1 to Orai1 that involves the putative coiled-coil domain in the C terminus of Orai1 (33). Furthermore, the C terminus of STIM1, in particular the essential cytosolic region 344–442 as narrowed down by SOAR, OASF, and CAD (20–22), has been established as the key fragment for CRAC as well as Orai1 activation, because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18, 32, 33). These fragments SOAR, OASF, and CAD when co-expressed with Orai1 (20–22) exhibit enhanced plasma membrane localization in comparison with the complete STIM1 C terminus in the presence of Orai1. Specificity of interaction of SOAR to the Orai1 C terminus has been shown by its disruption (20) employing the Orai1 L273S mutant (33). Park et al. (22) have provided additional, conclusive evidence for a direct binding by combining multiple biochemical approaches demonstrating CAD interaction with Orai1.This study focused specifically on the role of the putative coiled-coil domains of STIM1 as well as Orai proteins in their coupling. Coiled-coils generally function as protein-protein interaction sites with the ability of dynamic protein assembly and disassembly (35–37). We suggest the C-terminal, putative coiled-coil domains in all three Orai proteins and the second coiled-coil motif of STIM1 as essential for STIM1/Orai communication. Moreover, the single point coiled-coil STIM1 L373S mutant allowed for differential activation of Orai channels partially stimulating Orai2 as well as Orai3 but not Orai1.     

Neuropeptide signalling: a repository of targets for novel endectocides?

The only available parasiticides with a spectrum of action that includes a broad range of helminth and arthropod parasites are the macrocyclic lactones. Designated endectocides, these drugs have action against both endoparasitic nematodes and ectoparasitic arthropods. Unfortunately, the discovery of such drugs is exceedingly rare and there is no evidence that novel endectocidal agents will be identified and developed in the short to medium term. However, the discovery of neuropeptides with motor-modulatory activities in both arthropods and helminths, coupled with recent progress in the characterization of invertebrate neuropeptide receptors, has the potential to propel neuropeptide signalling to the forefront of efforts to develop a novel endectocide.     

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca(2+) release-activated Ca(2+) current (I(CRAC)) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.     

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

Transformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72 h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2 μM to 1.8 mM). Incubation in the absence of external calcium for 72 h had no significant effect on endoplasmic reticulum (ER) Ca^2 + contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G₂/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G₂/M reversible in less than 15 min. Our data showed that progression through G₂/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of I_CRAC and I_arc.     

Genetic dissection of tumor angiogenesis: are PlGF and VEGFR-1 novel anti-cancer targets?

Many proliferative diseases, most typically cancer, are driven by uncontrolled blood vessel growth. Genetic studies have been very helpful in unraveling the cellular and molecular players in pathological blood vessel formation and have provided opportunities to reduce tumor growth and metastasis. The fact that tumor vessels and normal blood vessels have distinct properties may help in designing more specific--and therefore safer--anti-angiogenic strategies. Such strategies may interfere with angiogenesis at the cellular or molecular level. Possible molecular targets include angiogenic growth factors and their receptors, proteinases, coagulation factors, junctional/adhesion molecules and extracellular matrix (ECM) components. Some anti-angiogenic drugs, i.e., vascular endothelial growth factor (VEGF) antibodies and VEGF receptor-2 (VEGFR-2) inhibitors, have progressed into clinical cancer trials. While the results of these trials support the potential of anti-angiogenic therapy to treat cancer, they also demonstrate the need for more effective and safer alternatives. Targeting placental growth factor (PlGF) or VEGFR-1 may constitute such an alternative since animal studies have proven their pleiotropic working mechanism and attractive safety profile. Together, these insights may bring anti-angiogenic drugs closer from bench to bedside.     

Purinergic receptors and neglected tropical diseases: why ignore purinergic signaling in the search for new molecular targets?

Purinergic receptors are widespread in the human organism and are involved in several physiological functions like neurotransmission, nociception, platelet aggregation, etc. In the immune system, they may regulate the expression and release of pro-inflammatory factors as well as the activation and death of several cell types. It is already described the participation of some purinergic receptors in the inflammation and pathological processes, such as a few neglected tropical diseases (NTDs) which affect more than 1 billion people in the world. Although the high social influence those diseases represent endemic countries, most of them do not have an efficient, safe or affordable drug treatment. In that way, this review aims to discuss the current literature involving purinergic receptor and immune response to NTDs pathogens, which may contribute in the search for new therapeutic possibilities.     

Adipocytokines - novel link between inflammation and vascular function?

Obesity and obesity related diseases are a major public health problem. Recent studies have shown that fat tissue is not a simple energy storage organ, but exerts important endocrine and immune functions. These are achieved predominantly through release of adipocytokines, which include several novel and highly active molecules released abundantly by adipocytes like leptin, resistin, adiponectin or visfatin, as well as some more classical cytokines released possibly by inflammatory cells infiltrating fat, like TNF-alpha, IL-6, MCP-1 (CCL-2), IL-1. All of those molecules may act on immune cells leading to local and generalized inflammation and may also affect vascular (endothelial) function by modulating vascular nitric oxide and superoxide release and mediating obesity related vascular disorders (including hypertension, diabetes, atherosclerosis, and insulin resistance) but also cancer or non-alcoholic fatty liver diseases. Present review, in a concise form, focuses on the effects of major adipocytokines, characteristic for adipose tissue like leptin, adiponectin, resistin and visfatin on the immune system, particularly innate and adaptive immunity as well as on blood vessels. Macrophages and T cells are populating adipose tissue which develops into almost an organized immune organ. Activated T cells further migrate to blood vessels, kidney, brain and other organs surrounded by infiltrated fat leading to their damage, thus providing a link between metabolic syndrome, inflammation and cardiovascular and other associated disorders. Ceretain treatments may lead to significant changes in adipocytokine levels. For example include beta-2 adrenoreceptor agonists, thiazolidinediones as well as androgens lead to decrease of plasma leptin levels. Moreover future treatments of metabolic system associated disorders should focus on the regulation of adipocytokines and their modes of action.     

A Highlights from MBoC Selection: Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions

Following endoplasmic reticulum (ER) Ca²⁺ depletion, STIM1 and Orai1 complexes assemble autonomously at ER–plasma membrane (PM) junctions to trigger store-operated Ca²⁺ influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER–PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools.     

Cereal phytochromes: targets of selection, targets for manipulation?

Plants respond to shading through an adaptive syndrome termed shade avoidance. In high-density crop plantings, shade avoidance generally increases extension growth at the expense of yield and can be at odds with the agronomic performance of the crop as a whole. Studies in Arabidopsis are beginning to reveal the essential role phytochromes play in regulating this process and to identify genes underlying the response. In this article, we focus on how phytochrome signaling networks have been targeted in cereal breeding programs in the past and discuss the potential to alter these pathways through breeding and transgenic manipulation to develop crops that perform better under typical high density conditions.     

Estrogen and mitochondria: a new paradigm for vascular protection?

Mitochondrial dysfunction has been implicated as a cause of age-related disorders, and the mitochondrial theory of aging links aging, exercise, and diet. Endothelial dysfunction is a key paradigm for vascular disease and aging, and there is considerable evidence that exercise and dietary restriction protect against cardiovascular disease. Recent studies demonstrate that estrogen receptors are present in mitochondria and that estrogen promotes mitochondrial efficiency and decreases oxidative stress in the cerebral vasculature. Chronic estrogen treatment increases mitochondrial capacity for oxidative phosphorylation while decreasing production of reactive oxygen species. The effectiveness of estrogen against age-related cardiovascular disorders, including stroke, may thus arise in part from hormonal effects on mitochondrial function. Estrogen-mediated mitochondrial efficiency may also be a contributing factor to the longer lifespan of women.     

Cell-derived microparticles in atherosclerosis: biomarkers and targets for pharmacological modulation?

Cardiovascular diseases remain an important cause of morbi-mortality. Atherosclerosis, which predisposes to cardiovascular disorders such as myocardial infarction and stroke, develops silently over several decades. Identification of circulating biomarkers to evaluate cardiovascular event risk and pathology prognosis is of particular importance. Microparticles (MPs) are small vesicles released from cells upon apoptosis or activation. Microparticles are present in blood of healthy individuals. Studies showing a modification of their concentrations in patients with cardiovascular risk factors and after cardiovascular events identify MPs as potential biomarkers of disease. Moreover, the pathophysiological properties of MPs may contribute to atherosclerosis development. In addition, pharmacological compounds, used in the treatment of cardiovascular disease, can reduce plasma MP concentrations. Nevertheless, numerous issues remain to be solved before MP measurement can be applied as routine biological tests to improve cardiovascular risk prediction. In particular, prospective studies to identify the predictive values of MPs in pathologies such as cardiovascular diseases are needed to demonstrate whether MPs are useful biomarkers for the early detection of the disease and its progression.     

Targeted therapeutics and nanodevices for vascular drug delivery: quo vadis?

This issue of the journal is dedicated to targeted delivery of therapeutics in the vasculature, an approach that holds promise to optimize treatment of diverse pathological conditions ranging from ischemia and tumor growth to metabolic and genetic diseases. From the standpoint of drug delivery, circulation system represents the natural route to the targets, whereas its components (blood and vascular cells) represent targets, carriers or barriers for drug delivery. Diverse nanodevices and targeted therapeutic agents that are designed and tested in animal and early clinical studies to achieve optimal and precise spatiotemporal control of the pharmacokinetics, destination, metabolism and effect of pharmacological agents will be discussed in this introductory essay and subsequent critical reviews in this series.     

An essential role of STIM1, Orai1, and S100A8-A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor.     

Influenza-virus-induced signaling cascades: targets for antiviral therapy?

Influenza viruses continue to pose a severe threat worldwide, causing thousands of deaths and an enormous economic loss every year. The major problem in fighting influenza is the high genetic variability of the virus, resulting in the rapid formation of variants that escape the acquired immunity against previous virus strains, or have resistance to antiviral agents. Every virus depends on its host cell and, hence, cellular functions that are essential for viral replication might be suitable targets for antiviral therapy. As a result, intracellular signaling cascades induced by the virus, in particular mitogen-activated protein kinase pathways, have recently come into focus.     

TRPV1: a therapeutic target for novel analgesic drugs?

The vanilloid receptor TRPV1 is now recognized as a molecular integrator of painful stimuli ranging from noxious heat to endovanilloids in inflammation. Pharmacological blockade of TRPV1 represents a new strategy in pain relief. TRPV1 antagonists are expected to prevent pain by silencing receptors where pain is generated rather than stopping the propagation of pain, as most-traditional pain killers do. This hypothesis has already being tested in the clinic by administering small molecule TRPV1 antagonists (e.g. GlaxoSmithKline SB-705498) for migraine and dental pain. Paradoxically, in some murine models of chronic pain, TRPV1-deficient mice exhibit more pain-related behavior than their wild-type littermates, indicating that the understanding of TRPV1 in pain is still incomplete. Moreover, there is mounting evidence to suggest the existence of functional TRPV1 both in the brain and in various non-neuronal tissues. The biological role of these receptors remains elusive, but their tissue distribution clearly indicates that they are involved in many more functions than just pain perception. Here, we review the potential therapeutic indications and adverse effects of TRPV1 antagonists.     

Therapy for CAG-repeat diseases?

Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretchIgarashi, S. et al. (1998)Nat. Genet. 18, 111–117