The Orai channel is characterized by voltage independence, low conductance, and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane (PM). How the channel is activated and promotes Ca2+ permeation is not well understood. This paper report the crystal structure and cryo-electron microscopy (cryo-EM) reconstruction of a Drosophila melanogaster Orai (dOrai) mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which 6 transmembrane 1 (TM1) helices in the center form the ion-conducting pore, and 6 TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation, and Ca2+ permeation (Liu, 2019).

Structural comparison between the open state and the closed state revealed a conformational transduction pathway from the peripheral TM4 helix to the innermost TM1 helix. Mutations that interfered with the pathway dramatically attenuated the STIM1-activated Orai function, as demonstrated by electrophysiology. Twisting of the basic section of the TM1 helix to face the cytosolic side may accommodate more anions to facilitate Ca2+ permeation. In the closed state of the channel, the latched TM4 helix closes the pore on the cytosolic side. Positive charge repulsion and anion plugs block Ca2+ permeation. Upon opening, the TM4 helix swing twists the basic section outward to accommodate more anions. These anions not only neutralize the positive charges to reduce charge repulsion but also increase the potential gradient across the membrane, thus facilitating Ca2+ permeation (Liu, 2019).

This model is consistent with many published functional studies. A 'nexus' site (amino acids 261-265) has been identified within the hOrai1 channel that is proposed to connect the peripheral C-terminal STIM1-binding site to the hOrai1 pore helices. Structural comparison of the dOrai channel structures between the open state and the published closed state clearly indicated a conformational transduction pathway (T4b helix -> T3 helix -> T1 helix basic section), providing further evidence that the 'nexus' site is most likely the trigger for channel activation. Furthermore, cholesterol has been reported to interact with the hOrai1 channel and inhibit its activity through residues hOrai1-L74 and hOrai1-Y80. These 2 residues are located within the interface between the TM1 helix and the TM3 helix. Cholesterol binding presumably interrupts the conformational transduction pathway, which explains why cholesterol did not affect the binding of STIM1 to the hOrai1 channel but attenuated hOrai1 activation (Liu, 2019).

How Orai channels conduct Ca2+ is a puzzling question. The Orai pore consists of an extracellular mouth, a selectivity filter, an unusually long hydrophobic cavity, and an intracellular basic region. The dOrai closed-state structure shows that the narrowest region of the pore has a diameter of 6.0 Å, wide enough for a dehydrated Ca2+ to pass through. Another study proposed rotating the pore helix upon channel activation. However, a third study reported that no rotation of the pore helix was observed in molecular dynamic simulation studies. In the current open-state structure of the dOrai channel, rotation of the pore helix was not observed. Recently, another crystal structure of the open state (H206A) of the dOrai channel at low resolution (6.7 Å ) was reported. That study did not observe pore helix rotation either. Therefore, the mechanism of pore helix rotation is inconsistent with the results of structural studies (Liu, 2019).

Another pore-dilation model has been proposed based on the structural findings of a dilated hydrophobic cavity and a wide open intracellular basic region. Another study reported that mutations in the TM2 helix may slightly increase the pore size in hydrophobic regions. However, the current structure showed the opening of the intracellular basic region but not the dilated hydrophobic cavity. Moreover, the pore-dilation model is inconsistent with the result that mutating the intracellular basic region of constitutively active hOrai1 abolished the channel activity. Therefore, the pore-dilation mechanism is less likely to account for Ca2+ permeation of the Orai channel (Liu, 2019).

The current proposed anion-assisted Ca2+ permeation model is reasonable for explaining these results. Furthermore, it has been reported that Orai currents are inhibited by acidic but potentiated by basic intracellular solutions in various cell types. This result is consistent with the current model because basic intracellular solutions provide more hydroxide anions, whereas acidic solutions provide more proton cations. Both the pore helix rotation and pore-dilation models are difficult to explain. In summary, these studies provide a reasonable model that clarifies the molecular details of the activation and Ca2+ permeation of the Orai channel (Liu, 2019).

Store-operated calcium release-activated calcium (CRAC) channels mediate a variety of cellular signaling functions. The CRAC channel pore-forming protein, Orai1, is a hexamer arranged with 3-fold symmetry. Despite its importance in moving Ca(2+) ions into cells, a detailed mechanistic understanding of Orai1 activation is lacking. In this paper a working model is proposed for the putative open state of Orai from Drosophila melanogaster (dOrai), which involves a 'twist-to-open' gating mechanism. The proposed model is supported by energetic, structural, and experimental evidence. Fluorescent imaging demonstrates that each subunit on the intracellular side of the pore is inherently strongly cross-linked, which is important for coupling to STIM1, the pore activator, and graded activation of the Orai1 channel. The proposed model thus paves the way for understanding key aspects of calcium signaling at a molecular level (Dong, 2019).

Ca²⁺-release-activated Ca²⁺ (CRAC) channels underlie sustained Ca²⁺ signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca²⁺ store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca²⁺ channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. This study shows, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca²⁺ store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca²⁺-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule (Penna, 2008).

Stimulation of immune cells triggers Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, olf186-F (Drosophila Orai, dOrai) and dStim have been identified as critical components of store-operated Ca²⁺ entry; dOrai and its human homologue Orai1 are pore subunits of the Ca²⁺ release-activated Ca²⁺ channel. This study reports that Orai1 is predominantly responsible for store-operated Ca²⁺ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca²⁺ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells (Gwack, 2007).

Ca²⁺ is a key second messenger in intracellular signaling pathways. In lymphocytes, specialized store-operated Ca²⁺ channels known as CRAC channels are required for sustained Ca²⁺ influx across the plasma membrane. The resulting prolonged elevation of intracellular free Ca²⁺ entry is essential for sustained nuclear translocation of the transcription factor NFAT, a small family of proteins whose activation is critical for a productive immune response. NFAT proteins reside in the cytoplasm of resting lymphocytes in a highly phosphorylated form and translocate to the nucleus upon dephosphorylation by the Ca²⁺/calmodulin-dependent serine/threonine phosphatase calcineurin. In the nucleus, NFAT proteins bind to promoters and regulatory regions of a large number of cytokine genes and other activation-associated genes, thereby mediating the activation, proliferation, and differentiation of T cells, B cells, and other immune system cells (Gwack, 2007).

Although the notion of Ca²⁺ influx through 'store-operated' Ca²⁺ channels was first proposed in 1986, the molecular identity of the proteins involved in this process remained unknown until the advent of large-scale RNAi-based screens. The first components of the pathway to be identified were Drosophila Stim (dStim) and its human homologues STIM1 and STIM2 through large-scale (albeit not genome-wide) RNAi-based screens in Drosophila and HeLa cells, respectively. STIM proteins are single-pass transmembrane proteins localized predominantly in the membrane of the endoplasmic reticulum (ER); they contain an N-terminal EF-hand located in the ER lumen and appear to function as sensors of ER Ca²⁺ levels. Upon store depletion, STIM1 relocalizes into puncta that were suggested to represent foci of insertion into the plasma membrane but are more likely points of apposition of the ER and plasma membranes. It is thought that within these puncta, STIM1 communicates with and opens CRAC channels located in the plasma membrane (Gwack, 2007 and references therein).

More recently, genome-wide RNAi screens performed in Drosophila cells have identified a CRAC channel component, olf186-F. This protein was renamed Drosophila Orai (dOrai). Its three human homologues, Orai1, Orai2, and Orai3 (also known as CRACM1, CRACM2 and CRACM3), are encoded by the genes TMEM142A, TMEM142B, and TMEM142C. Orai1 bears the causal mutation in a severe combined immunodeficiency (SCID) syndrome characterized by a defect in CRAC channel function and T cell cytokine expression. Combined overexpression of dOrai and dSTIM in Drosophila cells or Orai1 and STIM1 in Jurkat T cells, RBL cells, or HEK293 cells results in a dramatic increase in I_CRAC. Amino acid substitutions in either of two conserved glutamate residues, located in predicted transmembrane segments of dOrai and Orai1, changed the properties of I_CRAC, suggesting strongly that these proteins are pore subunits of the CRAC channel (Gwack, 2007).

This study compares the properties of the three mammalian Orai proteins. All three are widely expressed at the mRNA level and all can be incorporated into the plasma membrane when ectopically expressed. Orai1 forms homodimers and homomultimers in cells and in detergent solutions, can heteromultimerize with Orai2 and Orai3 as judged by co-immunoprecipitation, and has a predominant role in store-operated Ca²⁺ entry in HEK293 cells and human T cells and fibroblasts when stores are depleted with thapsigargin. Immunocytochemical analysis shows that ectopically expressed Orai1 and STIM1 colocalize partially in thapsigargin-stimulated T cells. Dominant-interfering forms of dOrai and human Orai1 have been generated by substituting glutamine residues in place of either of two highly conserved glutamates located in the first and third predicted transmembrane segments. Ectopic expression of the E106Q and E190Q mutants of Orai1 in primary murine T cells severely impairs store-operated Ca²⁺ influx, proliferation, and cytokine production, consistent with the conclusion that Orai1 is a major contributor to T lymphocyte function and the adaptive immune response (Gwack, 2007).

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca²⁺ influx and Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably 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 (also known as olf186-F), 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. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. This study shows that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca²⁺ store depletion. By site-directed mutagenesis, it was shown that 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 (Yeromin, 2006).

The results demonstrate that thapsigargin-triggered store depletion dynamically strengthens an interaction between Stim and Orai, supporting a model for CRAC channel activation in which Stim serves as the Ca²⁺ sensor to detect store depletion and as the messenger to activate CRAC channels in the plasma membrane. More importantly, it is concluded that Orai is a bona fide ion channel, based on the following: (1) RNA-interference-mediated knockdown of Orai expression suppresses thapsigargin-dependent Ca²⁺ influx and CRAC channel activity; (2) overexpression of Orai with or without Stim augments CRAC currents that exhibit biophysical properties identical to native CRAC current; and (3) mutations of negatively charged residues within the putative pore region of Orai significantly alter ion selectivity, current rectification and affinity to a charged channel blocker without altering channel activation kinetics. The marked alteration of these properties by a targeted point mutation provides definitive evidence that Orai embodies the pore-forming subunit of the CRAC channel (Yeromin, 2006).

The consensus sequence within the S1-S2 loop, 179VEVQLDxD186, contains the critical glutamate (bold) shown in this study to control ion selectivity properties of the CRAC channel, and two aspartates (underlined) that may help to attract Gd³⁺ (and Ca²⁺) towards the pore. It is not similar to pore sequences found in other channels. Unlike the pore regions of voltage-gated Ca²⁺ (CaV) channels, that contain a relatively long loop and a ring of critical glutamates from different domains that form a high-affinity Ca²⁺-binding site, the putative pore sequence of Orai is very short, and the key residue for ion selectivity (E180) is adjacent to the putative S1 segment. The corresponding residue is 178 in the Drosophila genome database (accession number AY071273), and 106 in the human Orai1 homologue (accession number BC015369). Because withdrawal of external divalent ions reveals permeability to monovalent cations in both CaV and CRAC channels, it is possible that the CRAC channel ion-selectivity filter is also formed by a ring of glutamates and that the mechanism of Ca²⁺ permeation is similar, although the single-channel conductance and maximum permeant ion size of the CRAC channel selectivity filter are smaller than that of the CaV channel. Negatively charged side chains also contribute to Ca²⁺ selectivity of TRPV6; in this instance, aspartate (at position 541) is proposed to coordinate with Ca²⁺ ions and line the selectivity filter in a ring structure formed by four subunits. The CRAC channel may be a multimer that includes several identical Orai subunits, as a non-conducting pore mutant (E180A) exerts a strong dominant-negative action on native CRAC current. Biochemical approaches and cysteine-scanning mutagenesis should be useful to elucidate better the unique pore architecture of the CRAC channel (Yeromin, 2006).

Recent studies have demonstrated a required and conserved role of Stim in store-operated Ca²⁺ influx and Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, 75 hits were identified that strongly inhibited Ca²⁺ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca²⁺ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca²⁺ pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca²⁺ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity (Zhang, 2006).

The genome-wide screen, based on direct Ca²⁺ influx measurements, validated Stim and identified several additional genes that are required for CRAC channel activity. olf186-F (Orai) was identified as essential for Ca²⁺ signaling and activation of CRAC current in S2 cells, confirming two recent reports (Feske, 2006; Vig, 2006). In addition, evidence is provided, based on overexpression, that Orai may form an essential part of the CRAC channel. In mammalian cells overexpression of STIM1 increases Ca²⁺ influx rates and CRAC currents by ~2-fold, but in S2 cells this study showed that overexpression of Stim alone does not increase CRAC current, consistent with Stim serving as a channel activator rather than the channel itself. In contrast, transfection of olf186-F by itself increased CRAC current densities 3-fold, and cotransfection of olf186-F with Stim resulted in an 8-fold enhancement and the largest CRAC currents ever recorded. These results support the hypothesis that olf186-F constitutes part of the CRAC channel and that Stim serves as the messenger for its activation. Consistent with this hypothesis, the CRAC channel activation kinetics during passive Ca²⁺ store depletion were significantly faster with cotransfected Stim. Many fundamental aspects of the mechanism of CRAC channel activation remain to be clarified, including the protein-protein interactions that underlie trafficking and channel activation. Site-directed mutagenesis in a heterologous expression system may help to define the putative pore-forming region (Zhang, 2006).

Similar to Stim, knockdown of olf186-F did not produce a severe cell growth phenotype. It was neither a hit in a previous screen of cell survival nor in any other published Drosophila whole-genome RNAi screen. The olf186-F gene is a member of a highly conserved gene family that contains three homologs in mammals, two in chicken, three in zebrafish, and one member only in fly and worm. C09F5.2, the only homolog in Caenorhabditis elegans, is expressed in intestine, hypodermis, and reproductive system as well as some neuron-like cells in the head and tail regions. Worms under RNAi treatment against C09F5.2 are sterile. Analysis of hydrophobic regions of the predicted protein from the fly gene and the three mammalian homologs suggested the presence of four conserved transmembrane segments. Cytoplasmic C termini are suggested by the presence of coiled-coil motifs in each sequence. Sequence alignment between members from human, chicken, and fly revealed strong sequence conservation in putative transmembrane regions and conserved negatively charged residues in loops between transmembrane segments. All three human members are expressed in the immune system. Mutation of a human homolog of Drosophila olf186-F, ORAI1 on chromosome 12, appears to be the cause of defective CRAC channel activity in severe combined immune deficiency patient T cells, consistent with a requirement for functional CRAC channels in the immune response. Interestingly, microarray data from public databases combined with tissue-specific EST counts show that all three human members are expressed in a variety of nonexcitable tissues including thymus, lymph node, intestine, dermis, and many other tissues including the brain, although expression patterns and levels are different among the three members (Zhang, 2006).

Ca-P60A has been proposed to be the only Drosophila SERCA gene. This study validated its ER pump function by showing that ionomycin did not induce significant store release from S2 cells pretreated with dsRNA against Ca-P60A. The elevation in resting [Ca²⁺]_i and rapidly changing Ca²⁺ transients during changes in external Ca²⁺ before addition of TG may indicate a low level of constitutive CRAC channel activity induced by store depletion. In addition, SERCA knockdown inhibited CRAC channel activity after passive store depletion in whole-cell patch recordings. These results are consistent with the SERCA pump being required for normal activity of CRAC channels but do not rule out indirect inhibition of CRAC current as a consequence of residual high resting [Ca²⁺]_i or store depletion. The role of SERCA in CRAC channel function merits further study (Zhang, 2006).

Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. Drosophila Orai and human Orai1 (also called TMEM142A) have been identified as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. This study shows that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel (Prakriya, 2006).

Antigen stimulation of immune cells triggers Ca2+ entry through 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. This study has identified the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that was called Orai1, which contains four putative transmembrane segments. [In Greek mythology, the Orai are the keepers of the gates of heaven: Eunomia (Order or Harmony), Dike (Justice) and Eirene (Peace)]. The 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 (I(CRAC)). It is proposed that Orai1 is an essential component or regulator of the CRAC channel complex (Feske, 2006).