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). CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions (Tiffner et al. 2020). Soluble αKlotho downregulates Orai1-mediated store-operated Ca²⁺ entry via PI3K-dependent signaling (Kim et al. 2021). Noble et al. 2020 have reviewed how numerous cell stimulation pathways lead to the mobilization of sarco/endoplasmic reticulum (S/ER) stored Ca²⁺, resulting in the propagation of Ca²⁺ signals through the activation of processes, such as store-operated Ca²⁺ entry (SOCE) (Noble et al. 2020). Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation (Baraniak et al. 2021).

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). STIM1 gene expression decreases with the progression of neurodegeneration in Alzheimer's disease, and STIM1 is essential for cell viability in some differentiated cells (Pascual-Caro et al. 2018). STIM1 deficiency simulates Ca_v1.2 and thereby triggers voltage-regulated Ca²⁺ entry-dependent cell death. Mitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.

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). The calcium influx mechanism is triggered after the activation of Gq protein-coupled receptors at the plasma membrane (PM) that activate phospholipase C which produces Inositol triphosphate (IP3) which diffuses throughout the cytosol, resulting in the binding and activation of IP3 receptors (IP3R) and the rapid efflux of calcium from the endoplasmic reticulum (ER) to the cytosol. The calcium depletion in the ER is sensed by STIM1 at the ER that binds intraluminal calcium through an EF-hand domain in its amino terminal region. The cytosolic portion of STIM1 contains multiple domains, and the region that interacts with and activates Orai channels is SOAR (the STIM1 Orai activating region). For SOAR to be accessible to Orai1, STIM1 must assume an extended conformation that unlocks SOAR from its coiled-coil 1 (CC1) region. The extended conformation is triggered by calcium depletion in the ER that oligomerizes STIM1. The oligomers of STIM1 then translocate to a close distance between two opposing membranes, forming ER-PM junctions. STIM1 accumulates at ER-PM junctions conforming the denominated STIM1 puncta (Pacheco et al. 2023).

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). STIM proteins sense a depletion of intramembrane Ca²⁺ and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes (Grabmayr et al. 2020).

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. CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions (Tiffner et al. 2020). CRAC channels control the differentiation of pathogenic B cells in Lupus Nephritis (Li et al. 2021).

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). 

Baraniak et al. 2020 described the unexpected unimolecular coupling of STIM with Orai and explained the observed variable stoichiometry of STIM-Orai interactions. They also defined the discrete C-terminal regions in Orai channels that initially latch onto STIM proteins and mediate allosteric activation of the channel. A critical 'nexus' region closely associated with the STIM-activated C-terminus of Orai1, propagates the STIM-binding signal through the four tightly-associated TMSs of Orai1, finally to modify the pore-forming helices and effect channel opening (Baraniak et al. 2020). 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 (Maltan et al. 2022).

How Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation has been reviewed. Tiffner et al. 2021 highlighted the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their roles in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening.
The three Orai channel isoforms, Orai1-3 ensure their versatile roles in a variety of cellular functions and tissues. While all three isoforms are activated in a store-operated manner by STIM1, they differ in diverse biophysical and structural properties ((Tiffner et al. 2021). Residues in TMS3 together with cytosolic loop2 maintain the closed state and the configuration of an opening-permissive channel conformation of Orai1 and Orai3.

The family of stromal interaction molecules (STIM) includes two widely expressed single-pass ER transmembrane proteins and additional splice variants that act as precise ER-luminal Ca²⁺ sensors. STIM proteins mainly function as one of the two essential components of the so-called Ca²⁺ release-activated Ca²⁺ (CRAC) channel. The second CRAC channel component is constituted by pore-forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation (Sallinger et al. 2023). Their activation commonly requires the emptying of the intracellular ER Ca²⁺ store. Using their Ca²⁺ sensing capabilities, STIM proteins confer this Ca²⁺ content-dependent signal to Orai, thereby linking Ca²⁺ store depletion to CRAC channel opening. Sallinger et al. 2023 reviewed the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease.

The transport reaction believed to be catalyzed by CRAC channels is:

Ca²⁺ (and other cations) (out) Ca²⁺ (and other cations) (in)