2.1. Introduction

Among all known second messengers in eukaryotic cells, Ca²⁺ is one of the most versatile and is involved in a multitude of physiological and cellular processes including cell proliferation, growth, gene expression, muscle contraction, and exocytosis/secretion [1,2]. To act as an intracellular signal molecule, Ca²⁺ has to enter the cell at specific physiological/cellular situations and time points. One major pathway that allows Ca²⁺ entry into the cells involves the Ca²⁺ release-activated Ca²⁺ (CRAC) channels, which belong to the group of store-operated channels (SOC) [3–14]. In the beginning of the CRAC/SOC channel analysis, these channels were studied and characterized using mainly cells of the immune system, that is, T-lymphocytes and mast cells [9,10,14,15]. Finally, in 2005–2006, the major key players forming the functional CRAC channel complex were identified [16–27]: first, the stromal interaction molecule (STIM), which represents the Ca²⁺ sensor in the endoplasmic reticulum (ER), and second, Orai, which is located in the plasma membrane (PM) and builds the ion-conducting transmembrane (TM) protein complex. Feske and colleagues [16] had studied a defect in CRAC channel function linked to one form of hereditary severe combined immune deficiency (SCID) syndrome, which allowed the identification of the Orai1 (also initially termed CRACM1) channel protein and its mutated form (Orai1 R91W) in SCID patients. By successfully employing and combining a modified linkage analysis with single-nucleotide polymorphism arrays and a Drosophila RNA interference screen, light was shed on the gene and protein that forms the Ca²⁺ conducting CRAC channel [16]. Furthermore, the search for homologous proteins using a sequence database research revealed Orai1, Orai2, and Orai3 in higher vertebrates. The three members of the Orai protein family have been analyzed with bioinformatics methods showing that they represent TM proteins with 4 PM spanning domains connected by one intracellular and two extracellular loops and cytosolic N-and C-termini [16,20,28,29]. Several research groups have concentrated on the electrophysiological examination and characterization of Orai proteins revealing the typical high Ca²⁺ selectivity and low single-channel conductance, concluding that these proteins unequivocally represent the pore-forming entity of the CRAC channel.

The CRAC channel-activating protein—stromal interaction molecule (STIM)—has been presented and published by Liou et al. as well as Roos et al. in 2005 [18,19]. Screening about 2300 signaling proteins in Drosophila S2 cells and HeLa cells using an RNA interference-based gene knockdown approach, 2 homologous proteins highly involved in ER store depletion-mediated Ca²⁺ influx were elucidated—STIM1 and STIM2. These proteins serve as ER-resident Ca²⁺ sensors, which closely communicate with the CRAC channels upon Ca²⁺ depletion of the ER [18,19]. Both STIM1 and STIM2 are single-pass TM proteins with the N-terminus in the ER lumen and the larger C-terminal part facing the cytosol. The ER luminal part, which functions as a Ca²⁺ sensor of [Ca²⁺]_ER, contains, among other parts, a Ca²⁺-sensing EF hand followed by the α-helical TM domain. The larger cytosolic part of STIM is responsible for coupling to and activation of Orai channels [6,30–33]. Confocal microscopy images reveal an intracellular tubular distribution of STIM1 under resting conditions with full ER Ca²⁺ stores; however, a small percentage of STIM1 has also been detected in the PM [34]. Lowering the ER-intraluminal Ca²⁺ concentration represents the initial trigger for STIM1 activation. In the course of store depletion, Ca²⁺ is released from the STIM1 EF hand domain followed by STIM1 homomerization and translocation to the cell periphery into the so-called ER-PM junctions—regions where the ER membrane is in tight proximity to the plasma membrane. Low [Ca²⁺]_ER finally leads to the formation of oligomeric STIM1 clusters/punctae in these microdomains where Orai channels localize as well. This physical coupling of STIM1 to Orai channels therefore induces Ca²⁺ influx linked to specific downstream signaling and ER store refilling [30,35–39]. Besides the activation of CRAC channels, STIM1 has been shown to play a role in arachidonate as well as leukotriene C4-stimulated Ca²⁺ channels (see Chapter 11) as well as TRP channel regulation [40].

After the initial characterization of STIM and Orai with limited structural knowledge based on bioinformatics predictions, in 2012, the crystal structures of cytosolic fragments of STIM1 and full-length Orai were reported, allowing new and more focused studies of STIM1 and Orai related to their intra- and intermolecular interactions [41].

2.2. STIM1

In resting cells, ER membrane-resident STIM1 reveals a dynamic constitutive movement along microtubules, whereas store depletion results in the redistribution of STIM1 into clusters/punctae at ER-PM junctions [42,43]. Dissection of STIM1 for examination of specific ER luminal as well as cytosolic STIM1 domains has shown that cytosolic coiled coil (CC) domains and the S/P domain are involved in the constitutive movement, whereas the luminal portion, in combination with cytosolic domains, is essential for STIM1 oligomerization and cluster formation upon store depletion [43–45]. Furthermore, STIM1 directly binds to EB1, a microtubule plus end tracking molecule. Both STIM1 and EB1 are required for tip attachment complex-mediated ER tubule extension where an ER tubule elongates with a growing microtubule [46,47]. The trigger preceding cytosolic CC interactions resulting in STIM1 oligomerization has been localized to the ER luminal STIM1 part as its substitution using an artificial luminal cross-linker clearly replaced the store depletion-dependent activation mechanism of STIM1 [48]. In conclusion, the initial publications on the activation of STIM1 showed that the initial step is induced by ER store depletion resulting in STIM1 oligomerization on its luminal side consequently leading to oligomerization on its cytosolic side [18,19,27,30,38,43,49–51]. The STIM1 luminal part contains a canonical and a hidden EF hand followed by a sterile-α motif (SAM) [44,45,52] (Figure 2.1a). The EF hand structurally represents a helix-loop-helix motif with negatively charged residues (Asp and Glu) binding Ca²⁺ (K_d ∼ 200–600 μM) in the high [Ca²⁺]_ER conditions. ER store depletion (lowering [Ca²⁺]_ER) results in Ca²⁺ dissociation from the STIM1 EF hand, therefore destabilizing the entire EF-SAM entity [44]. CD-spectroscopy measurements of that crucial domain performed by Stathopulos and colleagues revealed that EF-SAM with bound Ca²⁺ results in high α-helicity in contrast to the apo-EF-SAM (without Ca²⁺). Furthermore, the apo-EF-SAM domain proved to exist in at least dimeric form, whereas in the presence of Ca²⁺ the monomeric form is predominant [44,45]. An additional study by Covington and colleagues [53] using Förster resonance energy transfer (FRET) has shown that a STIM1 deletion mutant containing only the luminal part and the TM domain responds with FRET increase that depends on store depletion. Structurally, STIM2 is very similar to STIM1; however, comparisons of the EF-SAM domains in their ER luminal part reveal different degrees of structural stability explaining the observation that STIM2 reacts faster upon smaller decreases in [Ca²⁺]_ER [54–56]. Following the EF-SAM domain, a TM helix spans the ER membrane (Figure 2.1a). Ma et al. [57] were the first to identify a gain of function mutation in STIM1 TM. Screening the whole TM domain, the mutant STIM1 C227W exhibits the highest potency for eliciting constitutive Ca²⁺ influx. The function of C227W is to uncouple distinct activation steps, allowing the examination of STIM1 TM structural states without manipulating luminal or cytosolic functional components. Based on FRET and cross-linking experiments, it was concluded that C227W allows the close proximity of the N-terminal part of the TM domains, mimicking the store-depleted state with oligomerized EF-SAM domains. Furthermore, FRET analysis revealed a decreased CC1-SOAR (STIM-Orai activating region) interaction in the C227W mutant similar to store depletion-induced STIM1 activation [57] (Figure 2.2). By combining biochemical and bioinformatics approaches they further present a structural change of STIM1 TM domain dependent on the activation state. In their model, STIM1 TM dimers are not parallel but cross at a specific position, whereas the crossing angle is big enough to explain the separation of the luminal portions in a high ER Ca²⁺ condition. In contrast, store depletion results in a reduction of the crossing angle allowing the dimerization/oligomerization of the luminal STIM1 EF-SAM domains and consequent change of the relative positions of STIM1 cytosolic parts (Figure 2.2). Additionally, the TM domain contains three glycines (223, 225, 226) that yield high flexibility and are potentially involved in bend/kink formation representing a structural change in the course of signal transmission from the luminal to the cytosolic STIM1 parts [57,58].

Figure 2.1

Schematic depicting human STIM1 specifying essential structural/functional regions. Magnification of the NMR and crystal structures of the EF-SAM domain, the STIM1 CC1α3-CC2 fragment (312–387), and the STIM1 SOAR (344–444) fragment (more...)

Figure 2.2

Cartoon of a hypothetical model depicting STIM1 conformational changes in the process of STIM1 activation and coupling to Orai upon store depletion. (a) STIM1 quiescent state is accomplished by STIM1 CC1/CC3 interaction in the presence of full ER Ca²⁺ (more...)

The cytosolic portion following the TM domain includes three CC regions (CC1, CC2, and CC3), a CRAC modulatory domain (CMD), and a serine/proline- and a lysine-rich region (Figure 2.1a). Early studies by Huang et al. and Muik et al. demonstrated that the cytosolic expression of the STIM1 C-terminus is able to activate Orai1 [30,59]. As the STIM1 C-terminus (aa233-685) represents a large molecule, the goal has been laid on the identification of the STIM1 region representing the minimal fragment required to activate Orai channels. In a very narrow time frame, several Orai-activating STIM1 fragments were identified and published: (CRAC activation domain) CAD (aa342-448), SOAR (aa344-442), (Orai activating small fragment) OASF (aa233-450), and Ccb9 (aa339-444) [39,60–62] (Figure 2.1). All these Orai1-activating fragments contain CC2 (aa363-389), CC3 (aa399-423), and the STIM1 homomerization domain SHD (∼aa421-450) for correct oligomerization and coupling to and activation of Orai channels [60]. At that time, no detailed knowledge about the structures of small Orai-activating fragments and only large segments within SOAR were suggested to form CC domains. It took 3 more years until Yang et al. presented the crystal structure of hSOAR (345–444_{L374M, V419A, C437T}) [63] (Figure 2.1b). They reported a dimeric assembly of the SOAR molecules with multiple intra- and intermolecular interactions. However, it is still not clear which activation state this structure represents. The monomeric SOAR consists of an antiparallel arrangement of CC2 and CC3 with two short α-helices linking these domains, resembling the capital letter “R” (Figure 2.1b). These regions are named Sα1 (345–391), Sα2 (393–398), Sα3 (400–403), and Sα4 (409–437). The dimeric SOAR arrangement represents an overall V-shape with N-terminal amino acids (T354, L351, W350, L347) from an SOAR monomer interacting with C-terminal residues (R429, W430, I433, L436) of the other monomer, respectively. Furthermore, the dimer contains a crossing point at position Y361 of both CC2 domains and a cluster of positively charged amino acids (K382, K384, K385, K386, R387) located on either tip of the V-shaped dimeric SOAR structure [63] (Figure 2.1c).

The STIM1 CC1 (aa238-343) domain, which is located between the TM domain and SOAR, includes three α-helical segments (CC1α1 aa238-271, CC1α2 aa278-304, and CC1α3 aa308-337) [32,64,65] and has been demonstrated to play a key role in keeping STIM1 in a locked tight inactive conformation when ER stores are full [57,63,65–67]. The decrease of [Ca²⁺]_ER, which leads to luminal di-/oligomerization followed by closer proximity of the TM domains, triggers a conformational change in the cytosolic strand where CC1 adopts an extended conformation, thereby releasing SOAR [57,65,67] (Figure 2.2). Using FRET, Covington et al. examined the homomerization potential of different STIM1 C-terminal truncation mutants (i.e., STIM1-CC1 [aa1-344] and STIM1-CC1-CAD [aa1-448]), concluding that CC1 and CC3/SHD are both involved in homomerization [53]. However, further studies examining the role of CC1 have made clear that CC1 is a key domain in the STIM1 activation cascade integrating additional functions. One of the first hypothetical models of the control mechanism of CC1 has been proposed by Korzeniowski et al. [68]. They suggested a quiescent STIM1 state resulting from an autoinhibitory clamp based on attractive electrostatic interactions between a highly negatively charged segment within CC1α3 (aa308-337) and a positively charged region (aa382-387) within CC2 [68]. However, this hypothesis is not in line with the crystal structure of CC1-SOAR (from Caenorhabditis elegans) as the acidic region within CC1α3 (also called “inhibitory helix”) is too distant from the basic region at the tip of the structure to form the proposed electrostatic clamp [63]. Based on their crystal structure, Yang et al. describe intramolecular interactions involving residues of CC1α3 and residues at the beginning of CC2 as well as the end of CC3 in the SOAR dimer. This dimerized conformation is suggested to represent the inactive, resting state where SOAR is occluded by the inhibitory helix [63]. Another study focusing on the CC1α3 describes its role involving intramolecular shielding leading to the STIM1 quiescent state, however, without the contribution of electrostatic or CC interactions, as deletions or substitutions within this segment revealed no clear effect [69]. Further examination of CC1α3 by multiple substitutions affecting its amphipathic character has led to the conclusion that the amphipathic nature of this segment has an impact on the regulation of STIM1 activation [69]. Another FRET-based approach developed by Romanin’s group has allowed the monitoring of conformational changes within the cytosolic STIM1 region 233-474 (OASF) [66]. For this purpose, the OASF has been double labeled with yellow fluorescent protein (YFP) at the N-terminus and cyan fluorescent protein (CFP) at the C-terminus revealing different intramolecular FRET values depending on the OASF conformation. The wild-type OASF conformational sensor results in a robust FRET, suggesting a tightly packed structure representing the quiescent state of STIM1 when Ca²⁺ stores are full [66]. Another study performed by Hogan’s group reported a tight conformation of the STIM1 C-terminus (233-685) based on Tb³⁺-acceptor energy transfer measurements [67]. The obvious question arising is: what are the molecular steps that guide the cytosolic strand of STIM1 from the quiescent into the active form? To solve this task, several mutations as well as artificial cross-linking experiments have been performed. Point mutations within the OASF sensor with a high impact on the CC probability of CC1α1 (L251S) and CC3 (L416S L423S) result in decreased intramolecular FRET values consistent with an extended conformation representing the activated STIM1 state [66]. Introducing the same point mutations in full-length STIM1 and coexpressing these mutants with Orai1 in HEK cells has revealed constitutive inward Ca²⁺ currents independent of [Ca²⁺]_ER. These results suggest that intramolecular CC1-CC3 interactions within STIM1 lock the quiescent state that is released upon store depletion or point mutations affecting the CC formation [65,66]. In an alternative approach, Zhou et al. succeeded in extending STIM1-CT by cross-linking of the CC1 domains. They also showed the “activating” mutation L251S resulting in a conformational extension of STIM1-CT in line with the OASF conformational sensor [67]. In summary, these results point to the high impact of CC1α1 as a key structure in the transition of STIM1 from the tight quiescent state to the extended active state [65–67]. The tight state has been suggested to involve an interaction between CC1 and CC3, whereas the active state via CC1 homomerization releases the CC1-CC3 clamp, leading to extended conformation.

Another FRET-based approach called “FIRE” (FRET Interaction in a Restricted Environment) was developed by Romanin’s group to further dissect the STIM1 activation mechanism [65]. In that study, the role of CC1 in controlling the different cytosolic STIM1 activation states has been examined. The FIRE system has been engineered to mimic ER-targeted two-dimensional localization rather than using cytosolic expression. It consists of cytosolic STIM1 domains of interest linked via a flexible, 32 glycine linker to the ER STIM1 TM helix that is attached to a fluorescent protein (YFP or CFP) on the ER luminal side. The use of the FIRE system is accompanied by the advantages of a two-dimensional system, that is, low-affinity interactions of the protein domains of interest “survive” with higher probability in contrast to their cytosolic expression with three-dimensional degrees of freedom. Furthermore, FIRE allows the use of small fragments without the concern of steric hindrance as the fluorophores are attached on the ER luminal side. In case of an interaction of the peptides on the cytosolic side, the attached fluorophores on the ER luminal side achieve relatively close proximity, which is detected by FRET. In the study of Fahrner et al., using STIM1 fragments, the direct CC1α1-CC3 interaction has been established as a molecular determinant maintaining the quiescent STIM1 state. In line with the extended state, introduction of the activating L251S substitution in CC1α1 disrupted the interaction with CC3. CC1α3 is also involved in locking the inactive STIM1 state but not in a predominant manner as shown with deletion mutants [63,65]. Another result by this study has revealed a destabilizing role of CC1α2 that may be related to the Stormorken syndrome-associated mutant STIM1 R304W [65,70–72]. Possibly, this point mutation enhances the destabilizing force induced by CC1α2, leading to an extended and activated STIM1 that is able to couple to and activate Orai independent of [Ca²⁺]_ER.

Based on all recent functional and structural STIM1 data, a hypothetical STIM1 activation model has been designed, including distinct STIM1 conformations reflecting the different steps of STIM1 activation upon store depletion [65] (Figure 2.2). In the STIM1 activation model, the inactive state is represented by a dimer where the intramolecular CC1α1 and CC1α3 interact with CC2/CC3, forming a clamp keeping STIM1 quiescent and tightly packed. Ca²⁺ depletion from the ER is the first step affecting the luminal STIM1 conformation [57,65]. Decreased [Ca²⁺]_ER results in luminal EF-SAM domain destabilization, which represents a signal that is transmitted via the TM domain to the cytosolic portion of STIM1 [45,52,57]. Ultimately, the CC1-CC3 interactions are released most likely accompanied by an increased CC1 homomerization and a conformational rearrangement of CC2 and CC3 (CAD/SOAR) resulting in the formation of STIM-Orai association pocket (SOAP) and CC3 oligomerization and clustering [57,65–67,73]. The current model does not directly involve CC1α2; however, the Stormorken syndrome-relevant STIM1 R304W point mutation [70–72], which is at the very end of CC1α2, induces the extended conformation consistent with constitutive Orai1 activation (M. Fahrner et al., unpublished data). How exactly this substitution is able to induce the extended STIM1 structure is still under examination.

2.3. Orai

The Orai family consists of three homologous proteins named Orai1, Orai2, and Orai3, which reside in the PM and represent Ca²⁺-selective ion channels allowing Ca²⁺ influx upon stimulation [16,20,74,75]. Each Orai molecule contains a cytosolic N- and C-terminus and four TM helices that are connected via two loops on the extracellular and one loop on the intracellular side [40] (Figure 2.3a). TM1 has the highest sequence similarity between the three Orai proteins, whereas the non-TM domains are less conserved. Orai1 and Orai3 share 34% sequence identity in the N-terminus and 46% in the C-terminus [76,77]. Another obvious difference applies to the third loop, which is much longer in Orai3. It has been shown by several groups that both the Orai C-terminus and the N-terminus are involved in functional coupling to STIM1. Upon activation by STIM1, Orai channels open, giving rise to inwardly rectifying Ca²⁺currents with a low single-channel conductance [30,39,78–81].

Figure 2.3

Orai1:

The stoichiometry of Orai proteins forming a functional channel has been extensively investigated, suggesting that four Orai1 monomers constitute the Ca²⁺-selective CRAC channel [4,76,82–86]. A heteromeric pentameric assembly, consisting of three Orai1 and two Orai3 subunits, has also been proposed for a Ca²⁺-selective channel that is activated via arachidonic acid [87]. However, in 2012, dOrai (from Drosophila melanogaster) was crystallized, revealing an unexpected hexameric arrangement of the Orai subunits forming the functional channel [41]. In this architecture, three Orai dimers with crossing C-termini are combined to end in a quaternary structure with six Orai molecules per channel (Figure 2.3b). The ion-conducting pore is located right in the middle and is mainly created by TM1 of the six Orai subunits forming the first ring (Figure 2.3c). TM2 and TM3 form a ring surrounding TM1 and therefore separating TM1 from the PM lipid environment. TM4 contributes the third ring that is in tight contact with the components of the PM [41]. The cytosolic TM1 proximal N-terminal part of Orai represents the “Extended Transmembrane Orai1 N-terminal” region (ETON), which is an α-helical extension of TM1 reaching about 20 Å into the cytosol [78]. The helices of TM2 and TM3 expand into the cytosol too, to a smaller extent, and are connected via the intracellular loop 2, which is unfortunately not represented in the x-ray structure, as is the case for the two extracellularly located loops (loop 1 and loop 3), which are also missing in the structure [41]. Molecular modeling and molecular dynamics simulations have been performed for the missing parts of the crystal structure, suggesting a high flexibility of loop 3 [88]. The cytosolic extensions of TM4 representing the C-termini reveal two different kink angles in a highly conserved hinge region leading to an antiparallel dimeric interaction of the C-termini of two adjacent Orai molecules in the hexamer [41,73].

2.4. STIM1: Orai Interaction

ER store depletion results in the activation of STIM1 that unfolds into an extended conformation, homo-oligomerizes forming clusters, translocates to the cell periphery, and eventually couples to Orai, thereby opening this channel [40]. The small Orai-activating fragment CAD has been shown to directly bind to the Orai1 C-terminus, which seems to be the major interaction site between these two proteins [39,61]. CAD-Orai1 N-terminus interaction was determined as well, whereas the interaction with Orai1 loop 2 has not been detected [39]. NMR structure of a STIM1 fragment (312–387) including CC1α3 and CC2 has been presented with and without an Orai1 C-terminal fragment (272–292), respectively, which for the first time has revealed a potential STIM1-Orai1 C-terminus interaction on a structural basis [73]. Here, two STIM1 fragments form an antiparallel dimeric structure that interacts with two antiparallel Orai1 C-terminal fragments in line with the Orai C-terminal conformation presented in the hexameric dOrai crystal structure [41,73]. The NMR structure of SOAP reveals the residues that are in close proximity to amino acids of the Orai1 C-terminus. With respect to STIM1, the side chains of L347, L351, Y362, L373, and A376 are involved in hydrophobic interactions with Orai1 C-terminal residues. Another part of STIM1 comprising the positively charged residues K382, K384, K385, and K386 forms a highly charged region providing electrostatic complementarity to an acidic portion within the C-terminus. With respect to Orai1, on one hand, hydrophobic residues including L273 and L276 are involved in hydrophobic interactions, and on the other, negatively charged side chains of Orai1 (D284, D287, and D291) form electrostatic interactions with complementary STIM1 regions within SOAP, respectively. Point mutations of these hydrophobic as well as charged residues on either STIM1 or Orai1 have been reported to functionally disrupt the STIM1-Orai1 communication, however to a different extent, which is in agreement with the NMR structure [73]. Therefore, the STIM1-Orai1 C-terminal coupling is well described on a functional and structural level; however, the interaction between STIM1 and Orai1 N-terminus is only marginally defined. Possibly, F394 in STIM1 plays a critical role in binding to a hydrophobic counterpart of the ETON region as the point mutation F394H abolishes the activation of Orai1 [89].

Structurally, the Orai1 C-terminus adopts two different orientations in the crystal structure depending on the angles of the hinge region (aa261-265), which represents the link between TM4 and the cytosolic C-terminal part of Orai. With respect to an Orai dimer, the two C-termini cross each other at an angle of 152° [41]. The probability of forming CC for the Orai1 C-terminus is relatively weak, whereas for Orai2 and Orai3 it is about 17-fold higher [90]. Examination of strategically important positions of the CC domain (the hydrophobic “a” and “d” positions) of Orai1 using single point mutations (L273S and L276S) to decrease the CC probability, interferes with coupling to STIM1 [30,90]. Indeed, the NMR structure presenting SOAP as the STIM1-Orai1 binding domain is consistent with the previous functional experiments as positions L273 and L276 are engaged in hydrophobic interactions with STIM1 counterparts in SOAP [73]. Concerning Orai2 and Orai3, single point mutations do not reveal the same dominant effect, which may be explained by their higher C-terminal CC probabilities. Therefore, double mutations are required to robustly decrease the CC probability, resulting in full disruption of the communication with STIM1 [90]. Orai1 R281, L286, and R289 represent additional important residues in the Orai1 C-terminus contributing to SOAP. In accordance to the structural data, adjacent residues not involved in SOAP had no significant impact on channel activation [73]. The Orai1 C-terminal positions L273 and L276 are worth mentioning, as they are not solely involved in STIM1 binding within SOAP, but they also represent the crossing point of two antiparallel C-termini of each Orai dimer within the hexameric Orai. Substituting L273 and L276 in Orai1 with cysteines resulted in disulfide bond formation under oxidizing conditions in cross-linking experiments fully abolishing STIM1 binding. The addition of reducing agents breaking the disulfide bonds rescued STIM1 coupling [80,91]. The hinge region in Orai1 connecting TM4 and Orai C-terminus has a key role in defining the correct orientation and conformation of the C-terminus. As expected, mutations within the hinge region affect STIM1 coupling and Orai channel activation; however, to fully disrupt STIM1-Orai1 communication, additional Orai1 C-terminal mutations were necessary [80]. In summary, the hinge region is relevant for determining the optimal position of the C-termini for efficient coupling to STIM1, but the direct coupling of STIM1 to Orai C-terminal downstream regions is essential.

How do the crossing Orai C-termini reported in the crystal structure fit in with the NMR structure of SOAP? A closer look at the SOAP structure reveals antiparallel Orai C-termini with an angle of 136° [73] in contrast to 152° measured in the crystal structure without STIM1 [41]. Possibly, the different angles arise from the different origins of Orai as dOrai was crystallized for the x-ray structure determination, whereas the NMR structure is based on the human Orai1. However, it has to be pointed out that the two structures also represent different activation states of Orai1. The crystal structure corresponds to the closed Orai state without STIM1, whereas the NMR SOAP structure is derived from a complex of STIM1/Orai1 C-termini. Therefore, the observed difference of angles could hypothetically reflect the activation state, that is, Orai1 C-termini undergo a mild angle modification upon coupling to STIM1, resulting in a conformational shift.

Biochemically an Orai1 N-terminus-STIM1 interaction is detectable too, but to a weaker extent [39,78]. As the ETON region (Orai1 aa73-90) represents the elongated TM1 and incorporates relevant binding residues, Orai1 N-terminal deletion mutants reveal different effects depending on the size of truncation. Full channel activation is observed in case of Orai1 Δ1-72, whereas Δ1-74 results in only 50% activation and Δ1-76 abolishes current activation completely [78]. Several studies analyzing the ETON region within full-length Orai1 have revealed an inhibition of coupling to STIM1 as well as channel activation by substituting various residues in ETON [78,89,92]. The positively charged side chains within ETON seemingly have various functions, that is, interaction with STIM1, stabilization of the elongated pore, and an electrostatic barrier to cations [78,93]. Although Orai1, Orai2, and Orai3 all contain the conserved ETON region, equivalent N-terminal truncations have revealed different effects in the three Orai homologs. Strikingly, Orai3 store-operated activation is still preserved even with extensive truncations that fully abolish Orai1 function [78]. Probably, additional structures present in Orai3 are able to compensate for the large N-terminal deletions; however, their position still remains elusive. In summary, residues within ETON most likely are engaged in communicating with STIM1; however, a structural view of this interaction is still missing.

Based on structural and functional data, the STIM1-Orai1 interaction is predominantly achieved by the binding of STIM1 C-terminus to Orai1 C-terminus as well as N-terminus. The presence of both cytosolic Orai strands is absolutely necessary for channel activation by STIM1 as deletions or mutations of either Orai N- or C-terminus result in fully nonfunctional channels [78–80,94]. However, the C-terminus seems to have a superior role in this coupling, as a deletion/mutation of the Orai C-terminal binding sites completely inhibits the interaction with STIM1 or STIM1 C-terminal fragments, whereas Orai N-terminal deletions or mutations lead to nonfunctional channels but still allow partial coupling to STIM1 [30,31]. Structural and biochemical evidence point to the fact that the Orai C-terminus is indeed the primary STIM1 coupling site and the Orai N-terminus most likely is involved in gating [30,93,95]. In addition to STIM1, calmodulin and cholesterol have been reported to regulate Orai1 via its N-terminus [96–99]. In summary, both N- and C-terminal cytosolic Orai strands are necessary for Orai channel activation; however, the detailed molecular choreography of STIM1-Orai coupling and activation still remains a mystery. A commonly accepted hypothetical picture includes as the first step STIM1 coupling to the Orai C-terminus consequently attaching STIM1 to Orai, thereby allowing the next interaction involving the Orai N-terminus for channel gating. This finally results in a bridging of Orai1 N- and C-termini by the CAD/SOAR domain of STIM1. In addition to the hypothetical interaction sequence, the Orai C-terminus could not only be responsible for STIM1 coupling but also for discrete conformational changes propagating through the Orai TM domains via rearrangements of TM4 upon interaction with STIM1; however, this is still speculative.

To shed light on the interaction of Orai1 N- and C-termini with STIM1 upon store depletion, various Orai1 proteins with CAD fragments linked to the cytosolic Orai1 N-terminal and C-terminal strands, respectively, have been explored [79,80,94]. The direct linkage results in a local enrichment of STIM1-CAD close to Orai1. Combining the Orai1-CAD fusion with N-terminal (Δ1-76) and C-terminal (Δ276-301) loss of function deletions of Orai1, respectively, yields slight compensation resulting in partial channel activation. However, a tethered CAD cannot compensate for more severe deletions pointing to the fact that the presence of Orai1 residues 77–90 (N-terminus) and 267–275 (C-terminus) is necessary for preserved activation via tethered CAD. On the other hand, single loss of function point mutations in either Orai1 N- or C-terminus lose their dominant effect in the presence of attached CAD [80,94]. Obviously, the local CAD enrichment results in a higher likelihood of coupling to and activation of Orai1 channels even in the presence of point mutations that otherwise lead to a loss of function. To fully destroy the CAD-Orai1 interaction using the Orai1-CAD fusion protein, mutations in both Orai1 N- and C-termini are necessary, emphasizing that both Orai1 cytosolic strands contribute to the interaction with the STIM1 C-terminus. Hence, an alternative to the sequential STIM1-Orai coupling model has been proposed by Palty et al. proposing the formation of a distinct STIM1 binding pocket by Orai1 N- and C-terminal sites [80,94].

The NMR 3D structure representing a dimerized STIM1 fragment (312–387) with two Orai1 C-termini (272–292) shows a clear picture of the interacting residues within the SOAP. Based on this holo-structure, point mutations have been introduced in STIM1 as well as Orai1 for functional electrophysiological analysis revealing strong agreement with the presented structural data [73]. It is important to note, however, that full-length Orai1 involves both C-terminus and N-terminus for functional coupling and activation [30,31,39,78], whereas the NMR holo-structure only contains a C-terminal fragment of Orai1. Furthermore, the STIM1 fragment lacks residues 388–442, which represent an important domain within SOAR [61]. To gain more evidence for the physiological STIM1-Orai1 interaction, a structure containing a larger STIM1 fragment together with Orai1 cytosolic strands or even the full-length Orai1 channel complex would be of high benefit. Moreover, the cytosolic loop2 of Orai could play a cooperative role in CRAC activation too, however this possibility needs to be specifically examined and analyzed.

A comparison of the SOAR crystal and the NMR STIM1 312–387 reveals different tertiary and quaternary structures probably reflecting different conformational activation states of the protein. Both share only a small overlapping part (residues 344–387) containing the crossing pivotal point around Y361; however, the angles between CC2 and CC2′ in the dimer are different in the two structures. The NMR structure of STIM1 fragments within SOAP fits well with the antiparallel crossing C-termini of Orai1 suggesting a conformation close to the STIM1-activated state, whereas the SOAR crystal possibly reflects a structure close to the STIM1-inactivated tight state [63,73].

The NMR resolution of the STIM1 312–387 + Orai1 272–292 structures allows the interpretation that one STIM1 dimer interacts with one Orai1 dimer during the coupling and activation process. However, several studies have been performed analyzing the stoichiometry of the oligomeric STIM1-Orai1 complex revealing divergent results. Electrophysiological experiments suggest that the extent of Orai1 activation depends on the number of STIM1 molecules present [100–102]. Furthermore, the CRAC current inactivation depends on the STIM1 protein number, as less STIM1 proteins interacting with Orai1 result in less CRAC channel inactivation [103]. Various approaches have been performed to elucidate the STIM1-Orai1 stoichiometry, including varying STIM1/Orai1 ratios or direct fusion proteins of Orai1 connected to CAD dimers. Results point to the fact that eight STIM1 molecules per four Orai1 subunits lead to maximal CRAC channel activation and inactivation [100,101]. Analyses of Orai1 and constitutively active Orai1 V102A mutants connected to single or tandem CAD molecules result in increased Ca²⁺ selectivity with an increased number of STIM1 fragments, showing that CRAC channel activation is not an “all or none” phenomenon but rather a gradual process depending on the number of Orai1 subunits in a tetramer with an undisturbed C-terminal STIM1 binding site [101]. In the past, most studies pointed to an Orai1 tetramer forming the CRAC channel activated by eight STIM1 molecules ending in a 2:1 STIM1:Orai1 ratio. However, the crystallized dOrai structure reveals a hexameric assembly and it is difficult to explain how eight STIM1 proteins interact with six Orai subunits. Based on the NMR structure of interacting dimeric STIM1 fragments with two Orai1 C-terminal fragments a STIM1:Orai1 ratio of 1:1 is most likely present in the active complex [73]. Connecting this view to the hexameric Orai channel suggests an interaction of six STIM1 proteins with six Orai1 subunits displaying a picture where three STIM1 dimers couple to three Orai1 dimers in the hexameric CRAC channel complex. An alternative hypothesis has been presented by Zhou et al. suggesting a new STIM1-Orai1 coupling model based on experiments using a STIM1 F394H mutant, which disrupts STIM1-Orai1 coupling and activation [89,104]. Fusing two SOAR molecules either containing two wild type or one wild type and one F394H SOAR mutant has revealed a similar interaction with and activation of Orai1. To disrupt the coupling to and activation of the CRAC channel, both SOAR domains have to carry the F394H substitution. Another experiment using a PM-anchored Orai1 C-terminus reveals an interaction with the SOAR tandem construct and ∼50% decreased coupling in the case of the SOAR tandem with one F394H mutant monomer. These results suggest a unimolecular interaction involving one Orai monomer of the hexameric assembly and one STIM1 molecule within the STIM1 dimer [104]. This interpretation is based on the assumption that each SOAR monomer within a dimeric construct acts independently of the other in the Orai channel activation process, excluding a potential cooperative effect. This picture allows for the hypothetical interaction of the second STIM1 molecule of the dimer with an Orai subunit of an adjacent Orai hexameric channel. Connection of two Orai hexameric channels by one STIM1 dimer may result in a lattice structure of CRAC channels where STIM1 dimers grab neighboring Orai hexamers, consequently forming STIM1-Orai1 clusters at ER-PM junctions. However, one point difficult to explain is how the antiparallel crossing Orai C-termini reported in the dOrai crystal structure are released and structurally change position and orientation to fit the unimolecular interaction model proposed by Zhou et al. [104] (see Chapter 7). Potentially, one Orai dimer interacts with two STIM1 molecules but not from the same STIM1 dimer. The alternative to the unimolecular coupling and activation model is the bimolecular binding model presenting the interaction of one STIM1 dimer with one Orai1 dimer, as seen in the NMR structure [73]. This model would similarly enable cluster formation by the intrinsic ability of STIM1 to form higher-order oligomers via CC3_ext upon store depletion that link STIM1 dimers between adjacent Orai channel complexes, meaning that Orai is not necessary for cluster formation, which follows the inherent STIM1 clustering propensity. However, further functional and structural evidence is required to shed light on the STIM1-Orai1 coupling process and oligomeric cluster formation.

2.5. Ion Conduction Pathway of the Orai Pore

In the absence of STIM1 binding and store depletion, Orai1 channels remain in a closed conformation. However, specific mutations in the pore of Orai1 have been shown to yield constitutively active currents, suggesting that the pore itself is an essential gating domain [80,105–108]. To allow Ca²⁺ influx through the centrally located Orai1 pore by STIM1 requires energy to switch the Orai1 pore into a permeable state. The pore domain of Orai1 is formed by a ring of TM1 helices extending into the cytosol. The hexameric crystal structure of Orai1 reveals an external vestibule in the vicinity of the selectivity filter, a hydrophobic cavity, and a basic region [41] (Figure 2.3b and c). Such a long α-helical pore formation is further supported by cysteine cross-linking experiments [109,110]. Molecular dynamics simulations have revealed that Ca²⁺ ions can frequently bind to the external vestibule, named the Ca²⁺ accumulating region (CAR) [88] (Figure 2.3c). This external vestibule in Orai1 is formed by the TM1-TM2 loop, which includes three negatively charged residues (D110, D112, D114). D110 that is most centrally located to the pore contributes most frequently to Ca²⁺ ion binding. D112 instead not only binds Ca²⁺ but also basic residues of the longer extracellular loop 3, which affects Ca²⁺ binding and permeation [88]. Neither the negatively charged amino acids in loop 1 nor the positively charged residues in loop 3 are fully conserved among the three Orai isoforms [88]. Hence, these sequence differences may add to the plasticity of Ca²⁺ permeation of the Orai family members. The main role of the extracellular CAR in Orai channels is to raise local Ca²⁺ levels near the outer entrance of the pore and favor Ca²⁺ binding in the selectivity filter. CAR and the selectivity filter, which is exclusively formed by E106 residues, are only 1 nm apart [41,88] (Figure 2.3c). Even a conservative mutation E106 to D yields nonselective cation currents and widens the minimum pore diameter [111]. The relatively slow time of binding and unbinding of Ca²⁺ to the selectivity filter regulates the Ca²⁺ selectivity of the Orai1 channel [112]. This Ca²⁺ (un-) binding process likely also includes the closely located CAR segment. The more central part of the pore formed by hydrophobic residues hinders Ca²⁺ permeation in the closed conformation. Not surprisingly, a mutation of a rather bulky valine 102 to an alanine resulted in constitutively active Orai1 channels [105]. It is of note that these currents of Orai1-V102A are less Ca²⁺ selective, while the coexpression of STIM1 robustly increases their Ca²⁺ selectivity. These experiments suggest that the Ca²⁺ selectivity is not an intrinsic property of the Orai pore itself but rather is formed in a concerted manner with STIM1. The V102 residues may act as a hydrophobic gate upon the coupling of STIM1 with Orai1 to bend away from its pore blocking position and allow Ca²⁺ influx. Computer simulations of Orai1-V102 show that the hydrophobic barrier is lowered when this valine is changed to an alanine [113]. Consequently, water molecules can access the pore more easily and may help to shield Ca²⁺ charges and to allow Ca²⁺ influx. Additionally, STIM1 may induce additional pore conformations to the selectivity filter, as determined from terbium-binding experiments [114]. This observation also fits nicely with the altered Ca²⁺ selectivity of the pore mutant experiments in the absence and presence of STIM1 [105]. A second site that is essential for gating is the basic segment located at the cytosolic exit of the Orai1 TM1 pore. This is consistent with the disease-causing Orai1-R91W mutation that completely blocks the pore [16,95]. Remarkably, the crystal structure of dOrai was solved together with a small negatively charged plug that is surrounded by side chains of R91 and R87 [41]. It is yet unclear if such a negatively charged plug plays an essential physiological role, but Cl^- ions in the vicinity of the basic pore segment could play a similar role in preventing Ca²⁺ permeation. A second constitutive pore mutant, Orai1-G98P, has been shown to regulate the proposed R91 gate [106]. While the positive ring of R91 is, after the selectivity filter, the narrowest part of the wild-type Orai1 pore, the constitutively active Orai1-G98P extended the basic pore segment [106]. Again, the pore mutation not only affected gating but also the selectivity of the Orai1 channel [106]. Hence, an open Orai1 pore conformation might affect the whole pore, including the selectivity filter, the hydrophobic segment, and the basic segment. Within the conserved N-terminus of Orai1, a putative cholesterol binding site has recently been reported [99]. Upon cholesterol depletion, STIM1/Orai1-mediated as well as endogenous CRAC currents were enhanced without affecting the selectivity of the Ca²⁺ currents and retaining association of STIM1 to Orai1. Cholesterol binding to an Orai1 N-terminal peptide comprising the ETON region has been observed, and point mutations disrupting the cholesterol binding motif enhanced store-operated currents similar to cholesterol depletion. Since overall coupling of STIM1 to Orai1 remains unaffected by the amount of cholesterol in the plasma membrane, this lipid seems to interfere mainly with STIM1-dependent channel gating via the ETON region [99]. Clearly, further studies are required to resolve this unique channel modulation mechanism.

2.6. Perspectives

Within the past decade, major efforts have been made to identify the molecular components of the CRAC channel family together with the discovery of their 3D atomic structures. Nevertheless, several questions remain unresolved and need further examination. Regarding STIM1, different cytosolic C-terminal parts including a portion and the whole CAD/SOAR domain have been characterized by NMR and x-ray crystallography, revealing significant differences in their 3D atomic structures. In particular, larger STIM1 C-terminal fragments would be of benefit for the elucidation of intra- and intermolecular interactions that trigger the switch mechanism of STIM1, resulting in its tight, inactive or its extended, active conformation. The use of point mutations like L251S or R426L would help locking the STIM1 C-terminal structures in their extended or tight conformation, respectively.

With respect to Orai1, the crystal structure shows the closed state, and hence, Orai1 point mutants like P245L that yield a constitutively active Orai1 channel would lock the open state and may help elucidate the open channel conformation. How exactly STIM1 triggers the gating of Orai channels into the open state is only partially understood. NMR resolution and characterization of the SOAP, including a STIM1 fragment and part of the Orai1 C-terminus, have suggested a direct STIM1-Orai1 coupling mechanism. However, the gating mechanism, which may involve other Orai1 cytosolic domains, is still not fully understood and needs further efforts to obtain structural data including these cytosolic Orai1 domains together with STIM1 C-terminal parts. Possibly, these studies may elucidate conformational rearrangements within Orai1 cytosolic strands as well as TM domains revealing aspects of the molecular Orai1 gating mechanism. The emerging field of optogenetics will further allow to obtain more detailed mechanistic insights of the STIM1-Orai communication via a sophisticated control by light (see Chapter 8). Finally, proteins potentially modulating the STIM1-Orai interaction and communication, like STIMATE [115], Septin [116], SARAF [117], or CRACR2A [118] (see Chapters 4 and 10), have to be taken into consideration, pointing to the complexities of the native CRAC channel system.

Acknowledgments

We thank Isaac Jardin for editing the figures. This work was supported by the Austrian Science Fund (FWF project P28123 to M.F., FWF projects P26067 and P28701 to R.S., and FWF project P27263 to C.R.).

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Marc Fahrner

Life Science Center

Institute of Biophysics

Johannes Kepler University

Linz, Austria

Rainer Schindl

Life Science Center

Institute of Biophysics

Johannes Kepler University

Linz, Austria

Christoph Romanin

Life Science Center

Institute of Biophysics

Johannes Kepler University

Linz, Austria