Ion channels in innate and adaptive immunity.

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

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

Calcium 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.

Ca2+ Fluxes and Cancer.

Ca2+ ions are key second messengers in both excitable and non-excitable cells. Owing to the rather pleiotropic nature of Ca2+ transporters and other Ca2+-binding proteins, however, Ca2+ signaling has attracted limited attention as a potential target of anticancer therapy. Here, we discuss cancer-associated alterations of Ca2+ fluxes at specific organelles as we identify novel candidates for the development of drugs that selectively target Ca2+ signaling in malignant cells.

STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons.

Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage-dependent calcium (CaV ) channels. A second cold-activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co-expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store-operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold-induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues.

Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Suppression of Ca2+ signaling enhances melanoma progression.

The role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV-dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV-induced cholesterol biosynthesis was critical for UV-induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV-dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis-dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis.

Store-Operated Ca2+ Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.

Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.

PtdSer as a signaling lipid determined by privileged localization of ORP5 and ORP8 at ER/PM junctional foci to determine PM and ER PtdSer/PI(4)P ratio and cell function.

The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca2+ signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca2+ signaling and Ca2+-dependent gene regulation are not understood. Here, we show that STIM1-formed junctions are required for PI(4)P/PtdSer exchange by ORP5 and ORP8, which have reciprocal lipid exchange modes and function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. Targeting the ORP5 and ORP8 and their lipid transfer ORD domains to PM subdomains revealed that ORP5 sets low and ORP8 high junctional PI(4)P/PtdSer ratio that controls STIM1-STIM1 and STIM1-Orai1 interaction and the activity of the SERCA pump to determine the pattern of receptor-evoked Ca2+ oscillations, and consequently translocation of NFAT to the nucleus. Significantly, targeting the ORP5 and ORP8 ORDs to the STIM1 ER subdomain reversed their function. Notably, changing PI(4)P/PtdSer ratio by hydrolysis of PM or ER PtdSer with targeted PtdSer-specific PLA1a1 reproduced the ORPs function. The function of the ORPs is determined both by their differential lipid exchange modes and by privileged localization at the ER/PM subdomains. These findings reveal a role of PtdSer as a signaling lipid that controls the available PM PI(4)P, the unappreciated role of ER PtdSer in cell function, and the diversity of the ER/PM junctions. The effect of PtdSer on the junctional PI(4)P level should have multiple implications in cellular signaling and functions.

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.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

Differential functional role of Orai1 variants in constitutive Ca2+ entry and calcification in luminal breast cancer cells.

Resting cytosolic Ca2+ concentration is tightly regulated to fine-tune Ca2+-dependent cellular functions. Luminal breast cancer cells exhibit constitutive Ca2+ entry mediated by Orai1 and the secretory pathway Ca2+-ATPase, SPCA2, which result in mammary microcalcifications that constitute a prognostic marker of mammary lesions. Two Orai1 isoforms have been identified, the full-length Orai1α, consisting of 301 amino acids, and the short variant, Orai1ß, lacking the 63 or 70 N-terminal amino acids comprising residues involved in channel inactivation and binding sites with Orai1 partners. We show that only the mammalian-specific Orai1α rescues SPCA2-dependent constitutive Ca2+ entry in Orai1-knockout MCF7 cells, a widely used luminal breast cancer cell line. FRET analysis and immunoprecipitation revealed that Orai1α shows a greater ability to interact with SPCA2 than Orai1ß. Deletion of the first 38 amino acids in Orai1α reduced the interaction with SPCA2 to a similar extent as Orai1ß, thus suggesting that the N-terminal 38 amino acids play a relevant role in Orai1α-SPCA2 interaction. Finally, Orai1α, but not Orai1ß, rescue the ability of Orai1-deficient cells to form in vitro microcalcifications. These findings provide compelling evidence for a functional role of Orai1α in constitutive Ca2+ entry in MCF7 cells, which might be a target to prevent the development of mammary microcalcifications in luminal breast cancer.

STIM1-dependent store-operated calcium entry mediates sex differences in macrophage chemotaxis and monocyte recruitment.

Infiltration of monocyte-derived cells to sites of infection and injury is greater in males than in females, due in part, to increased chemotaxis, the process of directed cell movement toward a chemical signal. The mechanisms governing sexual dimorphism in chemotaxis are not known. We hypothesized a role for the store-operated calcium entry (SOCE) pathway in regulating chemotaxis by modulating leading and trailing edge membrane dynamics. We measured the chemotactic response of bone marrow-derived macrophages migrating toward complement component 5a (C5a). Chemotactic ability was dependent on sex and inflammatory phenotype (M0, M1, and M2), and correlated with SOCE. Notably, females exhibited a significantly lower magnitude of SOCE than males. When we knocked out the SOCE gene, stromal interaction molecule 1 (STIM1), it eliminated SOCE and equalized chemotaxis across both sexes. Analysis of membrane dynamics at the leading and trailing edges showed that STIM1 influences chemotaxis by facilitating retraction of the trailing edge. Using BTP2 to pharmacologically inhibit SOCE mirrored the effects of STIM1 knockout, demonstrating a central role of STIM/Orai-mediated calcium signaling. Importantly, by monitoring the recruitment of adoptively transferred monocytes in an in vivo model of peritonitis, we show that increased infiltration of male monocytes during infection is dependent on STIM1. These data support a model in which STIM1-dependent SOCE is necessary and sufficient for mediating the sex difference in monocyte recruitment and macrophage chemotactic ability by regulating trailing edge dynamics.

Loss of Calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy R1/2A (LGMD R1/2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wild-type (WT) mice, we determined whether loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD R1/2A pathology.

Inhibition of adenylyl cyclase 8 prevents the upregulation of Orai1 channel, which improves cardiac function after myocardial infarction.

The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.

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.

Sodium Houttuyfonate Alleviates Monocrotaline-induced Pulmonary Hypertension by Regulating Orai1 and Orai2.

An increased intracellular Ca2+ concentration ([Ca2+]i) is a key trigger for pulmonary arterial smooth muscle cell (PASMC) proliferation and contributes greatly to pulmonary hypertension (PH). Extracellular Ca2+ influx via a store-operated Ca2+ channel, termed store-operated Ca2+ entry (SOCE), is a crucial mechanism for [Ca2+]i increase in PASMCs. Calcium release-activated calcium modulator (Orai) proteins, consisting of three members (Orai1-3), are the main components of the store-operated Ca2+ channel. Sodium houttuyfonate (SH) is a product of the addition reaction of sodium bisulfite and houttuynin and has antibacterial, antiinflammatory, and other properties. In this study, we assessed the contributions of Orai proteins to monocrotaline (MCT)-enhanced SOCE, [Ca2+]i, and cell proliferation in PASMCs and determined the effect of SH on MCT-PH and the underlying mechanism, focusing on Orai proteins, SOCE, and [Ca2+]i in PASMCs. Our results showed that: 1) Orai1 and Orai2 were selectively upregulated in the distal pulmonary arteries and the PASMCs of MCT-PH rats; 2) knockdown of Orai1 or Orai2 reduced SOCE, [Ca2+]i, and cell proliferation without affecting their expression in PASMCs in MCT-PH rats; 3) SH significantly normalized the characteristic parameters in a dose-dependent manner in the MCT-PH rat model; and 4) SH decreased MCT-enhanced SOCE, [Ca2+]i, and PASMC proliferation via Orai1 or Orai2. These results indicate that SH likely exerts its protective role in MCT-PH by inhibiting the Orai1,2-SOCE-[Ca2+]i signaling pathway.

ER-Ca2+ stores and the regulation of store-operated Ca2+ entry in neurons.

Key components of endoplasmic reticulum (ER) Ca2+ release and store-operated Ca2+ entry (SOCE) are likely expressed in all metazoan cells. Due to the complexity of canonical Ca2+ entry mechanisms in neurons, the functional significance of ER-Ca2+ release and SOCE has been difficult to identify and establish. In this review we present evidence of how these two related mechanisms of Ca2+ signalling impact multiple aspects of neuronal physiology and discuss their interaction with the better understood classes of ion channels that are gated by either voltage changes or extracellular ligands in neurons. Given how a small imbalance in Ca2+ homeostasis can have strongly detrimental effects on neurons, leading to cell death, it is essential that neuronal SOCE is carefully regulated. We go on to discuss some mechanisms of SOCE regulation that have been identified in Drosophila and mammalian neurons. These include specific splice variants of stromal interaction molecules, different classes of membrane-interacting proteins and an ER-Ca2+ channel. So far these appear distinct from the mechanisms of SOCE regulation identified in non-excitable cells. Finally, we touch upon the significance of these studies in the context of certain human neurodegenerative diseases.

The store-operated Ca2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCß2.

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1ß, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1ß lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1ß is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCß activation. Our results indicate that Orai1α interacts with PKCß2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.