Transcripts expression
In immune cells, transcripts taken from isolated primary CD3⁺/CD4⁺ cells (Th-lymphocytes), CD3⁺/CD8⁺ cells (Tc-lymphocytes), CD19⁺ cells (B-lymphocytes) and BMMC showed that ORAI3 expression is readily detectable in Th^-, Tc^-, and B^- lymphocytes and BMMC (Gross et al., 2007).
mRNA expression in normal tissues has been assessed by different techniques (microarrays, RNAseq, SAGE). Microarrays analyses show that ORAI3 is overexpressed in prostate, lung, monocytes and whole blood (http://biogps.org/#goto=genereport&id=93129, with overexpression defined as 3 times the mean expression observed in the 83 tissues or cells tested in this study). ORAI3 mRNA expression is least important in pancreas, brain (especially the occipital lobe) and T cells (CD4⁺ as well as CD8⁺).

Post-translational modifications of the protein

Glycosylation:
Unlike ORAI1, ORAI3 does not have a glycosylation site on the asparagine residue (N223) situated between the transmembrane domains TM3 et TM4 (Frischauf et al., 2008; Prakriya et al., 2006).
ORAI1 has a putative N-glycosylation motif (NVS) in its extracellular loop between predicted transmembrane segments 3 and 4. This motif is absent in ORAI2 and 3 (Gwack et al., 2007). ORAI3 migration properties do not change by tunicamycin treatment. Indeed, HEK293 cells stably transfected with FLAG-tagged ORAI and treated with 2μg/ml tunicamycin, showed that ORAI3 migrated at positions close to their predicted molecular masse (32.5 kDa).
Phosphorylation:
Since ORAI3 is a tetraspanning plasma membrane protein, it contains three intracellular regions that can potentially be phosphorylated by intracellular protein kinases: the N-terminus, an intracellular loop between transmembrane domains 2 and 3, and the C-terminus, each intracellular region potentially contains one or more phosphorylation sites. Ser-27 and Ser-30 have been identified as the main phosphorylation sites in ORAI1 within its N-terminus. They are conserved throughout evolution in all mammalian ORAI1 proteins. Mutations at these phosphorylation sites increase store-operated Ca²⁺ entry (SOCE) and CRAC current suggesting that ORAI1 phosphorylation at these residues by protein kinase C (PKC) suppresses SOCE and CRAC channel activation. However, Ser-27 and Ser-30 are not present in ORAI2 and ORAI3.
A phosphorylation of ORAI3 peptide has been revealed by a phosphoproteome analysis of human liver cells (Sui et al., 2008). This phosphorylation site is located in the C-terminus of ORAI3 on a tyrosine residue (Y278). Experimental ORAI3 phosphorylation has also been demonstrated in HEK293 cells (Kawasaki et al., 2010).
To examine in vivo PKC-mediated phosphorylation, HEK293 cells expressing FLAG-tagged ORAI were incubated with ³²P monosodium phosphate, and then stimulated with thapsigargin in the presence of extracellular Ca²⁺. Thapsigargin mobilizes Ca²⁺ from the ER and the extracellular space and activates Ca²⁺/DAG-dependent PKC isoforms. ORAI1 phosphorylation is enhanced in response to thapsigargin. The levels of ORAI3 phosphorylation have been less than half of that observed for ORAI1 (Kawasaki et al., 2010).
Other phosphorylation sites on ORAI3 were predicted by NetPhos2.0: 13 serine sites (S20, S45, S50, S57, S64, S65, S68, S86, S191, S203, S213, S214 and S20), 3 threonine sites (T26, T183 and T190) and 2 tyrosine sites (Y146 and Y278).

ORAI3 activation and interaction with STIM proteins
ORAI channels are activated by STIM1 or STIM2, single-pass transmembrane proteins localized predominantly in the membrane of the endoplasmic reticulum. STIM proteins have a long C-terminal cytoplasmic region and contain an N-terminal EF-hand located in the ER lumen that functions as a sensor of ER Ca²⁺ levels (Roos et al., 2005; Liou et al., 2005; Williams et al., 2001; Stathopulos et al., 2006).
The activation of ORAI channels by STIM depends on Ca²⁺ store depletion and is reversible once the stores are refilled (Luik et al., 2008; Soboloff et al., 2006). STIM1 activates store-operated Ca²⁺ channels only when it is not fixing Ca²⁺, e.g. when the stores are depleted (Zhang et al., 2005). One minute after store depletion, STIM proteins are redistributed in puncta in close proximity to the plasma membrane (Liou et al., 2005; Luik et al., 2008; Várnai et al., 2007; Baba et al., 2006), where they co-localize with and activate ORAI channels, allowing Ca²⁺ influx (Liou et al., 2005; Wu et al., 2006; Muik et al., 2008). This process implies tetramerisation of STIM1 proteins using the N-terminus (Luik et al., 2008). It is thought that within these puncta, STIM1 communicates with and opens CRAC channels located to the plasma membrane (Luik et al., 2006; Parvez et al., 2008).
The initial interaction of STIM1 with the ORAI channels involves their cytosolic C-terminal region (Li et al., 2007; Muik et al., 2008; Frischauf et al., 2009). In all three ORAI subtypes, this region contains a predicted coiled-coil domain that is critical for interactions with STIM1 (Muik et al., 2008).
Truncation analysis identified a cytoplasmic region of STIM1, termed the CRAC activation domain (CAD)/STIM1 ORAI1 activating region (SOAR) to be sufficient to activate ORAI1 (Kawasaki et al., 2009; Muik et al., 2009; Park et al., 2009; Yuan et al., 2009). The cytoplasmic N and C termini of ORAI1 mediate channel opening by interaction with STIM1.
The activation of ORAI3-induced store-operated currents is significantly slower than that seen with ORAI1 and ORAI2 (Lis et al., 2007). Contrary to ORAI1, both ORAI2 and ORAI3 exhibit a 15-17 fold higher coiled-coil probability (Frischauf et al., 2009). A single point mutation in the ORAI1 coiled-coil domain (L273S) abrogates communication with STIM1 C-terminus (Frischauf et al., 2009; Muik et al., 2008). A single point mutation (L285S) within ORAI3 coiled-coil domain results in a partial inhibition of the interaction with STIM1 and subsequent activation of ORAI3 currents. Full inhibition of the ORAI3-induced currents requires incorporation of an additional mutation (L292S) in the coiled-coil domain.
According to Bergsmann, activation of ORAI channels requires coupling of the C terminus of STIM to the N and C termini of ORAI (Bergsmann et al., 2011), since increasing N-terminal truncations causes a progressive decrease of ORAI3 fast inactivation concomitant with diminished binding to calmodulin. Therefore, a fully conserved N-terminal ORAI region (aa 48-65 in ORAI3) is essential for STIM1-dependent STIMulation (Derler et al., 2009; Li et al., 2007; Yuan et al., 2009; Fahrner et al., 2009; Park et al., 2009; Lis et al., 2010). Moreover, a single lysine within this conserved region (K60E in ORAI3) represents a critical residue for store-operated activation (Lis et al., 2010).

Interaction between ORAI family members
ORAI3 can multimerize with ORAI1 to form cation channels that conduct Ca²⁺ to some degree, since HEK293 cells stably expressing FLAG-tagged ORAI2 and ORAI3 revealed co-immunoprecipitation of ORAI2 and ORAI3 with transiently overexpressed Myc-ORAI1. Thus, ORAI members form homomultimers and can also form heteromultimers (Gwack et al., 2007).

Protein Interactions other than STIM
In addition to STIM1, p45 renamed as CRACR2A (CRAC regulator 2A) is also shown to co-immunoprecipitate with ORAI1, ORAI2 and ORAI3, suggesting a conserved binding mechanism with all the ORAI proteins, and that the ORAI channels, STIM1 and CRACR2A may form a ternary complex though direct interaction.
Various other proteins and lipids have been identified to interact with either STIM1 or ORAI3 or both. Among them is calmodulin (Mullins et al., 2009; Parvez et al., 2008; Bergsmann et al., 2011). Calmodulin binds to ORAI3 and, together with STIM, contributes to fast calcium-dependent inactivation; the structural studies show that CRACR2A/B is also able to interact with ORAI3 (Srikanth et al., 2010) but to date there is no evidence of functional regulation, because ORAI3 is able to form some complex with STIM-1 (Faouzi et al., 2011). All proteins that interact with STIM1 are able to modulate ORAI3 function indirectly. Thus, SARAF (Palty et al., 2012), MS4A4B (Howie et al., 2009), Golli (Walsh et al., 2010), adenylyl cyclase type 8 (AC8) (Martin et al., 2009), the polycystin-1 cleavage product P100 (Woodward et al., 2010), caveolin (Yu et al., 2010), SPCA2 (Feng et al., 2010) and the L-type Ca²⁺ channel (Cav1.2) (Wang et al., 2010) or the phospholipids PIP2 and PIP3 (Korzeniowski et al., 2009; Walsh et al., 2009) are able to modulate indirectly ORAI3 activity.

ORAI3 inactivation
Fast inactivation of ORAI channels is mediated by cooperative interplay of several structures within ORAI proteins, by the CRAC modulatory domain (CMD) of STIM1, and via calmodulin binding to the ORAI N terminus (Parekh and Putney, 2005; Lee et al., 2009; Frischauf et al., 2011; Derler et al., 2009).
ORAI3 currents exhibit a marked fast inactivation within the first 100 ms, while that of ORAI2 or ORAI1 show less robust feedback regulation (Lis et al., 2007; Schindl et al., 2009; Lee et al., 2009). This effect depends on the presence of three conserved glutamates (E281, E283, E284) in the C-terminal region of ORAI3 (Lee et al., 2009). According to Yamashita et al. (2007), fast inactivation is determined by the same acidic residues involved in determining Ca²⁺ selectivity. A STIM1 C-terminus domain that include an acidic cluster (amino acids 475-483) termed CRAC Modulatory Domain (CMD) is also indispensable for fast ORAI channel inactivation (Derler et al., 2009; Mullins et al., 2009; Lee et al., 2009), since mutations in the CMD results in ORAI3 currents with attenuated or even abolished Ca²⁺-dependent inactivation (Derler et al., 2009; Lee et al., 2009). On the other hand, Litjens et al. (2004) suggest that fast inactivation may be calmodulin (CaM) dependent and involves a region in the cytosolic N-terminal domain of ORAI3 (S45-K62) that binds CaM in a Ca²⁺-dependent manner (Mullins et al., 2009; Frischauf et al., 2011). Transient CaM binding is assumed to mediate fast inactivation. The process may be that CaM transiently competes with STIM1 for the N-terminal interaction site on ORAI essential for channel gating.
Not only the C- but also the N-terminus and the second intracellular loop between TM2 and TM3 contribute to ORAI inactivation/gating in a cooperative manner (Frischauf et al., 2011) and modulate fast and slow inactivation as revealed by chimeric and mutational approaches (Srikanth et al., 2010). ORAI fast inactivation also involves the pore region since mutations of negatively charged residues within the pore of ORAI results in attenuation of Ca²⁺-dependent inactivation (Yamashita et al., 2007).

Pharmacology
To date there is no specific inhibitor of ORAI3 but ORAI3 channels can be blocked by generic blockers of calcium entry channels such as La³⁺ (50-100 μM) and Gd³⁺ (1-5 μM). Other non-specific blockers include SKF96365, the myosin light chain kinase inhibitor ML-9 (Smyth et al., 2008), and the bistrifluoromethyl-pyrazole derivative BTP2 (Zitt et al., 2004) can be used.
Another compound extensively studied is 2-aminoethoxydiphenyl borate (2-APB), originally characterized as an inhibitor of InsP₃ receptors (Maruyama et al., 1997; Bilmen and Michelangeli, 2002), later shown to have multiple diverse effects including both the inhibition and activation of various different members of the TRP channel family (Voets et al., 2001; Trebak et al., 2002; Chung et al., 2004; Hu et al., 2004; Li et al., 2006; Juvin et al., 2007), and the inhibition of SERCA pumps (Missiaen et al., 2001; Peppiatt et al., 2003), as well as to affect store-operated Ca²⁺ entry via CRAC channels (Gregory et al., 2001; Iwasaki et al., 2001; Prakriya and Lewis, 2001).
2-APB displays a bi-functional effect that is dependent on the concentration used. High concentrations of 2-APB were shown to increase store-operated currents in cells expressing STIM1 and ORAI3 (Lis et al., 2007; DeHaven et al., 2008; Peinelt et al., 2008; Schindl et al., 2008), accompanied by marked changes in ion selectivity by increasing ORAI3 channel pore size from ~3.8 Å to more than 5.34 Å, an effect that was apparently dependent on the E165 residue of ORAI3 that lies in the third transmembrane domain (Schindl et al., 2008). The residues that assist in formation of the 2-APB-activated ORAI3 pore are lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore (Amcheslavsky et al., 2014). Transmembrane domains 2 and 3, together with the linking intracellular loop, are required for 2-APB to directly activate ORAI3 channels (Zhang et al., 2008).
ORAI3 can be directly activated by high concentrations of 2-APB, in a STIM1- and store depletion-independent manner (DeHaven et al., 2008; Peinelt et al., 2008; Schindl et al., 2008; Zhang et al., 2008; Wang et al., 2009). These direct 2-APB induced currents display large inward and outward currents (i.e. they show double rectification) and a leftward shift in the reversal potential, features that indicate a marked reduction in Ca²⁺ selectivity, and an increased permeability to monovalent cations (DeHaven et al., 2008; Peinelt et al., 2008; Schindl et al., 2008; Zhang et al., 2008).
When ORAI3 forms a store operated channel, store-operated ORAI3 currents are potentiated by 2-APB at low concentrations (<10 μM) without affecting ion selectivity (Yamashita et al., 2011). This effect requires the presence of STIM1, and is strictly dependent on store depletion.
The most obvious unique property of the channels involving ORAI3 is their ability to be activated independently of store depletion, either pharmacologically by 2-APB or, physiologically, by agonist-generated increased levels of intracellular arachidonic acid.
A recent study by (Zeng et al., 2014) shows that the ryanodine receptor (RyR) agonist 4-chloro-3-ethylphenol (4-CEP) blocks ORAI1/3 store-operated channels. 4-CEP induces a significant Ca²⁺ release in rat L6 myoblasts, but inhibits SOCE. The inhibitory effect is concentration-dependent and more potent than the one of its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca²⁺ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly (Zeng et al., 2014).
GSK-7975A and GSK-5503A are selective CRAC channel blockers that inhibit both ORAI1 and ORAI3 currents by acting downstream of STIM1 oligomerization and STIM1/ORAI1 interaction, potentially via an allosteric effect on the selectivity filter of ORAI (Derler et al., 2012). Both GSK compounds fully inhibited ORAI3 currents. Similarly, Synta-66 inhibited ORAI3 currents at a similar rate as the GSK compounds. By contrast, 10 μM La³⁺ blocked ORAI3 currents more rapidly. The GSK compounds appeared to inhibit ORAI3 currents slightly faster than those of ORAI1. Overall these GSK compounds were equally effective at blocking ORAI1 and ORAI3, and inhibition occurred at a substantially slower rate than La³⁺. Inhibition of ORAI currents by GSK compounds is not readily reversible: neither ORAI1 nor ORAI3 currents showed substantial recovery from block by GSK-7975A or GSK-5503A over a 4-5 min wash-out period.
2-APB stimulated ORAI3 currents are less susceptible to GSK-7975A. 10 μM GSK-7975A was totally ineffective in inhibiting these ORAI3 currents in contrast to those activated via STIM1. 50 μM GSK-7975A caused 50% inhibition and 100 μM GSK-7975A caused full inhibition. The GSK CRAC channel blockers did not differentiate between ORAI1 and ORAI3 channels consistent with the conserved pore geometry and selectivity filter among the ORAI isoforms.