ORAI Ca2+ Channels in Cancers and Therapeutic Interventions.

The ORAI proteins serve as crucial pore-forming subunits of calcium-release-activated calcium (CRAC) channels, pivotal in regulating downstream calcium-related signaling pathways. Dysregulated calcium homeostasis arising from mutations and post-translational modifications in ORAI can lead to immune disorders, myopathy, cardiovascular diseases, and even cancers. Small molecules targeting ORAI present an approach for calcium signaling modulation. Moreover, emerging techniques like optogenetics and optochemistry aim to offer more precise regulation of ORAI. This review focuses on the role of ORAI in cancers, providing a concise overview of their significance in the initiation and progression of cancers. Additionally, it highlights state-of-the-art techniques for ORAI channel modulation, including advanced optical tools, potent pharmacological inhibitors, and antibodies. These novel strategies offer promising avenues for the functional regulation of ORAI in research and may inspire innovative approaches to cancer therapy targeting ORAI.

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

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

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

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

Emergence of broad cytosolic Ca2+ oscillations in the absence of CRAC channels: A model for CRAC-mediated negative feedback on PLC and Ca2+ oscillations through PKC.

The role of Ca2+ release-activated Ca2+ (CRAC) channels mediated by ORAI isoforms in calcium signalling has been extensively investigated. It has been shown that the presence or absence of different isoforms has a significant effect on store-operated calcium entry (SOCE). Yoast et al. (2020) showed that, in addition to the reported narrow-spike oscillations (whereby cytosolic calcium decreases quickly after a sharp increase), ORAI1 knockout HEK293 cells were able to oscillate with broad-spike oscillations (whereby cytosolic calcium decreases in a prolonged manner after a sharp increase) when stimulated with a muscarinic agonist. This suggests that Ca2+ influx through ORAI-mediated CRAC channels negatively regulates the duration of Ca2+ oscillations. We hypothesise that, through the activation of protein kinase C (PKC), ORAI1 negatively regulates phospholipase C (PLC) activity to decrease inositol 1,4,5-trisphosphate (IP3) production and limit the duration of agonist-evoked Ca2+ oscillations. Based on this hypothesis, we construct a new mathematical model, which shows that the formation of broad-spike oscillations is highly dependent on the absence of ORAI1. Predictions of this model are consistent with the experimental results.

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.

Functional differences in agonist-induced plasma membrane expression of Orai1α and Orai1ß.

Orai1 is the pore-forming subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1ß, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca2+ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca2+ concentration, as demonstrated by cell loading with the fast intracellular Ca2+ chelator dimethyl BAPTA in the absence of extracellular Ca2+ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1ß to the plasma membrane when expressed individually; by contrast, when Orai1ß is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1ß in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca2+ store depletion.

Photocrosslinking-induced CRAC channel-like Orai1 activation independent of STIM1.

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Role of Orai-family channels in the activation and regulation of transcriptional activity.

Store operated Ca2+ entry (SOCE) is a cornerstone for the maintenance of intracellular Ca2+ homeostasis and the regulation of a variety of cellular functions. SOCE is mediated by STIM and Orai proteins following the activation of inositol 1,4,5-trisphosphate receptors. Then, a reduction of the endoplasmic reticulum intraluminal Ca2+ concentration is sensed by STIM proteins, which undergo a conformational change and activate plasma membrane Ca2+ channels comprised by Orai proteins. STIM1/Orai-mediated Ca2+ signals are finely regulated and modulate the activity of different transcription factors, including certain isoforms of the nuclear factor of activated T-cells, the cAMP-response element binding protein, the nuclear factor κ-light chain-enhancer of activated B cells, c-fos, and c-myc. These transcription factors associate SOCE with a plethora of signaling events and cellular functions. Here we provide an overview of the current knowledge about the role of Orai channels in the regulation of transcription factors through Ca2+ -dependent signaling pathways.

Science CommuniCa2+tion Developing Scientific Literacy on Calcium: The Involvement of CRAC Currents in Human Health and Disease.

All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells' membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca2+ and its role in human health and disease with focus on CRAC channels.

Sex-dependent effect of aging on calcium signaling and expression of TRPM2 and CRAC channels in human neutrophils.

The vulnerability of older adults to bacterial infections has been associated with age-related changes in neutrophils. We analyzed the consequences of aging on calcium (Ca2+) mobilization and TRPM2 and CRAC channels expression in human neutrophils. The percentages of granulocytes, mature neutrophils, and neutrophil precursors were equivalent between young and older adults. However, neutrophil chemotaxis towards IL-8, C5a, or fMLP was lower in older adults of both sexes. Interestingly, a stronger Ca2+ transient followed by an identical Ca2+ influx to IL-8 was observed in older adult females. In addition, the Ca2+ response to LPS was delayed and prolonged in neutrophils of older adult males. There was no significant difference in Ca2+ response to fMLP, C5a, or store-operated Ca2+ entry in the older adults. There were also no differences in the expression of CXCR2, CD88, FPLR1, and TLR4. Interestingly, TRPM2- and ORAI1-mRNA expression was lower in neutrophils of older adults, mainly in females. Both channels were detected intracellularly in the neutrophils. TRPM2 was in late endosomes in young adults and in lysosomes in older adult neutrophils. In summary, defective neutrophil chemotaxis in aging seemed not to stem from alterations in Ca2+ signals; nevertheless, the low TRPM2 and ORAI1 expression may affect other functions.

PLC-IP3-ORAI pathway participates in the activation of the MRGPRB2 receptor in mouse peritoneal mast cells.

A novel mast cell-specific G-protein-coupled receptor (GPCR), known as Mas-related G protein-coupled receptor-B2 (MRGPRB2), plays important roles in immune response. However, the opening of ion channels mediated by MRGPRB2 activation remains unclear. In this study, we found that [Ca2+]i elevation and voltage-dependent current generated by MRGPRB2 activation were correlated with extracellular calcium concentration. The increases in [Ca2+]i and voltage-dependent current caused by MRGPRB2 activation were blocked by U73122 (PLC blocker) or 2-APB (IP3 blocker) or synta66 (ORAI blocker). The voltage-dependent current induced by MRGPRB2 was inhibited by calcium-activated chlorine channel (CACCS) blockers, DIDS, or NPPB. Our results indicated the involvement of the PLC-IP3-ORAI signaling pathway and CACCS in MRGPRB2-mediated mast cell 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.

Photopharmacological modulation of native CRAC channels using azoboronate photoswitches.

SignificanceCalcium release-activated calcium (CRAC) channels play key roles in the regulation of cellular signaling, transcription, and migration. Here, we describe the design, chemical synthesis, and characterization of photoswitchable channel inhibitors that can be switched on and off depending on the wavelength of light used. We use the compounds to induce light-dependent modulation of channel activity and downstream gene expression in human immune cells. We further expand the usage of the compounds to control seeding of cancer cells in target tissue and regulation of response to noxious stimuli in vivo in mice.

The Role of Lipids in CRAC Channel Function.

The composition and dynamics of the lipid membrane define the physical properties of the bilayer and consequently affect the function of the incorporated membrane transporters, which also applies for the prominent Ca2+ release-activated Ca2+ ion channel (CRAC). This channel is activated by receptor-induced Ca2+ store depletion of the endoplasmic reticulum (ER) and consists of two transmembrane proteins, STIM1 and Orai1. STIM1 is anchored in the ER membrane and senses changes in the ER luminal Ca2+ concentration. Orai1 is the Ca2+-selective, pore-forming CRAC channel component located in the plasma membrane (PM). Ca2+ store-depletion of the ER triggers activation of STIM1 proteins, which subsequently leads to a conformational change and oligomerization of STIM1 and its coupling to as well as activation of Orai1 channels at the ER-PM contact sites. Although STIM1 and Orai1 are sufficient for CRAC channel activation, their efficient activation and deactivation is fine-tuned by a variety of lipids and lipid- and/or ER-PM junction-dependent accessory proteins. The underlying mechanisms for lipid-mediated CRAC channel modulation as well as the still open questions, are presented in this review.

Contribution of STIM-Activated TRPC-ORAI Channels in Pulmonary Hypertension Induced by Chronic Sustained and Intermittent Hypoxia.

Sustained and intermittent hypoxia produce vasoconstriction, arterial remodeling, and hypertension in the lung. Stromal interaction molecule (STIM)-activated transient receptor potential channels (TRPC) and calcium release-activated calcium channel protein (ORAI) channels (STOC) play key roles in the progression of pulmonary hypertension in pre-clinical models of animals subjected to sustained and intermittent hypoxia. The available evidence supports the theory that oxidative stress and hypoxic inducible factors upregulate and activate STIM-activated TRPC-ORAI Ca2+ channels, contributing to the pulmonary remodeling and hypertension induced by sustained hypoxia. However, less is known about the effects of oxidative stress and hypoxic inducible factors on the modulation of STIM-activated TRPC-ORAI channels following chronic intermittent hypoxia. In this review, we examined the emerging evidence supporting the theory that oxidative stress and hypoxic inducible factors induced by intermittent hypoxia upregulate and activate STIM-activated TRPC-ORAI Ca2+ channels. In addition, we used bioinformatics tools to search public databases for the genes involved in the upregulation of STIMactivated TRPC-ORAI Ca2+ channels and compare the differential gene expression and biological processes induced by intermittent and sustained hypoxia in lung cells.

Highlighting the Multifaceted Role of Orai1 N-Terminal- and Loop Regions for Proper CRAC Channel Functions.

Orai1, the Ca2+-selective pore in the plasma membrane, is one of the key components of the Ca2+release-activated Ca2+ (CRAC) channel complex. Activated by the Ca2+ sensor in the endoplasmic reticulum (ER) membrane, stromal interaction molecule 1 (STIM1), via direct interaction when ER luminal Ca2+ levels recede, Orai1 helps to maintain Ca2+ homeostasis within a cell. It has already been proven that the C-terminus of Orai1 is indispensable for channel activation. However, there is strong evidence that for CRAC channels to function properly and maintain all typical hallmarks, such as selectivity and reversal potential, additional parts of Orai1 are needed. In this review, we focus on these sites apart from the C-terminus; namely, the second loop and N-terminus of Orai1 and on their multifaceted role in the functioning of CRAC channels.

Ca2+ Signaling Augmented by ORAI1 Trafficking Regulates the Pathogenic State of Effector T Cells.

Activation of the Ca2+ release-activated Ca2+ (CRAC) channel is crucial for T cell functions. It was recently shown that naked cuticle homolog 2 (NKD2), a signaling adaptor molecule, orchestrates trafficking of ORAI1, a pore subunit of the CRAC channels, to the plasma membrane for sustained activation of the CRAC channels. However, the physiological role of sustained Ca2+ entry via ORAI1 trafficking remains poorly understood. Using NKD2 as a molecular handle, we show that ORAI1 trafficking is crucial for sustained Ca2+ entry and cytokine production, especially in inflammatory Th1 and Th17 cells. We find that murine T cells cultured under pathogenic Th17-polarizing conditions have higher Ca2+ levels that are NKD2-dependent than those under nonpathogenic conditions. In vivo, deletion of Nkd2 alleviated clinical symptoms of experimental autoimmune encephalomyelitis in mice by selectively decreasing effector T cell responses in the CNS. Furthermore, we observed a strong correlation between NKD2 expression and proinflammatory cytokine production in effector T cells. Taken together, our findings suggest that the pathogenic effector T cell response demands sustained Ca2+ entry supported by ORAI1 trafficking.

The roles of transmembrane family proteins in the regulation of store-operated Ca2+ entry.

Store-operated Ca2+ entry (SOCE) is a major pathway for calcium signaling, which regulates almost every biological process, involving cell proliferation, differentiation, movement and death. Stromal interaction molecule (STIM) and ORAI calcium release-activated calcium modulator (ORAI) are the two major proteins involved in SOCE. With the deepening of studies, more and more proteins are found to be able to regulate SOCE, among which the transmembrane (TMEM) family proteins are worth paying more attention. In addition, the ORAI proteins belong to the TMEM family themselves. As the name suggests, TMEM family is a type of proteins that spans biological membranes including plasma membrane and membrane of organelles. TMEM proteins are in a large family with more than 300 proteins that have been already identified, while the functional knowledge about the proteins is preliminary. In this review, we mainly summarized the TMEM proteins that are involved in SOCE, to better describe a picture of the interaction between STIM and ORAI proteins during SOCE and its downstream signaling pathways, as well as to provide an idea for the study of the TMEM family proteins.

CRACR2A associated immunodeficiency - the supporting factor takes center stage.

Loss of function mutations in one of the core components of the calcium release-activated calcium (CRAC) channel complex, STIM1 and ORAI1, lead to severe immunodeficiency. Whether changes in regulatory components of the CRAC channel may also contribute to a disease state remained unknown. In a recent study, Wu et al. report a case of late onset immunodeficiency in a patient with bi-allelic mutation in CRACR2A, a regulatory component of the CRAC channel.