InteractiveFly: GeneBrief

olf186-F: Biological Overview | References

Gene name - olf186-F

Synonyms - Orai, dOrai

Cytological map position - 54F1-54F3

Function - channel

Keywords - store-operated Ca2+ channel, rhythmic firing of the flight motoneurons

Symbol - olf186-F

FlyBase ID: FBgn0041585

Genetic map position - 2R: 13,730,950..13,748,075 [+]

Classification - Orai-1

Cellular location - surface transmembrane

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Pathak, T., Agrawal, T., Richhariya, S., Sadaf, S. and Hasan, G. (2015). Store-operated calcium entry through Orai is required for transcriptional maturation of the flight circuit in Drosophila. J Neurosci 35: 13784-13799. PubMed ID: 26446229
Store operated calcium entry (SOCE) is thought to primarily regulate calcium homeostasis in neurons. Subsequent to identification of Orai as the SOCE channel in nonexcitable cells, investigation of Orai function in neurons demonstrated a requirement for SOCE in Drosophila flight. By analysis of an Orai mutant and by controlled expression of a dominant-negative Drosophila Orai transgene, this study shows that Orai-mediated SOCE is required in dopaminergic interneurons of the flight circuit during pupal development. Expression of dominant-negative Orai in dopaminergic neurons of pupae abolished flight. The loss of Orai-mediated SOCE alters transcriptional regulation of dopaminergic neurons, leading to downregulation of the enzyme tyrosine hydroxylase, which is essential for dopamine synthesis, and the dopamine transporter, which is required for dopamine uptake after synaptic release. These studies suggest that modulation of SOCE could serve as a novel mechanism for restoring dopamine levels in dopaminergic neurons.
Chakraborty, S., Deb, B. K., Chorna, T., Konieczny, V., Taylor, C. W. and Hasan, G. (2016). Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons. J Cell Sci [Epub ahead of print]. PubMed ID: 27591258
Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane (PM) Ca2+ channel, Orai. Inositol 1,4,5-trisphosphate receptors (IP3R) are assumed to regulate SOCE solely by mediating ER Ca2+ release. This study shows that in Drosophila neurons, mutant IP3R attenuate SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and IP3R to accumulate near the PM, association of STIM with Orai, clustering of STIM and Orai at ER-PM junctions, and activation of SOCE. These responses were attenuated in neurons with mutant IP3R and rescued by over-expression of STIM with Orai. It is concluded that after depletion of Ca2+ stores in Drosophila, translocation of IP3R to ER-PM junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE (Chakraborty, 2016).
Alavizargar, A., Berti, C., Ejtehadi, M. R. and Furini, S. (2018). Molecular dynamics simulations of Orai reveal how the third transmembrane segment contributes to hydration and Ca(2+) selectivity in Calcium Release-Activated Calcium Channels. J Phys Chem B 122(16): 4407-4417. PubMed ID: 29600712
Calcium release-activated calcium (CRAC) channels open upon depletion of Ca(2+) from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Molecular dynamics and Brownian dynamics simulations were employed to analyze how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and mutated channels revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca(2+) over Na(+). The crevices of water molecules, revealed by molecular dynamics simulations, are perfectly 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.
Hou, X., Burstein, S. R. and Long, S. B. (2018).. Structures reveal opening of the store-operated calcium channel Orai. Elife 7. PubMed ID: 30160233
The store-operated calcium (Ca(2+)) channel Orai governs Ca(2+) influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca(2+) stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Previous work revealed the structure of Orai with a closed pore. In this study, using a gain-of-function mutation that constitutively activates the channel, an X-ray structure is presented of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca(2+) selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, a sequence is proposed for store-operated activation.


Neuronal Ca2+ signals can affect excitability and neural circuit formation. Ca2+ signals are modified by Ca2+ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca2+ stores and their release to neuronal processes is poorly understood. This study shows, by neuron-specific siRNA depletion, that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca2+ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP3R) can partially compensate for their loss of flight. Ca2+ measurements show that Orai gain-of-function contributes to the quanta of Ca2+-release through mutant InsP3Rs and elevates store-operated Ca2+ entry in Drosophila neurons. These data show that replenishment of intracellular store Ca2+ in neurons is required for Drosophila flight (Venkiteswaran, 2009).

Several aspects of neuronal function are regulated by ionic calcium (Ca2+). Specific attributes of a Ca2+ 'signature' such as amplitude, duration, and frequency of the signal can determine the morphology of a neural circuit by affecting the outcome of cell migration, the direction taken by a growth-cone, dendritic development, and synaptogenesis. Ca2+ signals also determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors. Most of these Ca2+ signals have been attributed to the entry of extracellular Ca2+ through voltage-operated channels or ionotropic receptors. However, other components of the 'Ca2+ tool-kit' coupled to Ca2+ release from intracellular Ca2+ stores are also present in neurons. These molecules include the store-operated Ca2+ (SOC) channel, encoded by the Orai gene, identified in siRNA screens for molecules that reduce or abolish Ca2+ influx from the extracellular milieu after intracellular Ca2+ store depletion (Feske, 2006; Vig, 2006b; Zhang, 2006). Several reports have confirmed its identity as the pore forming subunit of the Ca2+-release activated Ca2+ (CRAC) channel (Prakriya, 2006; Vig, 2006b; Yeromin, 2006). Activation of this CRAC channel is mediated by the endoplasmic reticulum (ER) resident protein STIM (stromal interaction molecule), also identified in an RNAi screen for molecules that regulate SOC influx (Liou, 2005; Zhang, 2005). STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. STIM senses the drop in ER Ca2+ levels, and interacts with Orai by a mechanism which is only just being understood (Yuan, 2009). Orai and STIM function in conjunction with the sarco-endoplasmic reticular Ca2+-ATPase pump (SERCA) to maintain ER store Ca2+ and basal Ca2+. The importance of intracellular Ca2+ homeostasis and SOC entry (SOCE) in neural circuit formation and in neuronal function and physiology remains to be elucidated (Venkiteswaran, 2009).

This study reports how Orai and STIM mediated Ca2+ influx and Ca2+ homeostasis in Drosophila neurons contribute to cellular and systemic phenotypes. Reduced SOCE, measured in primary neuronal cultures, is accompanied by a range of defects in adults, including altered wing posture, increased spontaneous firing, and loss of rhythmic flight patterns. These phenotypes mirror the spontaneous hyperexcitability of flight neuro-muscular junctions and loss of rhythmic flight patterns observed in Drosophila mutants of the inositol 1,4,5-trisphosphate receptor (InsP3R, itpr gene). The InsP3R is a ligand gated Ca2+-channel present on the membranes of intracellular Ca2+ stores. It is thought to be critical for various aspects of neuronal function. Mutants in the gene coding for the mouse InsP3R1 are ataxic. Cerebellar slices from InsP3R1 knockout mice show reduced long-term depression, indicating that altered synaptic plasticity of the cognate neural circuits could underlie the observed ataxia (Venkiteswaran, 2009).

To understand the temporal and spatial nature of intracellular Ca2+ signals required during flight circuit development and function, dOrai (CG11430) and dSERCA (encoded by CaP-60A gene, CG3725) function was modulated by genetic means in itpr mutants [using a dominant mutant allele (Kum170) for the gene (Ca-P60A) encoding the SERCA]. This modulation can restore flight to flightless adults, by altering several parameters of intracellular Ca2+ homeostasis including SOCE. These results suggest that components of the central pattern generator (CPG) required for maintenance of normal rhythmic flight in adults have a stringent requirement for SOCE after InsP3R stimulation (Venkiteswaran, 2009).

This study shows that SOC entry through the Orai/STIM pathway and the rate of clearance of cytoplasmic Ca2+ by SERCA together shape intracellular Ca2+ response curves in Drosophila larval neurons. The phenotypic changes associated with altering Orai/STIM function on their own and in itpr mutant combinations suggest that these Ca2+ dynamics are conserved through development among neurons in pupae and adults. The development and function of the flight circuit appears most sensitive to these cellular Ca2+ dynamics, changes in which have a profound effect on its physiological and behavioral outputs. Direct measurements of Ca2+ in flight circuit neurons are necessary in future to understand why these cells are more sensitive to changes in intracellular Ca2+ signaling. Other circuits such as those required for walking, climbing and jumping remain unaffected. Possible effects of altering intracellular Ca2+ homeostasis on higher order neural functions have yet to be determined (Venkiteswaran, 2009).

The flow of information in a neural circuit goes through multiple steps within and between cells. Suppression experiments, such as the ones described in this study, present a powerful genetic tool for understanding the mechanisms underlying both the formation of such circuits and their function. The correlation observed between adult phenotypes and Ca2+ dynamics in populations of larval neurons from the various genotypes supports the following conclusions. Out-spread wings, higher spontaneous firing, and initiation of rhythmic firing on air-puff delivery in itprku (a heteroallelic mutant combination of itpr) are suppressed by either increasing the quanta (through hypermorphic alleles of dOrai and by dOrai+ overexpression) or by increasing the perdurance (through mutant Kum170) of the intracellular Ca2+ signal (Kum is ). Flight ability and maintenance of flight patterns requires SOCE in addition to increased quanta and perdurance of the Ca2+ signals, suggesting that SOCE in neurons contributes to recurring Ca2+ signals essential for flight maintenance (Venkiteswaran, 2009).

The signals that trigger InsP3 generation in Drosophila neurons and the nature of the downstream cellular response remain to be investigated. Previous work has shown that rescue of flight and related physiological phenotypes in itpr mutants require UASitpr+ expression in early to midpupal stages, indicating the InsP3R activity is necessary during development of the flight circuit (Banerjee, 2004). Due to perdurance of the InsP3R, its requirement in adults was not established. This study found that a basal level of dOrai+ expression through development followed by ubiquitous overexpression in adults can help initiate flight in itprku, indicating a requirement for SOCE in adult neurons that probably constitute the CPG for flight. The precise neuronal circuit and neurons in the flight CPG are under investigation (Frye, 2004). Aminergic, glutamatergic, and insulin producing neurons could assist in development and/or directly constitute the circuit. Similar patterns of neuronal activity in the flight circuit of itpr mutants, either by generating different combinations of Ca2+ fluxes (as shown in this study), or by UASitpr+ expression in nonoverlapping neuronal domains supports the idea that different aspects of neuronal activity can compensate for each other to maintain constant network output (Venkiteswaran, 2009).

Precisely how hypermorphic dOrai alleles modify itprku function to increase the quanta of Ca2+ release remains to be investigated. The ability of itprku to maintain elevated [Ca2+]ER at 25 °C suggests a possible interaction between this heteroallelic combination and Orai/STIM. The mutated residue in itprka1091 (Gly to Ser at 1891) lies in the modulatory domain, whereas in itprug3, it lies in the ligand binding domain (Ser to Phe at 224); both residues are conserved in mammalian InsP3R isoforms (Srikanth, 2004). The mutant residues could directly affect InsP3R interactions with a store Ca2+ regulating molecule like STIM (Taylor, 2006). Recent reports (Redondo, 2006) also demonstrate the formation of macromolecular assemblies of InsP3R, SERCA, and SOC channels, suggesting specific functional interactions between them (Venkiteswaran, 2009). Last, these results suggest new ways of treating diseases where altered intracellular Ca2+ signaling or homeostasis has been suggested as a causative agent. Perhaps, the best documented of these diseases are spino-cerebellar ataxia 15, which arises by heterozygosity of the mammalian IP3R1 gene (van de Leemput, 2007), severe combined immunodeficiency due to a mutation in Orai1 (Feske, 2006), and Darier's disease from a mutation in SERCA2 (Sakuntabhai, 1999). Based on the underlying changes in intracellular Ca2+ properties in these genetic diseases, this study suggests ways of deciding appropriate combination of drugs that might target the causative gene products and their functionally interacting partners (Venkiteswaran, 2009).

The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.

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).

Biochemical and functional characterization of Orai proteins

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 ICRAC. Amino acid substitutions in either of two conserved glutamate residues, located in predicted transmembrane segments of dOrai and Orai1, changed the properties of ICRAC, 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).

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

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).

Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity

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).

Orai1 is an essential pore subunit of the CRAC channel

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).

A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function

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).


Search PubMed for articles about Drosophila Orai

Feske, S., et al. (2006) A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441: 179-185. PubMed ID: 16582901

Frye, M. A. and Dickinson, M. H. (2004). Closing the loop between neurobiology and flight behavior in Drosophila. Curr. Opin. Neurobiol. 14: 729-736. PubMed ID: 15582376

Gwack, Y., et al. (2007). Biochemical and functional characterization of Orai proteins. J. Biol. Chem. 282(22): 16232-16243. PubMed ID: 17293345

Liou, J., et al. 2005). STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15: 1235-1241. PubMed ID: 16005298

Penna, A., et al. (2008). The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 456(7218): 116-20. PubMed ID: 18820677

Prakriya, M., et al. (2006). Orai1 is an essential pore subunit of the CRAC channel. Nature 443: 230-233. PubMed ID: 16921383

Redondo, P. C., et al. (2008). Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets. Biochim. Biophys. Acta 1783: 1163-1176. PubMed ID: 18191041

Sakuntabhai, A., et al. (1999). Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat. Genet. 21: 271-277. PubMed ID: 10080178

Srikanth, S., et al. (2004). Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants. Biophys. J. 86: 3634-3646. PubMed ID: 15189860

Taylor, C. W. (2006). Store-operated Ca2+ entry: A STIMulating stOrai. Trends Biochem. Sci. 31: 597-601. PubMed ID: 17029812

van de Leemput, J., et al. (2007). Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans. PLoS Genet 3: e108. PubMed ID: 17590087

Venkiteswaran, G. and Hasan, G. (2009). Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight. Proc. Natl. Acad. Sci. 106(25): 10326-10331. PubMed ID: 19515818

Vig, M., et al. (2006a). CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science 312: 1220-1223. PubMed ID: 16645049

Vig, M., et al. (2006b) CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr. Biol. 16: 2073-2079. PubMed ID: 16978865

Yeromin, A. V., et al. (2006) Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443: 226-229. PubMed ID: 16921385

Zhang, S. L., et al. (2005). STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 437: 902-905. PubMed ID: 16208375

Zhang, S. L., et al. (2006). Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity. Proc. Natl. Acad. Sci. 103: 9357-9362. PubMed ID: 16751269

Yuan, J, P., et al. (2009). SOAR and the polybasic STIM1 domains gate and regulate Orai channels. Nat. Cell Biol. 11: 337-343. PubMed ID: 19182790

Biological Overview

date revised: 10 June 2010

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.