ORAI1 gating and biophysical properties

ORAI1 N– and C–termini in channel gating

FRET experiments with STIM1–ORAI1 showed that the deletion of the ORAI1 C–terminus suppressed STIM1–ORAI1 interaction and SOCE (Muik et al., 2008). Further mutational studies confirmed the significance of the ORAI1 C–terminus and identified L273 and L276 as the two critical STIM1 binding residues (Calloway et al., 2010; Tirado-Lee et al., 2015). In addition, the Drosophila Orai1 crystal structure revealed that the channel hexamer is in fact a trimeric organization of dimers with a threefold symmetry, and that the two cytosolic TM4 extensions of a dimer adopt antiparallel orientations (Fig. 11B). Therefore, residues L273 and L276 of one subunit interplay with the equivalent residues of the neighboring subunit. Upon store depletion, hydrophobic interactions between STIM1 and L273/L276 would exert a pulling and rotational force on TM4 (Fig. 11C), followed by a conformational change of the ORAI1 overall structure and the subsequent channel opening. Upstream of L273/L276, the 261–265 LVSHK sequence in TM4 was identified as the ORAI1 “nexus”, a region connecting TM4 to TM3 and potentially involved in the transmission of the STIM1 gating signal from TM4 to the channel body (Zhou et al., 2016).

Several studies suggested that STIM1 also binds the N–terminus: purified peptides of the ORAI1 N–terminal portion 68–91 or 65–87 co–immunoprecipitated with CAD or C–terminal fragments of STIM1 in solution (Mullins et al., 2009; Zhou et al., 2010a). However, whether STIM1 oligomers bind to N–termini of intact ORAI1 channels needs to be confirmed. The FRET signal and clustering of STIM1 with an N–terminal deficient ORAI1 is conserved, but CRAC amplitude and selectivity are decreased, suggesting an important role of the ORAI1 N–

terminus in complex stability or in current modulation (Li et al., 2007; Muik et al., 2008; Lis et al., 2010; McNally et al., 2013). Again, based on the crystal structure of an ORAI1 peptide, CaM was shown to interact with the residues W76 and Y80 of the N–terminus, supporting the hypothesis that CaM mediates CDI (Liu et al., 2012). But the Drosophila model predicted W76 and Y80 to reside in the channel pore, a site far too narrow to be accessed by CaM. A recent publication revealed that truncated or mutated ORAI1 N–termini establish inhibitory interactions with loop II–III, which would be consistent with SOCE/CRAC suppression with ORAI1 N–terminal deletions (Fahrner et al., 2018).

- 34 - ORAI1 pore–lining residues

A vast library of ORAI1 mutants, where each individual amino acid was substituted to a cysteine, allowed the identification of the residues facing the channel pore. Treatment of these mutants with cross–linking reagents revealed that only the TM1 helix is exposed to the pore (McNally et al., 2009), an observation further supported by the Drosophila Orai1 crystal structure. The ORAI1 pore is about 55 Å long, with a variable diameter all along, and is formed by the hexameric arrangement of TM1 and four additional cytosolic helical turns of the N–

terminus (Fig. 12). The pore displays four regions with distinctive properties. The first extracellular negatively charged ring (E106), together with three aspartic acids on loop I–II were shown to confer the typically high Ca²⁺ selectivity to the channel (Prakriya et al., 2006;

Vig et al., 2006; McNally et al., 2009). This glutamic ring is very narrow (~ 6 Å) and might be responsible for the low unitary conductance of ORAI1. The selectivity filter is followed by a hydrophobic portion, whose van der Waals forces provide rigidity to the pore. Mutation of the valine at position 102 into serine, threonine or cysteine amino acids led to the constitutive opening of the channel, assigning V102 as the first ORAI1 gate (McNally et al., 2012). As the mutation R91W found in SCDI patients completely abolished Ca²⁺ influx, R91 was proposed to be the second gate of ORAI1 (Feske et al., 2006). Partially dehydrated Ca²⁺ ions travelling along the pore undergo rehydration in this rigid portion, before reaching a more flexible basic ring nearby the intracellular pore mouth. Crystal soaking experiments showed that these positively charged amino acids are able to bind anions, which is surprising considering that ORAI1 is a cation channel (Hou et al., 2012). This electrostatic barrier was then proposed to prevent Ca²⁺

ions backward reflux.

Figure 12. ORAI1 channel pore. Adapted from Hou et al. (2012).

Side view of the ORAI1 channel pore with TM1 pore-lining residues. The channel pore is ~ 55 Å long, and its diameter varies along the structure.

Drosophila residues are indicated with their human equivalent in parentheses.

E106 was identified as the selectivity filter. V102 and R91 in the hydrophobic and rigid portion were described as the channel gates. The supposed gating hinge (G98) is shown in purple.

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The glycine at position 98 first evoked the presence of a gating hinge, as glycine flexibility is often observed in bending helices (Zhang et al., 2011). STIM1 binding to ORAI1 would then exert a dragging force on the intracellular portion of the helix, inducing the flexion of TM1 around G98 and the dilatation of the R91 ring (Fig. 13A). But the recent publication of Yeung et al. suggested that STIM1 binding induces the rotation of TM1, which displaces the phenylalanine aromatic ring (F99) from the pore interface and facilitates Ca²⁺ ions permeation (Fig. 13B) (Yeung et al., 2017). In addition, the histidine 134 residing in the middle of TM2 and establishing hydrogen bonds with the TM1 S93 and S97 residues seems to be critical in ORAI1 gating (Frischauf et al., 2017). And mutations of the glycine 183 (middle of the TM3) or the proline 245 (middle of the TM4) also affect ORAI1 permeation (Srikanth et al., 2011; Nesin et al., 2014; Palty et al., 2015). Therefore, ORAI1 gating and selectivity might be controlled by both STIM1 binding to its N–terminus, and propagation of a conformational change from its C–terminus through the TM4, TM3, TM2 and finally TM1 helices (Ali et al., 2017).

Figure 13. ORAI1 gating models. Modified from Yeung et al. (2017).

(A) TM1 dilation model: STIM1 binding mediates the widening of the intracellular TM1 extension helix, destabilizing the electrostatic barrier and the steric interactions among pore-lining basic residues (R91, K87, R83). The gating hinge G98 (purple) would then facilitate TM1 bending to allow ion permeation. Drosophila and corresponding human residues (in parentheses) are shown. (B) TM1 rotation model: channel gating is achieved by the rotation of TM1, which moves the aromatic ring of phenylalanine F99 away from the pore interface and allows ion conduction.

- 36 - ORAI1’s essential residues

ORAI1 Ca²⁺ selectivity was intensively studied. E106 is highly conserved among species, and the mutation E106D in hORAI1 displayed constitutive activity, altered selectivity and resistance to GSK inhibition (Vig et al., 2006; Yamashita et al., 2007; Scrimgeour et al., 2012; Derler et al., 2013). Interestingly, mutation of the glutamic acid E190 in TM3, also altered channel selectivity and permeation, suggesting that residues far from the channel pore are involved in ORAI1 gating, probably by modifying intra–TM interactions (Zhou et al., 2010b). STIM1 contribution to ORAI1 selectivity is illustrated by the fact that poorly selective STIM1–free mutated channels such as V102C regain high Ca²⁺ selectivity when gated by STIM1. STIM1 interaction also narrowed the V102C pore size and reversed its constitutive activity (Muik et al., 2012). Actually, WT ORAI1 selectivity increases with overexpression of STIM1, illustrating the role of STIM1 in controlling ORAI1 properties.

While one can predict how mutations of TM1 residues affect the pore geometry, permeation and selectivity, more surprising was the association of synthetic or natural mutations in TM2 to TM4 with ORAI1 dysregulation. Mutation W176C in loop II–III conferred constitutive activity and, as G183A in TM3, conveyed large outward currents (Srikanth et al., 2011). Additionally, the TAM–associated gain–of–function mutations L138F in TM2, T184M in TM3 and P245L in TM4 respectively caused constitutive Ca²⁺ entry, mediated excessive SOCE, and suppressed SCDI (Nesin et al., 2014; Endo et al., 2015; Bohm et al., 2017). How residues in outer rings of the channel affect ORAI1 properties still needs to be elucidated, but the assumption that they destabilize ORAI1’s global structure or displace critical residues is tempting. Therefore, critical residues are not only confined to TM1 but are distributed along TM helices, intra/extracellular loops and N–/C– termini (Fig. 14). ORAI1 holds three reactive cysteines (C126, C142 and C195) described as ROS sensors. H2O2 inhibits WT ORAI1 clustering and Ca²⁺ entry but has no effect on the triple cysteine mutant (substitution by serine).

Molecular dynamic simulations (MDS) and electrophysiology recordings suggested that, in resting conditions, the oxidation of the TM3 C195 prompts interactions with S239 in TM4 that lock the channel in its closed conformation, even after store–depletion and STIM1 binding (Bogeski et al., 2010; Alansary et al., 2016). Additionally, these cysteines, especially C142 at the intracellular surface, are also good candidates for ORAI1 palmitoylation. However, how the attachment of palmitic acids to the channel alters its stability or gating remains to be investigated. As described previously, glycosylation of ORAI1 N223, or phosphorylation of the N–terminal S27 and S30 inhibit SOCE (Dorr et al., 2016). The acidification of intracellular or

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extracellular compartments is sensed by the residue H155 in loop II–III (intracellular) and glutamic/aspartic acids (E106, E190, D110, D112, extracellular) and decreases CRAC density (Beck et al., 2014; Tsujikawa et al., 2015). Consequently, natural mutations occurring in any portion of the ORAI1 channel could possibly disturb one of these biophysical features.