1.A.52 The Ca2+ Release-activated Ca2+ (CRAC) Channel (CRAC-C) Family
Antigen stimulation of immune cells triggers Ca2+ entry through tetrameric Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. Cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function (Zhou et al., 2010). The genetic defect in these patients appears to be in Orai1 (TM protein 142A; TMEM142a), which contains four putative transmembrane segments (Hogan and Rao, 2007). E106 residues in wild-type ORAI1 are positioned to form a Ca2+ binding site in the channel pore (Hogan and Rao, 2007). SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (ICRAC). Orai1 is an essential component of the CRAC channel complex (Feske et al., 2006). It is a teardrop-shaped molecule with a long, tapered cytoplasmic domain (Maruyama et al., 2009). The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers (Penna et al., 2008). Molecular determinants of fast Ca2+-dependent inactivation and gating of the Orai channels has been examined by Lee et al. (2009). A single lysine in the N-terminal region of store-operated CRAC channels 1 and 3 is critical for STIM1-mediated gating (Lis et al., 2010). The 4TMS CRAC channels arose by loss of 2TMSs from 6TMS CDF carriers, an example of 'reverse' evolution (Matias et al., 2010). Structure-function and subunit composition of Orai/STIM channels have been reviewed (PMID# 30299645).
The human CRAC channel protein Orai1 has homologues in all animals with sequenced genomes. It may be found exclusively in animals. Almost all homologues are about 250 residues long, but some are up to 100 residues longer (e.g., the Drosophila melanogaster Olf186-F (CG11430-PA isoforms A, B and C)) and the human CRAC (Feske et al., 2006) or CRACM1 protein (Vig et al., 2006). These proteins interact with the stromal interaction molecule 1 precursor (STIM1) to form the functional channel (Mercer et al., 2006; Peinelt et al., 2006; Soboloff et al., 2006; Vig et l., 2006). One study concluded that Orai1 forms a homotetramer (Mignen et al., 2008a). Coupling of STIM1 to store-operated Ca2+ entry depends on its movement in the endoplasmic reticulum (Baba et al., 2006). The intracellular loop of Orai1 plays a central role in fast inactivation of Ca2+ release-activated Ca2+ channels (Srikanth et al., 2010). Park et al. (2009) identified a highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N- and C-termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants revealed that channel clustering is not sufficient for channel activation (Park et al., 2009). These studies establish a molecular mechanism for store-operated Ca2+ entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions. STIM/Orai signaling complexes and their regulation have been described in vascular smooth muscle (Trebak, 2012).
CRAC channels (Orai1) exhibit high Ca2+ selectivity, low Cs+ permeability, and small unitary conductances. The architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TMS1-TMS2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca2+ selectivity is conferred mainly by E106 (McNally et al., 2009). Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91 (Zhang et al., 2011). Human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca2+-selective pore during store-operated activation, and Orai3 forms a dimeric nonselective cation pore upon activation by 2-aminoethyldiphenyl borate (2-APB) (Demuro et al., 2011). Ion selectivity requires conserved charged residues in TMSs 1 and 3 plus 3 residues in the first extracellular loop of mammalian Orai proteins (Hull et al. 2010).
Orai1 and TRPC1 are core components of store-operated CRAC and SOC channels, respectively. STIM1, a Ca2+-sensor protein in the ER, interacts with and mediates store-dependent regulation of both channels. TRPC1+STIM1-dependent store operated current (SOC) requires functional Orai1 (Cheng et al., 2008). 2-Aminoethyldiphenyl borate (2-APB) activates and then inhibits SOCE and the underlying calcium-release-activated Ca2+ current (ICRAC) (Dehaven et a., 2008). 2-APB effects SOCE due to effects on both STIM1 and Orai channel subunits. A phospholipase A2, iPLAβ of Homo sapiens (O60733) is an essential component of the signal transduction pathway from the stores to the plasma membrane channels (Bolotina 2008). STIM 1 is the mechanistic 'missing link' between the ER and the plasma membrane. STIM proteins sense the depletion of Ca2+ from the ER, oligomerize, translocate to junctions adjacent to the plasma membrane, organize Orai or TRPC (transient reeptor potential cation) channels into clusters and open these channels to bring about SOC entry (Cahalan, 2009).
STIM1 is a calcium sensor specilized for digital signaling (Bird et al., 2009). It functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai, ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. Interaction between wild-type Stim and Orai is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. A point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel (Hogan and Rao, 2007). Mutations of acidic residues in TMSs 1 and 3 and in the I-II loop decrease Ca2+ flux and increase Cs+ flux (Yamashita et al., 2007). The structural elements involved in ion permeation were proposed to overlap with those involved in the gating of CRAC channels.
STIM1 regulates the activity of the store-independent, arachidonic acid-regulated Ca2+ (ARC) channels, but does so in a manner distinct from its regulation of CRAC channels. While the levels of Orai1 alone determine the magnitude of the CRAC channel currents, both Orai1 and the closely related Orai3 are critical for the corresponding currents through ARC channels. Thus, in cells stably expressing STIM1, overexpression of Orai1 increases both CRAC and ARC channel currents. But overexpression of Orai3 in cells specifically increased ARC channel currents (Mignen et al., 2008b). Direct binding of the ER protein STIM to tetramers of the Orai1 calcium channel in the plasma membrane triggers opening of this channel (Clapham, 2009). Ali et al. 2016 summarized discoveries on the structure-function relationship of Orai1, as well as its interaction with the native channel opener STIM1 and chemical modulator 2-aminoethoxydiphenyl borate (2-APB).
CRACM1 proteins multimerize and bind STIM1. Both CRACM2 and CRACM3, when overexpressed with STIM1, potentiate CRAC currents. A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca2+ and Na+, differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca2+. Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca2+ signals (Lis et al., 2007). Mechanistic insights into the Orai channel by molecular dynamics simulations have been summarized (Bonhenry et al. 2019).
2-aminoethoxydiphenyl borate (2-APB) has emerged as a useful pharmacological tool in the study of store-operated calcium entry (SOCE). It has been shown to potentiate store-operated CRAC currents at low micromolar concentrations and inhibit them at higher concentrations. Experiments with the three CRAC channel subtypes CRACM1, CRACM2, and CRACM3 have indicated that they are differentially affected by 2-APB. It activates CRACM3 channels in a store-independent manner without the requirement of STIM1, wheras CRACM2 by itself is completely unresponsive to 2-APB, and CRACM1 is only weakly activated (Peinett et al., 2008; Zhang et al., 2008). 2-APB probably facilitates CRAC channels by altering pore architecture (Zhang et al., 2008). 2-aminoethoxydiphenyl borate has been reported to alter the selectivity of Orai3 channels by increasing their pore sizes (Schindl et al., 2008).
Two chromosomal loci have been identified for the murine orai2 gene, one is an intronless gene and a second locus gives rise to the splice variants ORAI2 long (ORAI2L) and ORAI2 short (ORAI2S). Prominent expression of the ORAI2 variants occurs in the brain, lung, spleen, and intestine, while ORAI1, ORAI3, and STIM1 appear to be nearly ubiquitously expressed in mouse tissues. Co-expression experiments with STIM1 and either ORAI1 or ORAI2 variants showed that ORAI2L and ORAI2S enhance CRAC currents (Gross et al., 2007). Native store-operated calcium (Ca2+) entry (SOCE) and I(CRAC) in estrogen receptor-positive (ER(+)) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER(-)) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER(+) breast cancer cells represent the first example where the native SOCE pathway and I(CRAC) are mediated by Orai3 (Motiani et al., 2010).
The primary mechanism of extracellular Ca2+ entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca2+ concentration in the ER and an activator of the Ca2+ selective channel ORAI1 in the plasma membrane. Yarkoni and Cambier (2011) reported that STIM1 expression differs in murine T and B lymphocytes; mature T cells express ∼4 times more STIM1 than mature B cells. Through the physiologic range of expression, STIM1 levels determine the magnitude of Ca2+ influx responses that follow BCR-induced intracellular store depletion.
Store operated calcium entry (SOCE) is used to regulate basal calcium, refill intracellular Ca2+ stores, and execute a wide range of specialized activities. STIM and Orai are as the essential components enabling the reconstitution of Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE. Palty et al. (2012) reported the molecular identification of SARAF as a negative regulator of SOCE. It is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca2+-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca2+ signals and determining the content of the major intracellular Ca2+ stores, a role that is likely to be important in protecting cells from Ca2+overfilling (Palty et al., 2012).
The crystal structure of Orai from Drosophila melanogaster has been determined at 3.35 angstrom resolution (Hou et al. 2012). The calcium channel is composed of a hexameric assembly of Orai subunits arranged around a central ion pore. The pore traverses the membrane and extends into the cytosol. A ring of glutamate residues on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs markedly from other ion channels and provides insight into the principles of selective calcium permeation and gating (Hou et al. 2012).
Local rearrangement of STIM1, rather than alteration in the oligomeric state of STIM1, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region (Ma et al. 2015). Critical autoinhibitory residues within the cytoplasmic domain of STIM1 were identified. Thus, the transmembrane region of STIM1 reorganization switches the cytoplasmic domain of STIM1 into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels (Ma et al. 2015).
Mitochondria exert control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation was examined by Ben-Kasus Nissim et al. 2017. They showed that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevented the cytosolic Na+ rise, inhibited NCLX activity, and impaired SOCE and Orai1 channel current. They showed further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1.
Orai1 requires Ca2+ store depletion in the ER and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. Frischauf et al. 2017 screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation, suggesting that certain Orai1 mutations were clustered in transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region (Frischauf et al. 2017).
The transport reaction believed to be catalyzed by CRAC channels is:
Ca2+ (and other cations) (out) Ca2+ (and other cations (in)
The CRAC channel protein, Orai1 (CRACM1) (Prakriya et al. 2006), complexed with the STIM1 or STIM2 protein (Feske et al., 2006). Replacement of the conserved glutamate in the first TMS with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na+, Ba2+, and Sr2+ permeation relative to Ca2+. Mutating E190Q in TMS3 also affects channel selectivity, suggesting that glutamate residues in both TMS1 and TMS3 face the lumen of the pore (Vig et al. 2006). The Orai1:Stim stoichiometry = 4:2 (Ji et al., 2008). Human Orai1 and Orai3 channels are dimeric in the closed resting state and open states. They are tetrameric when complexed with STIM1 (Demuro et al., 2011). A dimeric form catalyzes nonselective cation conductance in the STIM1-independent mode. STIM1 domains have been characterized (How et al. 2013). Alternative translation initiation of the Orai1 message produces long and short types of Ca2+ channels with distinct signaling and regulatory properties (Desai et al. 2015). STIM2 plays roles similar to STIM1 in regulating basal cytosolic and endoplasmic reticulum Ca2+ concentrations by controling Orai1, 2 and 3. STIM2 may inhibit STIM1-mediated Ca2+ influx. It also regulates protein kinase A-dependent phosphorylation and trafficking of AMPA receptors (TC# 1.A.10) (Garcia-Alvarez et al. 2015). A mechanistic model for ROS (H2O2)-mediated inhibition of Orai1 has been determined (Alansary et al. 2016). Regions that are important for the optimal assembly of hetero-oligomers composed of full-length STIM1 with its minimal STIM1-ORAI activating region, SOAR, have been identified (Ma et al. 2017). Orai1 may be multifunctional (Carrell et al. 2016). Activatioin of Orai1 requires communication between the N-terminus and loop 2 (Fahrner et al. 2017). STIM1 dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions (Zhou et al. 2018). SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. In addition to being an ER Ca2+ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca2+-regulated processes including secretion, contraction, metabolism, cell division, and apoptosis (Spassova et al. 2006). STIM1 also contributes to smooth muscle contractility (Feldman et al. 2017). STIM1-mediated Orai1 channel gating, involves bridges between TMS 1 and the surrounding TMSs 2/3 ring, and these are critical for conveying the gating signal to the pore (Yeung et al. 2018). A review article summarizes the current high resolution structural data on specific EF-hand, sterile alpha motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels (Novello et al. 2018). Orai1 and STIM1 are involved in tubular aggregate myopathy (Wu et al. 2018). Knowledge of the structure-function relationships of CRAC channels, with a focus on key structural elements that mediate the STIM1 conformational switch and the dynamic coupling between STIM1 and ORAI1 has been discussed (Nguyen et al. 2018). While STIM1 is the native channel opener, a chemical modulator is 2-aminoethoxydiphenyl borate (2-APB) (Ali et al. 2017). ORAI1 channel gating and selectivity iare differentially altered by natural mutations in the first and third transmembrane domains (Bulla et al. 2018). Stim1 responds to both ER Ca2+ depletion and heat, mediates temperature-induced Ca2+ influx in skin keratinocytes via coupling to Orai Ca2+ channels in the plasma membrane, and thereby brings about thermosensing (Liu et al. 2019). Possibly, the interplay between STIM1 alpha3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation (Butorac et al. 2019).
Orai1/STIM1 complex of Homo sapiens
The ARC (Arachidonate-regulated Ca2+-selective) channel, a complex of STIM1, Orai1 and Orai3 (Mignen et al., 2008). It is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits (Mignen et al., 2009). (But see Demuro et al., 2011; 1.A.52.1.1). Molecular determinants within the N-terminus control channel activation and gating (Bergsmann et al., 2011). Specifically activated by high concentrations (>50 microM) of 2-aminoethyl diphenylborinate (2-APB) (Amcheslavsky et al. 2014).
Orai3 of Homo sapiens (Q9BRQ5)
The CRAC channel Orai2 (DUF 1650) (264 aas) (Gross et al., 2007).
Orai2 of Mus musculus (Q8BH10)
Insect STIM1/Orai1 (Hull et al., 2010). Influences sex pheromone production in moths.
Stim1/Orai1A or B of Bombyx mori
Orai1, splice form A (B5BRC5)
Orai1, splice form B (B5BRC4)
Ca2+ release-activated Ca2+ (CRAC) channel subunit, Orai, which mediates Ca2+ influx following depletion of intracellular Ca2+ stores. In Greek mythology, the 'Orai' are the keepers of the gates of heaven. The crystal structure (3.35 Å), revealed a hexameric assembly of Orai subunits arranged around a central ion pore which traverses the membrane and extends into the cytosol. A ring of glutamate residues on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs from those of other solved ion channels (Hou et al. 2012). Residues in the third TMS of orai affect the conduction properties of the channel (Alavizargar et al. 2018); a conserved glutamate residue (E262) contributes to selectivity. Mutation of this residue affected the hydration pattern of the pore domain, and impaired selectivity of Ca2+ over Na+. The crevices of water molecules are located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function (Alavizargar et al. 2018).
Orai (Olf186-F) of Drosophila melanogaster
Orai homologue (494aas; 4 or 5 TMSs)
Orai homologue in Ostreococcus tauri (Q012G5)
Orai homologue (244aas; 4 TMSs)
Orai homologue in Phytophthora infestans T30-4 (D0NKP9)