Calcium Release-activated Calcium Channel Activation Research Articles

Agrimonia coreana Extract Exerts Its Therapeutic Effect through CRAC Channel Inhibition for Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (ACext) can be used in treating AD-related dermatologic symptoms. ACext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon ACext treatment. In a mouse model of AD, ACext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD.

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

SummaryCalcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2’s recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

Defects in the STIM1 SOAR\u03b12 domain affect multiple steps in the CRAC channel activation cascade

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Graphic abstractLegend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins.

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.

Abstract C045: RP4010, a small molecule inhibitor of Store-Operated Calcium Entry (SOCE), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML

Abstract Background: Store-operated calcium entry (SOCE) through calcium release activated calcium (CRAC) channels contribute to calcium influx in non-excitable cells. Elevation of cytosolic calcium through SOCE mediates an array of cellular responses including metabolism and gene expression, cell growth, and proliferation. Aberrant CRAC activity has been linked to various auto-immune disorders and certain cancers via the NFAT pathway. Calcium signaling via CRAC channels also mediates several physiological processes including cell proliferation and apoptosis. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. The objective of this study was to evaluate the single agent activity and the combination of RP4010 and standard of care drugs in AML models in vitro and in vivo. Methods: Cell growth following incubation with RP4010 (5 μM), Gilteritinib (0.25-0.75 μM), or a combination of both agents was determined in the AML lines: THP-1, HL-60, KG-1, and U-937. Apoptosis was evaluated following incubation of cell lines (THP-1 and U-937) with compounds for 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. For cell cycle, cells were incubated with compounds for 72 h, fixed with 70% ethanol, incubated at 4°C for 3 h, and stained with Propidium Iodide prior to analysis by flow cytometry. AKT and Stat-5 phosphorylation was determined by Western blotting. In vivo efficacy was evaluated in a MV-4-11 mouse xenograft model. Results: Store-operated calcium entry with RP4010 potentiated Gliteritinib activity manifested by a significant (P<0.05) reduction in cell growth when compared to the activity of the individual agents. Similarly, the combination (3 µM RP4010 + 1 or 2 µM Gilteritinib) led to a 13% increase in the number of apoptotic cells besides increasing cells in the G0/G1 phase by 17%. Combining RP4010 (1 µM) and Gilteritinib (0.1 µM) reduced AKT and Stat-5 phosphorylation by 45 and 57%, respectively, when compared to Gilteritinib in the THP-1 cell line. In the MV-4-11 mouse xenograft model, RP4010 when administered orally at 30 mg/kg/BID for 21 days demonstrated significant (P<0.001) anti-tumor activity both as a single agent and in combination with Cytarabine with tumor growth inhibitions of 42 and 68 %, respectively. Both RP4010, as well as the combination of RP4010 + Cytarabine, were well tolerated throughout the study period with no appreciable change in body weight. Conclusions: RP4010, a potent SOCE inhibitor, attenuated AML tumor growth as a single agent besides demonstrating remarkable potentiation in activity of approved/standard of care drugs for treating AML. Findings provide a rationale for use of this molecule in clinical trials involving naïve or relapsed/refractory AML patients. RP4010 is currently being evaluated in a Phase-1 dose-escalation study in patients with relapsed/refractory lymphomas. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. RP4010, a small molecule inhibitor of Store-Operated Calcium Entry (SOCE), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C045. doi:10.1158/1535-7163.TARG-19-C045

Ion Permeation through ORAI Proteins

Under depleted calcium levels in the endoplasmic reticulum, calcium release-activated calcium (CRAC) channels generate a sustained calcium influx crucial for several cellular responses such as gene expression, cell migration, and growth. Abnormal CRAC channel activity has been linked to various diseases, including severe combined immunodeficiency and autoimmunity disorders, allergy, and inflammatory bowel disease. CRAC channels are composed of two proteins: Orai proteins, that form the pore subunit, and Stim proteins, that activate the pore. Recent crystal structures of Orai proteins have shed light onto the nature of the permeation pathway of CRAC channels but details of the ion permeation mechanism are yet to be revealed. In this study, we use Umbrella Sampling and the Weighted Histogram Analysis Method to investigate the permeation of calcium, potassium and sodium ions through Orai pores. We extend the calculations to both Orai hexamer and tetramer assemblies, since previous studies have implicated different multimeric states of Orai as functional CRAC channels. In addition, we investigate the effect of rotation of the pore-forming transmembrane helices (suggested to be responsible for gating) on the free energy cost of ion permeation. Our simulations indicate that both hexamer and tetramer assemblies form conducting ion pores in which the free energy barrier is mainly set by the hydrophobic core of the pore. In all cases, helix rotation lowers the energetic cost of translocating an ion.

Abstract B127: Antitumor activity of RP4010, a novel small-molecule inhibitor of the calcium release-activated calcium (CRAC) channel pathway

Abstract Background: Store operated calcium channels, namely calcium release activated calcium (CRAC) channels, contribute to calcium influx in nonexcitable cells. Elevation of cytosolic calcium through activation CRAC channels mediates an array of cellular responses including metabolism and gene expression, cell growth, and proliferation. Aberrant CRAC activity has been linked to various autoimmune disorders and certain cancers via the NFAT pathway. Herein, we describe the preclinical profile of RP4010, a novel small-molecule inhibitor of the CRAC channel pathway. Methods: Modulation of CRAC channel activity upon addition of RP4010 was determined in a thapsigargin-induced calcium influx assay using Jurkat cells. Inhibition of downstream NFAT activity in Jurkat cells was measured using a reporter-gene luciferase assay. Effect of RP4010 on viability was determined using an OncoPanel multiplexed cytoxicity assay that measures multiple parallel responses of cancer cell lines to drug treatments through high-content fluorescence imaging. Change in migratory behavior upon addition of RP4010 was studied in a fetal bovine serum (FBS) induced scratch wound assay in A549 cells. Pharmacokinetic behavior of RP4010 in plasma after single dose oral administration or IV injection was determined in mouse and dog. Results: RP4010 demonstrated remarkable potency at inhibiting CRAC channel activity manifested by a reduction in thapsigargin-induced calcium influx into Jurkat cells (IC50 = 60 nM). Subsequent to CRAC channel inhibition, RP4010 dose-dependently attenuated the NFAT-regulated signalling in Jurkat cells with a half-maximal inhibition observed at 100 nM. Data from the OncoPanel testing indicated that RP4010 inhibited growth of cancer cells across diverse tumor sets ranging from breast, lung, lymphomas, skin, colon, prostate, stomach, and kidney with EC50 ranging from 1-10 μM. Interestingly, viability of Wi38, a normal lung cell line, was not affected by RP4010, indicating the specificity of the compound towards cancerous cells. Additionally, increasing concentrations of RP4010 caused a dose-dependent reduction in the migration of FBS induced A549 cells, demonstrating its antimetastatic potential. Pharmacokinetic studies indicated good oral bioavailability of RP4010 with a favorable half-life and peak plasma concentrations in mouse and dog. Conclusions: Results demonstrate the therapeutic potential of RP4010 across various cancer types via inhibition of the CRAC channel pathway and subsequent modulation of the downstream marker, NFAT. Achieving antitumor efficacy through inhibition of CRAC channels represents a novel approach primarily aimed at alleviating the deleterious effects associated with cytotoxic agents. Citation Format: Srikant Viswanadha, Seeta Nyayapathy, Sridhar Veeraraghavan, Swaroop Vakkalanka. Antitumor activity of RP4010, a novel small-molecule inhibitor of the calcium release-activated calcium (CRAC) channel pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B127.

Abstract B128: Efficacy of RP4010, a calcium release-activated calcium (CRAC) inhibitor, in preclinical models of DLBCL

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is an aggressive, rapidly growing type of lymphoma that comprises 30-40% of NHL cases. Chemotherapy remains the most widely used treatment for DLBCL, thereby necessitating the need to develop targeted and safer drugs. Calcium signaling via CRAC channels mediates several physiologic processes including cell proliferation and apoptosis. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. The objective of this study was to evaluate the effect of RP4010 as a single agent or in combination with known agents in preclinical models of DLBCL. Methods: Growth inhibition and apoptosis was estimated following incubation with RP4010 for 48-72 h using representative DLBCL cell lines. Besides single-agent activity, the effect of combining RP4010 with emerging and marketed therapies for DLBCL such as Ibrutinib and GA-101 was evaluated. Translation of in vitro activity to in vivo efficacy was assessed in mouse xenograft models representative of DLBCL. The effect of RP4010 (30 mg/kg/BID) as a single agent or in combination with Ibrutinib (30 mg/kg/QD) was tested in a DOHH2 human non-Hodgkin lymphoma cancer model using CB17/SCID mice. In a follow-up study, RP4010 at 30 mg/kg/BID was tested in combination with Rituximab (2.5 mg/kg BIW x 2W) or Rituximab + Bendamustine (10 mg/kg; QD x 2, 12 days off i.v.). Results: Channel blockade resulted in reduction in cell viability (EC50 = 0.3 -18 μM) with a corresponding dose-dependent induction of apoptosis. Adding RP4010 to Ibrutinib or GA-101 resulted in a synergistic effect on OCI-LY-1 and OCI-LY-10 cell proliferation. Single-agent RP4010 and the combination of RP4010 + Ibrutinib or RP4010 + Rituximab + Bendamustine were well tolerated throughout the study period with no appreciable change in body weight. IN DOHH-2 mouse xenografts, RP4010 demonstrated significant (p<0.001) antitumor activity both as a single agent and in combination with Ibrutinib with tumor growth inhibitions of 68 and 86%, respectively. Similar antitumor activity was observed upon combining RP4010 with Rituximab or Rituximab + Bendamustine with tumors remaining regressed for the duration of 2-week follow-up post withdrawal of treatment. Conclusions: RP4010, a potent CRAC channel inhibitor, attenuated DLBCL tumor growth as a single agent besides demonstrating remarkable synergy with known agents. Findings provide a rationale for use of this molecule in clinical trials involving naïve or relapsed/refractory DLBCL patients. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Swaroop Vakkalanka. Efficacy of RP4010, a calcium release-activated calcium (CRAC) inhibitor, in preclinical models of DLBCL [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B128.

Coordinate control of cytoskeletal remodeling and calcium mobilization during T‐cell activation

Ca(2+) mobilization and cytoskeletal reorganization are key hallmarks of T-cell activation, and their interdependence has long been recognized. Recent advances in the field have elucidated the molecular pathways that underlie these events and have revealed several points of intersection. Ca(2+) signaling can be divided into two phases: initial events leading to release of Ca(2+) from endoplasmic reticulum stores, and a second phase involving STIM 1 (stromal interaction molecule 1) clustering and CRAC (calcium release activated calcium) channel activation. Cytoskeletal dynamics promote both phases. During the first phase, the actin cytoskeleton promotes mechanotransduction and serves as a dynamic scaffold for microcluster assembly. Proteins that drive actin polymerization such as WASp (Wiskott-Aldrich syndrome protein) and HS1 (hematopoietic lineage cell-specific protein 1) promote signaling through PLCγ1 (phospholipase Cγ1) and release of Ca(2+) from endoplasmic reticulum stores. During the second phase, the WAVE (WASP-family verprolin homologous protein) complex and the microtubule cytoskeleton promote STIM 1 clustering at sites of plasma membrane apposition, opening Orai channels. In addition, gross cell shape changes and organelle movements buffer local Ca(2+) levels, leading to sustained Ca(2+) mobilization. Conversely, elevated intracellular Ca(2+) activates cytoskeletal remodeling. This can occur indirectly, via calpain activity, and directly, via Ca(2+) -dependent cytoskeletal regulatory proteins such as myosin II and L-plastin. While it is true that the cytoskeleton regulates Ca(2+) responses and vice versa, interdependence between Ca(2+) and the cytoskeleton also encompasses signaling events that occur in parallel, downstream of shared intermediates. Inositol cleavage by PLCγ1 simultaneously triggers both endoplasmic reticulum store release and diacylglycerol-dependent microtubule organizing center reorientation, while depleting the pool of phosphatidylinositol-4,5-bisphosphate, an activator of multiple actin-regulatory proteins. The close interdependence of Ca(2+) signaling and cytoskeletal dynamics in T cells provides positive feedback mechanisms for T-cell activation and allows for finely tuned responses to extracellular cues.

The Regulation of Calcium Homeostasis in T Lymphocytes

The Regulation of Calcium Homeostasis in T Lymphocytes

Hypoxia Triggers AMPK Activation through Reactive Oxygen Species-Mediated Activation of Calcium Release-Activated Calcium Channels

AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ.

B‐lymphocyte calcium inFlux

Dynamic changes in cytoplasmic calcium concentration dictate the immunological fate and functions of lymphocytes. During the past few years, important details have been revealed about the mechanism of store-operated calcium entry in lymphocytes, including the molecular identity of calcium release-activated calcium (CRAC) channels and the endoplasmic reticulum (ER) calcium sensor (STIM1) responsible for CRAC channel activation following calcium depletion of stores. However, details of the potential fine regulation of CRAC channel activation that may be imposed on lymphocytes following physiologic stimulation within an inflammatory environment have not been fully addressed. In this review, we discuss several underexplored aspects of store-operated (CRAC-mediated) and store-independent calcium signaling in B lymphocytes. First, we discuss results suggesting that coupling between stores and CRAC channels may be regulated, allowing for fine tuning of CRAC channel activation following depletion of ER stores. Second, we discuss mechanisms that sustain the duration of calcium entry via CRAC channels. Finally, we discuss distinct calcium permeant non-selective cation channels (NSCCs) that are activated by innate stimuli in B cells, the potential means by which these innate calcium signaling pathways and CRAC channels crossregulate one another, and the mechanistic basis and physiologic consequences of innate calcium signaling.

Essential Roles for Coiled-coil Domains in STIM1 Oligomerization and CRAC Channel Activation

The calcium release-activated calcium (CRAC) channel is activated by depletion of Ca2+ from the ER. Store depletion causes the ER Ca2+ sensor, STIM1, to translocate to sites of close ER-plasma membrane apposition, where it interacts with Orai1, the pore-forming subunit of the CRAC channel and activates Ca2+ entry. STIM1 self-associates in resting cells (Baba et al., PNAS 103:16704, 2006) and further oligomerizes after store depletion (Liou et al., PNAS 104:9301, 2007), an event that triggers the self-assembly and activation of STIM1-Orai1 clusters at ER-PM junctions (Luik et al., Nature 454:538, 2008). STIM1 has several protein interaction domains, including a lumenal sterile alpha motif (SAM) and two putative cytosolic coiled-coil regions. The isolated lumenal EF hand - SAM region is known to oligomerize upon Ca2+ removal in vitro (Stathopulos et al., JBC 281:35855, 2006), but the roles of the coiled-coil domains in the functions of STIM1 in situ are not as well understood. Using fluorescence recovery after photobleaching, co-immunoprecipitation, and blue native PAGE analysis on truncated mutants of STIM1 we show that the two coiled-coil domains of STIM1 affect STIM1 oligomerization in different ways. The ER-proximal coiled-coil is sufficient for the self-association of STIM1 in resting cells but does not by itself support oligomerization in response to store depletion. The distal coiled-coil is required for depletion-induced oligomerization. Mutation of specific residues within the predicted hydrophobic interface of the distal coiled-coil prevents the formation of STIM1 puncta and the activation of CRAC channels. These results reveal an essential role for the distal coiled-coil of STIM1 in the oligomerization step that controls store-operated Ca2+ entry.

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

Calcium Inhibition and Calcium Potentiation of Orai1, Orai2, and Orai3 Calcium Release-activated Calcium Channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells.

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation.