CRAC Channel Activation Research Articles

A multiple-oscillator mechanism underlies antigen-induced Ca2+ oscillations in Jurkat T-cells

T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.

A single amino acid deletion in the ER Ca2+ sensor STIM1 reverses the in vitro and in vivo effects of the Stormorken syndrome-causing R304W mutation.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

S417 in the CC3 region of STIM1 is critical for STIM1-Orai1 binding and CRAC channel activation.

Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE is controlled by the interaction of the ER Ca2+ sensor STIM1 with the plasma membrane Ca2+ channel Orai1. S417 is located in the third coiled-coil (CC3) domain of the C-terminus of STIM1. We found that single-point mutation of this residue (S417G) abolished STIM1 C-terminus interactions with Orai1. Mutation of S417 also abolished CAD-Orai1 binding and Orai1 channel activation, eliminated STIM1 puncta formation, and co-localization with Orai1 and SOCE. 2-APB was found to restore the binding of the STIM1 C-terminus mutant (S417G) to Orai1 and dose-dependently activate Orai1 channel. Both CBD and NBD of Orai1 are required for 2-APB-induced coupling between the Orai1 and STIM1 C-terminus mutant (S417G) and CRAC channel activation. We also demonstrated that 2-APB led to delayed activation of Orai1-K85E channel, although Orai1-K85E obviously impairs 2-APB-induced STIM1 C-terminus mutant (S417G)-Orai1 coupling. Our results suggest S417 in the CC3 domain of STIM1 is essential for STIM1-Orai1 binding and CRAC channel activation.

Plasma Membrane Ca2+ ATPase Activity Enables Sustained Store-operated Ca2+ Entry in the Absence of a Bulk Cytosolic Ca2+ Rise.

In many cell types, the rise in cytosolic Ca2+ due to opening of Ca2+ release-activated Ca2+ (CRAC) channels drives a plethora of responses, including secretion, motility, energy production, and gene expression. The amplitude and time course of the cytosolic Ca2+ rise is shaped by the rates of Ca2+ entry into and removal from the cytosol. However, an extended bulk Ca2+ rise is toxic to cells. Here, we show that the plasma membrane Ca2+ ATPase (PMCA) pump plays a major role in preventing a prolonged cytosolic Ca2+ signal following CRAC channel activation. Ca2+ entry through CRAC channels leads to a sustained sub-plasmalemmal Ca2+ rise but bulk Ca2+ is kept low by the activity of PMCA4b. Despite the low cytosolic Ca2+, membrane permeability to Ca2+ is still elevated and Ca2+ continues to enter through CRAC channels. Ca2+-dependent NFAT activation, driven by Ca2+ nanodomains near the open channels, is maintained despite the return of bulk Ca2+ to near pre-stimulation levels. Our data reveal a central role for PMCA4b in determining the pattern of a functional Ca2+ signal and in sharpening local Ca2+ gradients near CRAC channels, whilst protecting cells from a toxic Ca2+ overload.

Mapping interactions between the CRAC activation domain and CC1 regulating the activity of the ER Ca2+ sensor STIM1

Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca2+ sensor and activator of Orai1 channels. In resting cells with replete Ca2+ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca2+ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1–CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1–CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca2+ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1–CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease–linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.

On the Connections between TRPM Channels and SOCE.

Plasma membrane protein channels provide a passageway for ions to access the intracellular milieu. Rapid entry of calcium ions into cells is controlled mostly by ion channels, while Ca2+-ATPases and Ca2+ exchangers ensure that cytosolic Ca2+ levels ([Ca2+]cyt) are maintained at low (~100 nM) concentrations. Some channels, such as the Ca2+-release-activated Ca2+ (CRAC) channels and voltage-dependent Ca2+ channels (CACNAs), are highly Ca2+-selective, while others, including the Transient Receptor Potential Melastatin (TRPM) family, have broader selectivity and are mostly permeable to monovalent and divalent cations. Activation of CRAC channels involves the coupling between ORAI1-3 channels with the endoplasmic reticulum (ER) located Ca2+ store sensor, Stromal Interaction Molecules 1-2 (STIM1/2), a pathway also termed store-operated Ca2+ entry (SOCE). The TRPM family is formed by 8 members (TRPM1-8) permeable to Mg2+, Ca2+, Zn2+ and Na+ cations, and is activated by multiple stimuli. Recent studies indicated that SOCE and TRPM structure-function are interlinked in some instances, although the molecular details of this interaction are only emerging. Here we review the role of TRPM and SOCE in Ca2+ handling and highlight the available evidence for this interaction.

Physiological Functions of CRAC Channels.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity.

Metabolic Screening of Cytotoxic T-cell Effector Function Reveals the Role of CRAC Channels in Regulating Lethal Hit Delivery.

Cytotoxic T lymphocytes (CTL) mediate cytotoxicity toward tumor cells by multistep cell-cell interactions. However, the tumor microenvironment can metabolically perturb local CTL effector function. CTL activity is typically studied in two-dimensional (2D) liquid coculture, which is limited in recapitulating the mechanisms and efficacy of the multistep CTL effector response. We here developed a microscopy-based, automated three-dimensional (3D) interface coculture model suitable for medium-throughput screening to delineate the steps and CTL effector mechanisms affected by microenvironmental perturbation. CTL effector function was compromised by deregulated redox homeostasis, deficient mitochondrial respiration, as well as dysfunctional Ca2+ release-activated Ca2+ (CRAC) channels. Perturbation of CRAC channel function dampened calcium influx into CTLs, delayed CTL degranulation, and lowered the frequency of sublethal hits (i.e., additive cytotoxicity) delivered to the target cell. Thus, CRAC channel activity controls both individual contact efficacy and CTL cooperativity required for serial killing of target cells. The multistep analysis of CTL effector responses in 3D coculture will facilitate the identification of immune-suppressive mechanisms and guide the rational design of targeted intervention strategies to restore CTL effector function.

The Novel Calcium Release-Activated Calcium (CRAC) Channel Inhibitor RP4010 Exerts Potent Antitumor Effects in NOD/SCID/IL2Rg-/- Mice with Diffuse Large B Cell Lymphoma (DLBCL) Cell Line Xenografts

INTRODUCTION: Calcium ions (Ca2+) act as second messengers in cell signaling triggering various cellular processes, including gene transcription, secretion, cell proliferation, migration, and apoptosis. Store-operated Ca2+ entry (SOCE) is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and Orai1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways. As emerging evidence points to the involvement of aberrant CRAC channel activity in human diseases including Diffuse Large B Cell Lymphoma (DLBCL), we hypothesize that the CRAC channel inhibitorRP4010 might represent a unique therapeutic opportunity for DLBCL patients. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro . This study aimed at investigating the activity of RP4010 in preclinical DLBCL models.METHODS: The DLBCL cell lineswith GCB phenotype (SUDHL-4, SUDHL-6, SUDHL-16, and SUDHL-10) and ABC phenotype (SUDHL-8, RCK-8, RIVA, and SUDHL-2) were used to investigate in vitro the effects of RP4010 on cell growth and cell death and gene expression. The antitumor efficacy and of RP4010 was also analyzed in NOD/SCID/IL2Rg-/-mice bearing DLBCL cell line xenografts.RESULTS: Incubating GCB-DLBCL and ABC-DLBCL cell lines for up to 72 hours with increasing doses of RP4010 (1.25 - 10 µM)resulted in a significant dose- and time-dependent decrease in cell proliferation. For all cell lines, the peak of the cytostatic effect was detected upon incubation with 10 µM of RP4010 for 72 hours, when an average 80% decrease in cell proliferation over vehicle-treated controls (P ≤ 0.0001) was detected. RP4010 treatment resulted in a significant time- and dose-dependent increase in cell death, as assessed by AnnexinV/PI staining, in GCB-DLBCL cell lines and in two out of four ABC-DLBCL cell lines (SUDHL-8 and SUDHL-2), whereas modest or no effects were detected in the RCK-8 and RIVA ABC-DLBCL cell lines. Exposure to 5 µM RP4010 for 72 hours significantly increased cell death over vehicle-treated controls in SUDHL-4 (90 ± 4% vs. 11 ± 2%), SUDHL-6 (39 ± 4% vs. 16 ± 3%), SUDHL-16 (70 ± 5% vs. 18 ± 4%), SUDHL-10 (49 ± 5% vs. 15 ± 1%), SUDHL-8 (78 ± 4 vs. 8 ± 1%) and SUDHL-2 (57 ± 5 vs. 26 ± 4%) cell lines (P ≤ 0.0001). Interestingly, RP4010 (1.25 - 10 µM) failed to induce any cytotoxic effects on the in vitro growth of hematopoietic colony-forming cells from healthy volunteers. While no activation of caspase-8, -9, -3, or PARP were observed in ABC-DLBCL cell lines treated with RP4010, a potent caspase cleavage was observed upon RP4010 treatment in GCB-DLBCL cell lines. Pretreating cells with the pan-caspase inhibitor Z-VAD-fmk abrogated apoptosis only in GCB-DLBCL cell lines, but not in the ABC-DLBCL cell lines, supporting that RP4010 induces both caspase-dependent and caspase-independent cell death. Gene expression analysis revealed a strong correlation between Orai1 expression and sensitivity to RP4010-induced cell death. Thus, Orai1 has a pivotal role in the establishment of an apoptosis-resistant phenotype in ABC-DLBCL cells. Finally, in vivo experiments were conducted to investigate the antitumor activity of RP4010. Treatment of NOD/SCID/IL2Rg-/-mice bearing DLBCL tumor nodules with increasing dose of RP4010 (30, 60, 90 mg/Kg body weight, PO, 5 days/3 weeks) resulted in a dose-dependent inhibition of tumor growth (TGI) of SUDHL-8 [TGI = 35%, 74%, and 83% at 30, 60, and 90 mg/kg, respectively], RIVA and SUDHL-6[TGI = 66%, 52% at 90 mg/kg, respectively], compared to vehicle-treated controls. No mice experienced any apparent treatment-related toxicity. Despite the marked antitumor activity observed in DLBCL xenografts, RP4010 treatment did not induce any tumor necrosis in healthy tissues (including lungs, livers, spleens, and kidneys), suggesting that RP4010-induced growth inhibition was tumor restricted.CONCLUSIONS: RP4010 is associated with a potent anti-lymphoma activity, suggesting its therapeutic potential in DLBCL. A Phase 1/1b study in Relapsed/Refractory Non-Hodgkin Lymphoma (NHL) patients is currently ongoing in the US (ClinicalTrials.gov Identifier: NCT03119467). DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; ADC Therapeutics: Research Funding; Rhizen Pharmaceuticals: Research Funding.

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

The short isoform of extended synaptotagmin-2 controls Ca2+ dynamics in T cells via interaction with STIM1

Ca2+ release-activated Ca2+ (CRAC) channels elevate cytoplasmic Ca2+ concentration, which is essential for T cell activation, differentiation and effector functions. T cell receptor stimulation induces depletion of the endoplasmic reticulum (ER) Ca2+ stores, which is sensed by stromal interaction molecule 1 (STIM1). STIM1 translocates to the ER-plasma membrane (PM) junctions to interact with ORAI1, the pore subunit of the CRAC channels. Here, we show that two members of the extended synaptotagmin (E-Syt) family, E-Syt1, and the short isoform of E-Syt2 (E-Syt2S), contribute to activation of CRAC channels in T cells. Knockdown or deletion of both ESYT1 and ESYT2 reduced store-operated Ca2+ entry (SOCE) and ORAI1-STIM1 clustering in Jurkat T cells. Further, depletion of E-Syts in primary T cells decreased Ca2+ entry and cytokine production. While the ER-PM junctions were reduced in both HeLa and Jurkat T cells deleted for ESYT1 and ESYT2, SOCE was impaired only in Jurkat T cells, suggesting that the membrane-tethering function of E-Syts is distinct from their role in SOCE. Mechanistically, E-Syt2S, the predominant isoform of E-Syt2 in T cells, recruited STIM1 to the junctions via a direct interaction. This study demonstrates a membrane-tethering-independent role of E-Syts in activation of CRAC channels in T cells.

Ca2+ signaling in T lymphocytes: the interplay of the endoplasmic reticulum, mitochondria, membrane potential, and CRAC channels on transcription factor activation

T cell receptor stimulation initiates a cascade of reactions that cause an increase in intracellular calcium (Ca2+) concentration mediated through inositol 1,4,5-trisphosphate (IP3). To understand the basic mechanisms by which the immune response in T cells is activated, it is useful to understand the signaling pathways that contain important targets for drugs in a quantitative fashion. A computational model helps us to understand how the selected elements in the pathways interact with each other, and which component plays the crucial role in systems. We have developed a mathematical model to explore the mechanism for controlling transcription factor activity, which regulates gene expression, by the modulation of calcium signaling triggered during T cell activation. The model simulates the activation and modulation of Ca2+ release-activated Ca2+ (CRAC) channels by mitochondrial dynamics and depletion of endoplasmic reticulum (ER) store, and also includes membrane potential in T-cells. The model simulates the experimental finding that increases in Ca2+ current enhances the activation of transcription factors and the Ca2+ influx through CRAC is also essential for the NFAT and NFκB activation. The model also suggests that plasma membrane Ca2+-ATPase (PMCA) controls a majority of the extrusion of Ca2+ and modulates the activation of CRAC channels. Furthermore, the model simulations explain how the complex interaction of the endoplasmic reticulum, membrane potential, mitochondria, and ion channels such as CRAC channels control T cell activation.

TRPM7 activation potentiates SOCE in enamel cells but requires ORAI

Calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediated by STIM1/2 and ORAI (ORAI1-3) proteins form the dominant store-operated Ca2+ entry (SOCE) pathway in a wide variety of cells. Among these, the enamel-forming cells known as ameloblasts rely on CRAC channel function to enable Ca2+ influx, which is important for enamel mineralization. This key role of the CRAC channel is supported by human mutations and animal models lacking STIM1 and ORAI1, which results in enamel defects and hypomineralization. A number of recent reports have highlighted the role of the chanzyme TRPM7 (transient receptor potential melastanin 7), a transmembrane protein containing an ion channel permeable to divalent cations (Mg2+, Ca2+), as a modulator of SOCE. This raises the question as to whether TRPM7 should be considered an alternative route for Ca2+ influx, or if TRPM7 modifies CRAC channel activity in enamel cells. To address these questions, we monitored Ca2+ influx mediated by SOCE using the pharmacological TRPM7 activator naltriben and the inhibitor NS8593 in rat primary enamel cells and in the murine ameloblast cell line LS8 cells stimulated with thapsigargin. We also measured Ca2+ dynamics in ORAI1/2-deficient (shOrai1/2) LS8 cells and in cells with siRNA knock-down of Trpm7. We found that primary enamel cells stimulated with the TRPM7 activator potentiated Ca2+ influx via SOCE compared to control cells. However, blockade of TRPM7 with NS8593 did not decrease the SOCE peak. Furthermore, activation of TRPM7 in shOrai1/2 LS8 cells lacking SOCE failed to elicit Ca2+ influx, and Trpm7 knock-down had no effect on SOCE. Taken together, our data suggest that TRPM7 is a positive modulator of SOCE potentiating Ca2+ influx in enamel cells, but its function is fully dependent on the prior activation of the ORAI channels.

Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells.

Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.

Structural and Mechanistic Insights of CRAC Channel as a Drug Target in Autoimmune Disorder.

Calcium (Ca2+) ion is a major intracellular signaling messenger, controlling a diverse array of cellular functions like gene expression, secretion, cell growth, proliferation, and apoptosis. The major mechanism controlling this Ca2+ homeostasis is store-operated Ca2+ release-activated Ca2+ (CRAC) channels. CRAC channels are integral membrane protein majorly constituted via two proteins, the stromal interaction molecule (STIM) and ORAI. Following Ca2+ depletion in the Endoplasmic reticulum (ER) store, STIM1 interacts with ORAI1 and leads to the opening of the CRAC channel gate and consequently allows the influx of Ca2+ ions. A plethora of studies report that aberrant CRAC channel activity due to Loss- or gain-of-function mutations in ORAI1 and STIM1 disturbs this Ca2+ homeostasis and causes several autoimmune disorders. Hence, it clearly indicates that the therapeutic target of CRAC channels provides the space for a new approach to treat autoimmune disorders. This review aims to provide the key structural and mechanical insights of STIM1, ORAI1 and other molecular modulators involved in CRAC channel regulation. Understanding the structure and function of the protein is the foremost step towards improving the effective target specificity by limiting their potential side effects. Herein, the review mainly focusses on the structural underpinnings of the CRAC channel gating mechanism along with its biophysical properties that would provide the solid foundation to aid the development of novel targeted drugs for an autoimmune disorder. Finally, the immune deficiencies caused due to mutations in CRAC channel and currently used pharmacological blockers with their limitation are briefly summarized.

Review: Structure and Activation Mechanisms of CRAC Channels.

Ca2+ release activated Ca2+ (CRAC) channels represent a primary pathway for Ca2+ to enter non-excitable cells. The two key players in this process are the stromal interaction molecule (STIM), a Ca2+ sensor embedded in the membrane of the endoplasmic reticulum, and Orai, a highly Ca2+ selective ion channel located in the plasma membrane. Upon depletion of the internal Ca2+ stores, STIM is activated, oligomerizes, couples to and activates Orai. This review provides an overview of novel findings about the CRAC channel activation mechanisms, structure and gating. In addition, it highlights, among diverse STIM and Orai mutants, also the disease-related mutants and their implications.

A novel STIM1-Orai1 gating interface essential for CRAC channel activation

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400–403), which is located within the CAD/SOAR domain. We determined this segment’s specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

The whole-cell Ca2+ release-activated Ca2+ current, ICRAC , is regulated by the mitochondrial Ca2+ uniporter channel and is independent of extracellular and cytosolic Na.

Key points Ca2+ entry through Ca2+ release‐activated Ca2+ channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca2+ buffering, mitochondrial Ca2+ uptake regulates CRAC channel activity.Knockdown of the mitochondrial Ca2+ uniporter channel prevented the development of I CRAC in weak buffer but not when strong buffer was used instead.Removal of either extracellular or intra‐pipette Na+ had no effect on the selectivity, kinetics, amplitude, rectification or reversal potential of whole‐cell CRAC current.Knockdown of the mitochondrial Na+–Ca2+ exchanger did not prevent the development of I CRAC in strong or weak Ca2+ buffer.Whole cell CRAC current is Ca2+‐selective.Mitochondrial Ca2+ channels, and not Na+‐dependent transport, regulate CRAC channels under physiological conditions. Ca2+ entry through store‐operated Ca2+ release‐activated Ca2+ (CRAC) channels plays a central role in activation of a range of cellular responses over broad spatial and temporal bandwidths. Mitochondria, through their ability to take up cytosolic Ca2+, are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca2+ buffering. The mitochondrial Ca2+ transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca2+ uniporter (MCU) channel or the Na+–Ca2+–Li+ exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca2+ transporters in supporting the CRAC current (I CRAC) under a range of conditions in RBL mast cells. Knockdown of the MCU channel impaired the activation of I CRAC under physiological conditions of weak intracellular Ca2+ buffering. In strong Ca2+ buffer, knockdown of the MCU channel did not inhibit I CRAC development demonstrating that mitochondria regulate CRAC channels under physiological conditions by buffering of cytosolic Ca2+ via the MCU channel. Surprisingly, manipulations that altered extracellular Na+, cytosolic Na+ or both failed to inhibit the development of I CRAC in either strong or weak intracellular Ca2+ buffer. Knockdown of NCLX also did not affect I CRAC. Prolonged removal of external Na+ also had no significant effect on store‐operated Ca2+ entry, on cytosolic Ca2+ oscillations generated by receptor stimulation or on CRAC channel‐driven gene expression. In the RBL mast cell, Ca2+ flux through the MCU but not NCLX is indispensable for activation of I CRAC.

Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits.

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

Phosphorylation-mediated structural changes within the SOAR domain of stromal interaction molecule 1 enable specific activation of distinct Orai channels

Low-conductance, highly calcium-selective channels formed by the Orai proteins exist as store-operated CRAC channels and store-independent, arachidonic acid-activated ARC channels. Both are activated by stromal interaction molecule 1 (STIM1), but CRAC channels are activated by STIM1 located in the endoplasmic reticulum membrane, whereas ARC channels are activated by the minor plasma membrane-associated pool of STIM1. Critically, maximally activated CRAC channel and ARC channel currents are completely additive within the same cell, and their selective activation results in their ability to each induce distinct cellular responses. We have previously shown that specific ARC channel activation requires a PKA-mediated phosphorylation of a single threonine residue (Thr389) within the cytoplasmic region of STIM1. Here, examination of the molecular basis of this phosphorylation-dependent activation revealed that phosphorylation of the Thr389 residue induces a significant structural change in the STIM1-Orai-activating region (SOAR) that interacts with the Orai proteins, and it is this change that determines the selective activation of the store-independent ARC channels versus the store-operated CRAC channels. In conclusion, our findings reveal the structural changes underlying the selective activation of STIM1-induced CRAC or ARC channels that determine the specific stimulation of these two functionally distinct Ca2+ entry pathways.