STIM and ORAI

STIM isoforms and splicing variants

The ubiquitous STIM family members are ER Ca²⁺ sensors highly conserved among species.

They are type I single–pass ER proteins with a luminal N–terminus and a cytosolic C–terminus.

In humans, the family includes two isoforms, STIM1 and STIM2 that share most of their sequence (Fig. 6). Their luminal portion holds an N–terminal ER targeting signal sequence (SS), two EF hand motifs, one canonical (cEF) and one non–canonical “hidden” (hEF), followed by a sterile alpha motif (SAM) and the transmembrane domain. The cytosolic portion possesses three coiled–coil domains (CC1, CC2 and CC3), an inhibitory domain (ID) involved in CDI, and a polybasic (lysine–rich) tail interacting preferentially with PIP2 to anchor the protein at the PM upon store depletion (Liou et al., 2007; Derler et al., 2009; Korzeniowski et al., 2009; Walsh et al., 2009; Soboloff et al., 2012; Bhardwaj et al., 2013; Bhardwaj et al., 2016). The main differences between STIM1 and STIM2 lie in the affinity of their respective EF hands for Ca²⁺

ions, and the affinity of their polybasic tail for PIP2. The high Ca²⁺ dissociation constant (Kd) of STIM2 (~ 0.4 mM, compared to ~ 0.2 mM for STIM1) predispose this isoform to sense very small changes in ER Ca²⁺ content (Zheng et al., 2008). Additionally, the high affinity of the STIM2 polybasic tail for lipids stabilizes the protein’s localization at the PM in resting conditions (Parvez et al., 2008), thereby allowing STIM2 to quickly respond to discrete ER Ca²⁺ variations and assigning STIM2 to more homeostatic roles.

Alternative splicing of STIM1 produces a longer isoform (STIM1L) with an additional cytosolic actin–binding domain (ABD) that influences the protein’s distribution (Darbellay et al., 2011; Sauc et al., 2015). STIM1L is mainly expressed in the heart, in the brain and in skeletal muscles. This isoform was shown to mediate rapid SOCE activation and, together with TRPC1 and TRPC4, seems to be involved in muscle differentiation and maturation (Antigny et al., 2013;

Antigny et al., 2017). Recent studies also identified a new STIM2 splicing variant with inhibitory properties: STIM2.1 or STIM2β (Miederer et al., 2015; Rana et al., 2015). STIM2.1 is ubiquitously but poorly expressed and its binding to ORAI channels is defective. This isoform travels to the PM by forming heterodimers with STIM1 or STIM2 proteins and exerts a direct allosteric inhibition on the channel. As STIM2.1 affinity for CaM is very high, an additional indirect modulation of channel gating by STIM2.1 via CaM in not excluded. Because STIM1 is

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the most studied isoform, and for simplicity reasons, this manuscript will further refer to STIM1 only.

STIM1 activation and SOCE

In resting conditions, Ca²⁺ ions are trapped in EF hand pockets and STIM1 dimers are kept in a folded “closed” conformation by intra– and intermolecular interactions (Fahrner et al., 2014). A recent study proposed that “sentinel” residues in CC1 prevent STIM1 extension and activation (Hirve et al., 2018). Upon store depletion, Ca²⁺ dissociation from the EF hands induces a luminal rearrangement of STIM1, the pairing of transmembrane domains (TMD) and CC1 regions, the protein elongation, and a subsequent high–order STIM1 oligomerization (Fig.

7) (Stathopulos et al., 2008; Stathopulos et al., 2013).This “open” STIM1 conformation exposes the coiled–coil CC2 and CC3 domains forming the CRAC activation domain (CAD; also referred

Figure 6. Stromal interaction molecules (STIM). Adapted from Bhardwaj et al. (2016).

Schematic outline of human STIM1, STIM1L (A) and STIM2 (B) isoforms, with 685, 791 and 746 amino acids respectively. N-termini are luminal, C-termini are cytosolic. SS: signal sequence, cEF and hEF: canonical and hidden EF-hand domains, SAM: sterile alpha motif, TMD: transmembrane domain, CC: coiled-coil domains 1 to 3, SOAR: STIM-ORAI activating region (referred as CAD in the text), ID: inactivation domain, ABD: actin-binding domain in STIM1L, S/P: serine/proline-rich regions, P/H: proline/histidine-rich region in STIM2, K: lysine-rich domains (with amino acid sequences). Cysteine residues are indicated with yellow circles.

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as STIM–ORAI activation region, SOAR), that will interact with and aggregate ORAI Ca²⁺

channels in clusters at the PM (Wu et al., 2006; Muik et al., 2009; Park et al., 2009; Yuan et al., 2009).

The interaction of ER resident proteins with PM channels requires the close apposition of the ER membrane to the PM (both separated by only 10–20 nm). Following stores depletion, ER remodeling and the organization of ER–PM membrane contact sites (MCS) were indeed described (Orci et al., 2009). Total internal reflection fluorescence (TIRF) microscopy, allowing Figure 7. Store-operated Ca²⁺ entry (SOCE). From Bhardwaj et al. (2016), redrawn from Soboloff et al. (2012).

1) At rest, inactive STIM1 and ORAI1 homodimers are homogenously distributed in ER and PM membranes respectively. Intra- and intermolecular interactions of STIM1 CC domains keep the dimer in a “closed” conformation.

2) The agonist-mediated activation of PLC induces PM PIP2 hydrolysis and results in IP3 production.

3) IP3 further binds to its respective receptor channel IP3R on the ER membrane, prompting Ca²⁺

release from the stores.

4) Ca²⁺ dissociation from STIM1 EF-hands activates STIM1 dimers: the interaction of their luminal EF-SAM domains and their CC domains, leading to the extension of the proteins and the exposure of CAD/SOAR domains and polybasic tails.

5) Activated STIM1 dimers oligomerize and translocate to ER-PM membrane contact sites (MCS), where they bind to PM PIP2.

6) STIM1 further binds and clusters ORAI1 channels.

7) The interaction of STIM1 CAD with ORAI1 induces channel gating and subsequent Ca²⁺ entry into the cytosol.

8) SERCA pumps recruited at ER-PM contact sites allow ER refilling and termination of the process.

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the study of events at close proximity to the PM, showed STIM1 diffusion and formation of puncta co–localizing with ORAI1 clusters (Fig. 8A and B) (Liou et al., 2005; Navarro-Borelly et al., 2008). These findings were further confirmed by electron microscopy and the observation of thin cortical ER (cER) structures facing the PM and enriched in STIM1 (Fig. 8C and D) (Orci et al., 2009). These MCS do not only occur at the PM, but were also described among ER and mitochondria, the Golgi apparatus, endo/lysosomes and phagosomes, and are thought to be major sites for Ca²⁺ signals and lipid transfer (Phillips and Voeltz, 2016).

ORAI isoforms and splicing variant

The Ca²⁺ selective ORAI channels (also known as CRACM – CRAC modulators) are largely present in all eukaryotic cells. Their name comes from Greek mythology, where Orai (or Horae) are the “gatekeepers of heaven”. The family comprises 3 isoforms, ORAI1, ORAI2 and ORAI3, Figure 8. ER-PM membrane contact sites (MCS). From Liou et al. (2005), Navarro-Borelly et al. (2008) and Orci et al. (2009).

(A) Redistribution of YFP-STIM1 into punctae at the PM following store depletion in HeLa cells, and assessed with TIRF. (B) Tg-induced ORAI1-CFP and STIM1-YFP molecular interactions assessed by FRET. (C) Electron micrograph showing the apposition of thin cortical ER (cER) sheets (asterisk) close to the PM (circle) in untransfected HeLa cells treated with Tg. (D) Cryo-immuno electron micrograph of YFP-STIM1 transfected HeLa cells. STIM1, detected with an anti-GFP antibody and gold particles, was enriched in cER (asterisk) facing the PM (circle).

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with high sequence homology but different tissue distribution, electrophysiological features and regulation mechanisms (Gwack et al., 2007; Hoth and Niemeyer, 2013).They are ~ 30 kDa proteins with four transmembrane domains (TM), cytosolic N– and C–termini, and assemble as homo– or heteromers to form a highly selective Ca²⁺ channel (Lis et al., 2007; Schindl et al., 2009). ORAI isoforms mainly differ in their sensitivity to the SOCE inhibitor 2–APB (2–

aminoethoxydiphenyl borate): while ORAI1 and ORAI2 are inhibited by high concentrations of the compound, 2–APB activates ORAI3 in a STIM–independent manner, alters the channel selectivity and abolishes CDI (Schindl et al., 2008; Zhang et al., 2008). ORAI3 and ORAI1 heteromerization forms the ARC (arachidonate–regulated Ca²⁺–selective) channel, related to SOCE and regulated by arachidonic acid (Mignen et al., 2008a). A recent study identified a shorter form of ORAI1 (ORAI1β) resulting from an alternative translation initiation and suggested that, by loosing a phospholipid binding domain, ORAI1β’s diffusion ability is increased (Fukushima et al., 2012). Overall, the ORAI1 isoform is the most studied and best described homolog, and is particularly investigated in the immune system as T lymphocytes, B lymphocytes and natural killer (NK) cells almost exclusively rely on ORAI1 activity (Feske et al., 2006). The thesis focuses on ORAI1, whose structure and properties are further developed in section 3.

Channel gating

ORAI1 gating is triggered by the interaction with the STIM1 CAD domain (Muik et al., 2009;

Park et al., 2009) and endogenous SOCE can be easily assessed with Ca²⁺ imaging experiments.

However, the extremely low unitary conductance of the channel (10–35 fS compared to > 20 pS for voltage–gated Ca²⁺ channels) makes single channel or endogenous Ca²⁺ current recording very difficult in most cell types (Prakriya and Lewis, 2006). In a divalent free solution, the Na⁺ unitary conductance though can reach ~ 1 pS and is sometimes used as an alternative for the evaluation of ORAI1 activity. ORAI1 and STIM1 overexpression increases the current amplitude up to 100 times and whole–cell configuration patch clamp is usually required to follow ORAI1–STIM1 mediated currents in isolated cells (Peinelt et al., 2006). STIM1–ORAI1 stoichiometry is also a strong determinant of channel trapping and gating. A minimum of two STIM1 molecules per ORAI1 subunit seems to be optimal to record maximal SOCE or ICRAC in Ca²⁺ imaging or patch clamp experiments. At high levels of ORAI1 expression, SOCE amplitude and ICRAC density drastically decrease (Hoover and Lewis, 2011). Overexpression of ORAI1 alone without STIM1 co–expression actually abolishes endogenous Ca²⁺ entry in several cell types

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(Soboloff et al., 2006; Cheng et al., 2008). Interestingly, CDI also depends on the STIM1:ORAI1 ratio, with stronger inhibition observed as the ratio increases (Scrimgeour et al., 2009).