STIM/Orai discovery, and their structure

Two independent studies using either Drosophila S2 cells or Hela cells for RNAi based screening, revealed STIM1 and STIM2 as the ER Ca²⁺ sensors involved in the SOCE pathway (Liou, Kim et al. 2005, Roos, DiGregorio et al. 2005). One year later, 3 other studies based also on genome wide RNAi screen in Drosophila S2 cells identified the channel responsible for the Icrac current associated with SOCE (Feske, Gwack et al. 2006, Vig, Peinelt et al. 2006, Zhang, Yeromin et al. 2006). Feske and co-workers named the 3 mammalian homologous to the Drosophila channel for the keepers of the gates of heaven: Orai1, Orai2 and Orai3.

Additionally, a genome-wide single-nucleotide polymorphism analysis associated a missense mutation (R91W) in Orai1 gene with the hereditary severe combined immune deficiency (SCID) syndrome, in which T lymphocytes were defective for SOCE, confirming Orai1 as key player in this pathway (Feske, Gwack et al. 2006). Finally, overexpression of STIM1 together with Orai1 recapitulating ICRAC current in heterologous system validated definitively that these 2 molecules were necessary and sufficient to mediate SOCE (Peinelt, Vig et al. 2006, Soboloff,

Current Conductance Selectivity Permeability ratio Cell type

ICRAC 0.02 pS; 110 Ca²⁺

Ba²⁺> Ca²⁺≥Sr²⁺ Ca²⁺:Na⁺; 1000:1

Mast cell RBL-1/-2H3 Jurkat T cells Hepatocytes Dendritic cells Megakaryocytes MDCK cells

ISOC

11 pS; 10 Ca²⁺ Ca²⁺>Na⁺ Ca²⁺:Na⁺; >10:1 Endothelia 1-2 pS; 100-160 Ca²⁺ Ba²⁺≥Ca²⁺>>K⁺ Ca²⁺:K⁺; 1000:1 A431 epidermal cells

2.7 pS; 90 Ca²⁺ Ca²⁺=Ba²⁺= Na⁺ Ca²⁺:Na⁺ :K⁺; 1 :1:1 Aortic myocyte 2.3 pS; 1.5 Ca²⁺ Ca²⁺>Na⁺ Ca²⁺:Na⁺; 50:1 Portal vein myocyte

5.4 pS; 20 Ca²⁺ ? ? Pulmonary artery myocytes

0.7 pS; 90 Ca²⁺ ? ? Mesangial cells

43 pS; 1.3 Ca²⁺ K⁺, Na⁺>Ca²⁺ Ca²⁺:Na⁺; 13:1 Pancreatic acinar cells

Spassova et al. 2006). Subsequent publications allowed a deep characterization of STIM and Orai structure and function as well as of the mechanism of interaction between the 2 molecules.

3.1.1. Structure of STIM1 (figure III.5)

STIM1 is a transmembrane (TM) protein with two EF-hand binding site in the luminal part of the protein: a canonical one that binds Ca²⁺ and a hidden EF-hand that stabilizes the canonical binding site. These 2 domains form a stable 3D conformation with a sterile alpha motif (or SAM) that maintains the molecule in a quiescent state (Stathopulos, Zheng et al. 2008). In the cytosolic part of STIM1, 3 coiled-coil domains are juxtaposed. This region is involved in STIM1 higher order oligomers formation upon activation as well as Orai1 gating (Covington, Wu et al. 2010). Several studies have mapped more precisely the STIM1-Orai1 interacting domain and named it OASF (Orai-activating small fragment)(Muik, Frischauf et al. 2008), CAD (CRAC activating domain)(Park, Hoover et al. 2009), SOAR (STIM Orai-activating region)(Yuan, Zeng et al. 2009), and Ccb9 (coiled-coil domain region containing region b9)(Kawasaki, Lange et al.

2009). More precisely, the minimal domain for STIM1 dependent Orai1 gating is the CC2-CC3 region that is folded and maintained in this position through CC1 interaction when STIM1 is in inactive conformation (Muik, Fahrner et al. 2011, Fahrner, Muik et al. 2014). STIM1 possesses also a CRAC modulatory domain (CMD) also called inactivation domain of STIM (IDSTIM) involved in Ca²⁺-dependent inactivation (Derler, Fahrner et al. 2009, Lee, Yuan et al. 2009, Mullins, Park et al. 2009, Muik, Fahrner et al. 2011), a serine/proline domain and a poly-lysine region at the C terminal that participates in STIM1 targeting at the PM (Liou, Kim et al. 2005, Baba, Hayashi et al. 2006, Huang, Zeng et al. 2006, Smyth, Dehaven et al. 2006).

Figure III.5: Functional domains within human STIM1.

Adapted from Muik M., et al. (2012).

From left to right: cEF, canonical EF-hand motif; nEF, hidden EF-hand motif; SAM, sterile alpha motif;

TM, transmembrane domain; CC1/CC2/CC3, coiled-coil domains 1-3; CMD, CRAC modulatory domain;

IDSTIM,inactivation domain of STIM1; S/P, serine/proline-rich region; K or PDB, polybasic cluster. The minimal functional regions within STIM1 are highlighted on the top: CAD, CRAC activating domain;

SOAR, stim activating region; Ccb9 coiled-coil domain region containing region b9; OASF Orai-activating small fragment. Note STIM1L supplementary domain at position 515 aa.

3.1.2. Structure of Orai1 (figure III.6)

Orai1 is composed of 4 transmembrane domains (TM) with the N and C terminus of the protein localized within the cell and implicated in Orai1-STIM1 interaction. The N terminus segment together with TM1 constitute the pore of the channel with the E106 residue being the selectivity filter (Vig, Peinelt et al. 2006, Gwack, Srikanth et al. 2007, McNally, Yamashita et al. 2009) and G98/R91 the gate of the pore (Zhang, Yeromin et al. 2011) (see figure III.6).

The channel itself is comprised of several Orai1 subunits of which TM1 faces the pore while TM2-4 constitute the outer part of the channel. The number of Orai1 molecules necessary to form a functional channel is still a matter of debate as some studies favors tetrameric configuration while crystallography of the Drosophila Orai1 channel revealed a hexameric structure in active state (Hou, Pedi et al. 2012). By recording ICRAC current in cells over-expressing preassembled tandem Orai1 multimers of 2, 3 or 4 subunits together with a dominant negative, Mignen’s study suggests tetrameric conformation as the functional channel (Mignen, Thompson et al. 2008). Others studies based on single-molecule imaging of 2 or 4 Orai1 subunits assembled in concatemers followed by counting of photo-bleaching step necessary to extinguish concatemers fluorescence leads to comparable conclusion (Ji, Xu et al. 2008, Penna, Demuro et al. 2008). However, regarding the high amino acid sequence

homology between mammalian and Drosophila channel, it is likely that mammalian Orai1 channel would be also formed by 6 Orai1 subunits. Tetrameric configuration found in concatemer-based studies would result of rearrangement of tetrameric concatemeres into hexamers in the PM or artefacts as photo-bleaching imaging is not always precise due to fluorophore instability that can easily leads to under-estimation.

Figure III.6: Functional organization of Orai1.

Adapted from Prakriya M., et al. (2015).

The topology of Orai1 is illustrated, with selected functional domains noted by colored bars or circles.

The thick yellow bars in the NH2 and COOH termini depict putative STIM1 binding sites on Orai1. Yellow and red lines show the locations of mutations causing gain-of-function (i.e., STIM1-independent Orai1 activation) or loss-of-function phenotypes, respectively. Black lines indicate mutations affecting ion selectivity. Intracellular residues important for CDI are marked in purple (NH2 terminus and II–III loop) and selected residues important for STIM binding and gating are marked in red (NH2 and COOH termini).

3.2. Sequential events from store depletion to Ca²⁺ entry