SOCE modulation

Ca²⁺–dependent inactivation (CDI)

The first type of modulation is Ca²⁺ itself, through what is called Ca²⁺–dependent inhibition (CDI). Electrophysiological studies describe two different processes in which ICRAC density decreases over time, one fast (in the msec range, FCDI) and one slow (sec to min, SCDI) (Zweifach and Lewis, 1995a, b; Parekh, 1998). FCDI is revealed by imposing negative voltage ramps to store–depleted cells, and by blocking SERCA–mediated refilling with Tg. FCDI is observed even when the intracellular compartment is dialyzed with 10 mM EGTA. As EGTA only buffers Ca²⁺ ions at a minimal distance of 0.1 μm from the PM (Fierro and Parekh, 1999), it was proposed that FCDI is mediated by the accumulation of Ca²⁺ ions in the close vicinity of the channel intracellular pore that exert a direct negative feedback on the current. SCDI not only differs by its slower kinetics, but also by the fact that intracellular EGTA abolishes ICRAC slow inactivation. Although the underlying molecular mechanisms of SCDI are still unclear, this process is considered to finely tune CRAC activity by sensing more global intracellular Ca²⁺

elevations.

SOCE termination

While FCDI and SCDI occur in the absence of store refilling, SOCE termination requires the replenishment of Ca²⁺ stores. This step is essential but not sufficient to initiate STIM1 de–

oligomerization and dissociation from the PM, and only an additional local cytosolic Ca²⁺

elevation close to the STIM1–ORAI1 interface allows the deactivation of the SOCE process (Shen et al., 2011). Mitochondria play a central role in the regulation of Ca²⁺ levels in such microdomains. Their capacity to take up Ca²⁺ ions essentially depends on high Ca²⁺ gradients, and mitochondria were shown to relocate to T lymphocytes immune synapses where SOCE takes place (Quintana et al., 2007; Quintana and Hoth, 2012). Mitochondria are thought to decrease CDI, store refilling and impair SOCE termination, as blocking mitochondrial migration along microtubules towards the PM fails to sustain Ca²⁺ influx (Quintana et al., 2006).

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In addition to their ability to capture Ca²⁺ ions, mitochondria also regulate SOCE by producing reactive oxygen species (ROS). The oxidation of STIM1 cysteine residues enhances the protein multimerization and PM translocation, and is followed by Ca²⁺ entry, even in the absence of store depletion (Bhardwaj et al., 2016). In contrast, the addition of hydrogen peroxide (H2O2) to the external environment inhibits ORAI1 clustering and reduces SOCE (Bogeski et al., 2010; Alansary et al., 2016). Along with redox modulation, external and internal pH changes severely impact on ICRAC density, with current potentiation at basic pH and decline upon acidification (Beck et al., 2014; Tsujikawa et al., 2015). Besides, increasing temperature boosts ICRAC, probably by affecting lipid membrane composition. The current density – temperature relationship is non–linear and ICRAC drastically decays under 21°C (Somasundaram et al., 1996). One should carefully consider this property as most ICRAC whole–cell patch–clamp recordings are performed at room temperature (23°C) for simplicity.

Post–translational modifications

Post–translational modifications of STIM1 and ORAI1 alter their stability and function.

STIM1 glycosylation seems to be necessary for the protein translocation to the PM and the initiation of SOCE (Williams et al., 2002). On the contrary, the addition of a glycan to the unique ORAI1 glycosylation site reduces SOCE, probably by altering the channel interaction with other modulating proteins (Dorr et al., 2016). The high turnover of ORAI1 channels at the PM is due to their internalization in sub–plasmalemal endocytic vesicles, and their enrichment at ER–PM junctions by a STIM1–mediated trapping (Hodeify et al., 2015). And ORAI1 glycosylation might also promote this high turnover (Niemeyer, 2016). In addition, STIM1 phosphorylation prejudices STIM1–ORAI1 interaction and is thought to be a determinant of ER redistribution during the cell cycle (Preston et al., 1991). PKC–mediated phosphorylation of ORAI1 inhibits the channel activity in a Ca²⁺–dependent manner, consigning this post–transcriptional modification to the Ca²⁺ negative feedback loop (Kawasaki et al., 2010). Finally, ORAI1 and STIM1 ubiquitination by the ubiquitin ligases Nedd4-2 and POSH respectively, reduce their surface expression and limit SOCE (Keil et al., 2010; Lang et al., 2012).

Interacting proteins

Many STIM1–ORAI1 interacting molecules complete the set of positive and negative SOCE regulators (Srikanth et al., 2013). Among them is Junctate, an ER resident protein interacting

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with STIM1 and enhancing its recruitment to the PM, depending on the ER luminal Ca²⁺

content. Invalidating Junctate EF–hands induces puncta formation and SOCE activation in store–replete cells, and is an alternative mechanism to trigger STIM1 translocation (Srikanth et al., 2012). Another ER transmembrane protein named STIMATE (STIM–activating enhancer) directly interacts with STIM1 and supports its conformational change (Jing et al., 2015).

CRACR2A (CRAC channel regulator 2A) and its homolog CRACR2B are cytosolic Ca²⁺ sensors that associate with STIM1 and ORAI1 to form a stable ternary complex (Srikanth et al., 2010).

In its Ca²⁺–bound form, CRACR2A destabilizes the complex and ends SOCE. When Ca²⁺ binding is prevented by mutations in the respective motif, the excessive activity of the SOCE machinery leads to Ca²⁺ overload and cell death. The organization of STIM1–ORAI1 clusters at the PM appears therefore to be critical for SOCE regulation. The GTP–binding cytoskeleton filaments Septins (in particular Septin 4) were described as SOCE coordinators that assemble in ring structures to scaffold SOCE partners and prevent their diffusion along the plasma lipid bilayer.

Septins’ rearrangement organizes PIP2 rich membrane microdomains at ER–PM junctions, promotes STIM1 recruitment and stabilizes ORAI1 clusters (Sharma et al., 2013). The Ca²⁺

effector CaM is a prevalent negative modulator of SOCE/CRAC that, in the Ca²⁺–bound form, initiates STIM1–ORAI1, STIM1–PM and STIM1 multimers dissolution, hence resulting in SOCE termination (Li et al., 2017). CaM was proposed to associate with the CAD domain of STIM1 and to interact with the ORAI1 N–terminus (residues W76 and Y80, in the channel pore) to mediate CDI (Mullins et al., 2009; Liu et al., 2012; Bhardwaj et al., 2013). However the poor accessibility of the predicted CaM–binding motif to CaM questions this hypothesis. The ER Ca²⁺

binding protein SARAF (SOCE–associated regulatory factor) was suggested to elicit SCDI (Palty et al., 2012; Jha et al., 2013). SARAF travels to the PM together with STIM1 upon store depletion, senses global Ca²⁺ elevations and destabilizes STIM1–ORAI1 interactions to prevent stores overfilling. Golli is a member of the myelin basic protein family shown to be essential in T lymphocytes (Feng et al., 2004). By interacting with the STIM1 C–terminus, Golli negatively regulates SOCE and preserves Ca²⁺ homeostasis. However, this stranglehold can be reversed by the overexpression of STIM1 (Walsh et al., 2010). Upon store–depletion, POST (partner of STIM), mainly located at the ER membrane, associates with STIM1, ORAI1, SERCA and PMCA in large complexes, and sustains the accumulation of Ca²⁺ ions in the ER–PM cleft by inhibiting the PMCA pump (Krapivinsky et al., 2011). This short list of SOCE regulators is not exhaustive and new STIM1-ORAI1 partners are continuously identified. In the end, the combination of all of these molecular mechanisms and physical parameters are thought to support Ca²⁺ signaling

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and maintain Ca²⁺ homeostasis by providing a negative feedback that avoids Ca²⁺ overload and subsequent cell death.

Chemical SOCE modulators

The continuous identification of diseases associated with SOCE dysregulation, raised the need of developing specific pharmacological SOCE modulators. The first described ICRAC

inhibitors are lanthanides such as the trivalent ions La³⁺ and Gd³⁺ that are potent blockers of endogenous and exogenous Ca²⁺ currents (Ross and Cahalan, 1995; Aussel et al., 1996). By sticking in the pore mouth of ORAI channels, lanthanides prevent Ca²⁺ influx. However, their specificity to ORAI channels is poor, as they also block TRP and voltage–gated Ca²⁺ channels (Lansman et al., 1986; Clapham et al., 2001). The noncompetitive IP3R antagonist 2–APB is extensively used to study CRAC properties. However, 2–APB displays bimodal properties: at low concentrations (< 20 µM), the drug potentiates ORAI1–mediated currents, and at higher concentrations (> 50 µM), 2–APB inhibits Ca²⁺ influx (Maruyama et al., 1997; Prakriya and Lewis, 2001; Ma et al., 2002). How 2–APB alters ORAI1 gating is still unclear, and its multifaceted characteristics together with a poor specificity for ORAI channels (2–APB also modulates IP3R and TRP channels, (Hu et al., 2004)) makes the use of 2–APB delicate. BTP2 (3,5–bis(trifluoromethyl)pyrazole) treatment of immune cells was shown to inhibit Ca²⁺ influx, proliferation and cytokine release (Trevillyan et al., 2001). But, as for 2–APB, its weak specificity (also described as a K⁺, TRP and voltage–gated channels inhibitor, (Ishikawa et al., 2003; Zitt et al., 2004; He et al., 2005)) limits the use of BTP2 for the study of ICRAC. The lack of specificity of usual CRAC inhibitors encouraged the development of more–specific 2–APB or BTP2 derivatives such as Synta66, GSK–5503A and GSK–7975A. Synta66 inhibition is restricted to ORAI1, ORAI3 and TRPV6. However, with very slow inhibition kinetics necessitating long pre–

incubation periods, and because of its irreversible effects, Synta66 popularity declined in favor of GSK compounds (Chen et al., 2013). GSK–7975A and Synta66 specificities are comparable, but GSK–7975A can acutely and more potently inhibit CRAC current (Derler et al., 2013). GSK–

7975A efficiency was established in mouse models of acute pancreatitis and the drug recently entered clinical trials (Gerasimenko et al., 2013). The latest progress in establishing highly potent and specific blockers was brought by the generation of ORAI1 monoclonal antibodies (Lin et al., 2013). Nevertheless, how most of these modulators affect STIM1–ORAI1 choreography and channel gating still needs to be elucidated, and envisaging their application

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for therapeutic purposes remains delicate as ORAI channels are ubiquitously expressed in human tissues.