4.1. Introduction

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous mechanism in eukaryotic cells to elevate the intracellular Ca²⁺ concentration and stimulate downstream signaling pathways. SOCE is especially important for Ca²⁺ entry in cells with immune receptors, including T cells, B cells, and mast cells. Under resting conditions, cytoplasmic Ca²⁺ concentration ([Ca²⁺]) is very low (∼100 nM) in T cells, while that in the endoplasmic reticulum (ER), which serves as an intracellular Ca²⁺ store, is much higher (∼4,000–10,000-fold higher—0.4–1.0 mM) [1,2]. Extracellular [Ca²⁺] reaches almost 2 mM concentration, establishing a huge [Ca²⁺] gradient between the extracellular space and the cytoplasm (∼20,000-fold). Therefore, dynamic regulation of Ca²⁺ flow occurs constantly to maintain these gradients even in resting T cells. When T cells are activated, there is a sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), which is initiated by emptying of the ER Ca²⁺ stores. The increase in cytoplasmic [Ca²⁺] by depletion of ER Ca²⁺ stores can be minor especially in T cells, due to small volume of the ER. Instead, ER Ca²⁺ depletion induces Ca²⁺ entry via store-operated Ca²⁺ (SOC) channels, which raises [Ca²⁺]_i up to micromolar concentrations [3–6]. Therefore, SOCE via the Ca²⁺ release-activated Ca²⁺ (CRAC) channels is a primary mechanism for activation of Ca²⁺ signaling in T cells.

Upon pathogen infection or self-peptide presentation in autoimmunity, antigen-presenting cells (APCs, e.g., dendritic cells or B cells) present antigens on their surface together with major histocompatibility complex class II to activate CD4+ helper T cells. Antigen engagement of T cell receptors (TCRs) triggers a conformational change of TCRαβ chain, which induces a cascade of tyrosine phosphorylation events mediated by CD3ζ chain-ZAP70 (zeta chain-associated protein kinase 70) complexes [7,8]. This results in phosphorylation of a signaling adaptor Lat, which dissociates from CD3ζ chain-ZAP70 complex and activates phospholipase C-γ (PLCγ). In turn, PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol. IP₃ binds to the IP₃ receptor on the ER membrane and releases Ca²⁺ from the ER into the cytoplasm, and this depletion leads to activation of CRAC channels. The CRAC channel is a prototype SOC channel, well characterized in immune cells. Because ER Ca²⁺ store especially in T cells is limited, SOCE via CRAC channels is important to maintain elevated levels of [Ca²⁺]_i, which are required for activation of downstream signaling pathways including the protein kinase C, extracellular signal-regulated kinases, or nuclear factor of activated T cell (NFAT) pathways to affect the transcriptional programs for generating a productive immune response (see Chapter 5). Defective function or lack of expression of the CRAC channel components causes severe combined immune deficiency in humans [9]. Hence, an in-depth understanding of CRAC channel-mediated Ca²⁺ signaling in T cells is crucial for developing drug therapies for immune deficiency or inflammatory disorders.

Identification of essential components of CRAC channels revealed a unique mechanism of its activation, which is mediated by protein interactions. Genome-wide RNAi screens identified Orai1 as a pore subunit of the CRAC channels [10–13]. Prior to identification of Orai1, limited RNAi screens in Drosophila and HeLa cells had identified STIM1, a Ca²⁺-binding protein localized predominantly in the ER as an important regulator of SOCE [14–16]. STIM1 senses [Ca²⁺]_ER via its N-terminal EF-hands and gates Orai1 by direct interaction. The EF-hand of STIM1 has a low affinity for Ca²⁺, between 0.2 and 0.6 mM [17], and remains Ca²⁺ bound at rest. Under resting conditions, Orai1 and STIM1 are homogeneously distributed at the plasma membrane (PM) and the ER membrane, respectively. Upon ER Ca²⁺ depletion triggered by TCR stimulation, STIM1 loses Ca²⁺ binding, multimerizes, translocates to the ER-PM junctions, mediates clustering of Orai1 on the PM, and stimulates Ca²⁺ entry (Figure 4.1a) [14–16]. Detailed studies have identified a minimal domain of STIM1 necessary for activation of Orai1 as the CRAC activation domain (CAD)/STIM1-Orai1 activating region (SOAR) that directly binds to the cytosolic N and C termini of Orai1 [18,19] (see Chapter 2). This region, containing coiled-coil (CC) domains 2 and 3 of STIM1 is located in its cytoplasmic C terminus (Figure 4.1a). Further studies showed that Ca²⁺-bound STIM1 under resting conditions exhibits a folded structure mediated by intramolecular protein interaction between the positively charged residues within its CAD/SOAR domain and the negatively charged, autoinhibitory region preceding the CAD/SOAR domain, located in the CC1 region [20]. STIM1 activation requires unfolding of this intramolecular interaction to allow the basic residues within the CAD/SOAR domain to interact with the acidic residues within the C terminus of Orai1 [20]. While Orai1 and STIM1 are the major components of CRAC channels, multiple auxiliary proteins have been shown to regulate CRAC channel function. Some of these have been reviewed in detail before [21,22] and are only briefly summarized in Table 4.1. In this chapter, we focus on the recently identified molecules regulating the function of Orai1 and STIM1.

Figure 4.1

Molecular mechanisms of CRAC channel regulation.

Table 4.1

Summary of Associating Partners of Orai1 and STIM1.

4.2. Modulators of Orai1 via Protein Interaction

As demonstrated by STIM1, protein interactions broadly regulate gating, Ca²⁺ selectivity, intracellular localization, and clustering of Orai1. The pore subunit of the CRAC channels comprises of Orai1 multimers with each monomer containing four transmembrane segments (TM1-TM4). The monomers contain the N and C termini facing the cytoplasm as well as an intracellular loop between TM2 and TM3, which is important for channel inactivation (Figure 4.1b) [23–27]. The residues R91, G98, V102, and E106 within TM1 of Orai1 and D¹¹⁰xD¹¹²xD¹¹⁴ facing the extracellular milieu line the pore and are important for Ca²⁺ selectivity and gating [3–6,28,29]. Therefore, interacting partners can potentially induce conformational changes to regulate gating and Ca²⁺ selectivity in a STM1-dependent or STM1-independent manner. In addition to direct regulation of channel gating, it is also possible that interacting partners of Orai1 regulate its localization, clustering, posttranslational modification, or degradation. Various aspects of these regulatory mechanisms are currently under investigation.

It was previously proposed that calmodulin (CaM) binds to the N terminus of Orai1 at elevated [Ca²⁺]_i and potentiates a negative feedback to induce Ca²⁺-dependent inactivation (CDI) to inhibit SOCE [30]. However, recent crystal structure of the Drosophila melanogaster Orai protein showed that side chains of the residues corresponding to W76 and Y80 of Orai1, previously shown to interact with CaM, are facing the lumen of the pore, and this can potentially create steric hindrance between CaM and other Orai TMs [31]. Careful analyses of CDI of Orai1 in the presence of various CaM mutants defective in Ca²⁺ binding suggested that CaM binding may not be important for CDI [32]. Mullins et al. found that the residues W76 and Y80 are actually involved in conformational changes within the pore and accordingly proposed a conformational gating model for induction of CDI in Orai channels [32]. Hence, the role of CaM in regulating CRAC channels, particularly CDI, needs further investigation. In addition to STIM1 and CaM, other proteins interacting with the N terminus of Orai1 include a novel cytoplasmic EF-hand-containing protein, CRAC channel regulator 2A (CRACR2A, EFCAB4B or FLJ33805), which was identified from large-scale affinity protein purification using Orai1 as bait [33]. CRACR2A has two splice isoforms, CRACR2A-a (∼80 kDa) and CRACR2A-c (45 kDa). The short isoform CRACR2A-c is cytoplasmic and forms a ternary complex with Orai1 and STIM1 to stabilize their interaction after store depletion. Accordingly, its depletion decreases STIM1 clustering at the ER-PM junctions and, hence, SOCE. This interaction with Orai1 and STIM1 is [Ca²⁺]_i dependent, with low [Ca²⁺]_i favoring association and high [Ca²⁺]_i favoring its dissociation, and thus fine-tunes Ca²⁺ entry. The long isoform CRACR2A-a encodes a large Rab GTPase (Figure 4.2) [34,35]. Recently, α-SNAP was identified as a cytosolic factor that interacts with both Orai1 and STIM1 [36]. The original function of α-SNAP is disassembly of the SNARE (NSF attachment protein receptor) complex, a cellular machinery used for vesicle fusion. Different from its original function, α-SNAP, a predominantly cytoplasmic protein, physically interacts with the cytosolic CAD/SOAR domain of STIM1 and the C-terminal tail of Orai1. Through this interaction, α-SNAP regulates an active molecular rearrangement within Orai1-STIM1 clusters to obtain the STIM1/Orai1 ratio required for optimal activation of CRAC channels, without affecting the rate of STIM1 translocation into the ER-PM junctions. Accordingly, after store depletion, α-SNAP-deficient cells stably expressing Orai1 and STIM1 exhibited an increase in density of Orai1 in clusters without altering STIM1 density, leading to a reduced ratio of STIM1/Orai1 in individual clusters, thereby reducing SOCE. A direct binding to the C terminus of Orai1 and the CAD/SOAR domain of STIM1 suggests that α-SNAP may regulate CRAC channel gating, which has not been investigated so far.

Figure 4.2

Schematic depicting the different isoforms of CRACR2A proteins. Schematic showing the predicted domain structure of human CRACR2A proteins. CRACR2A gene encodes two splice isoforms, CRACR2A-a and CRACR2A-c. Both proteins share EF-hand motifs (dotted box), (more...)

Protein interactions are not only important for CRAC channel regulation but also essential for activation of signaling pathways downstream of Orai1. Cyclic adenosine monophosphate (cAMP), an important second messenger, is generated by adenylyl cyclase (AC)-mediated cleavage of adenosine triphosphate and in turn activates downstream protein kinase A pathway. A recent study demonstrated a direct interaction between AC8 and Orai1 N terminus using Forster resonance energy transfer (FRET) and GST pulldown and immunoprecipitation analyses [37]. This interaction places AC8 close to Ca²⁺ microdomains to mediate its Ca²⁺-dependent activation. These studies reveal that the interaction with Orai1 is important not only for positive or negative feedback to regulate CRAC channels but also for crosstalk between SOCE and other signaling pathways.

4.3. Vesicular Components in Regulation of Orai1

Orai1-interacting molecules also play a role in its trafficking. Machaca and colleagues used Xenopus oocyte as a model and showed that during meiosis, SOCE is inactivated due to internalization of Orai1 into an intracellular vesicular compartment and inhibition of STIM1 clustering [38]. In a follow-up study, the authors showed that Orai1 internalization occurred via a caveolin- and dynamin-dependent endocytic pathway. The authors mapped a caveolin-binding site in the N terminus of Orai1 and showed that a significant fraction of total Orai1 is intracellular, and at rest, Orai1 actively recycles between an endosomal compartment and the PM [39]. Vesicles are also involved in translocation of Orai1 from the intracellular pool into the PM after store depletion. Recently, the same group showed that a large portion of Orai1 (∼60%) exists in intracellular vesicles rather than the PM during the steady state in CHO and HEK293 cells [40]. A subset of these vesicles localize in close proximity to the PM and fuse to it after store depletion, increasing surface Orai1 levels. Furthermore, this study also showed that Orai1 trafficking to the PM after store depletion is dependent on interaction with STIM1 (in a “trafficking trap” mechanism). Accordingly, depletion of STIM1 blocked enrichment of surface Orai1 after store depletion. Conversely, overexpressed STIM1 caused intracellular trapping of Orai1-containing vesicles, resulting in reduced SOCE. Therefore, an optimal ratio between Orai1 and STIM1 is not only important for gating but also for enrichment of Orai1 to the PM.

Recently, we identified a long isoform of CRACR2A, CRACR2A-a, which is localized to the proximal Golgi area and vesicles, and uncovered its role in TCR signaling pathways including the SOCE pathway [34]. While the regions for Ca²⁺ binding and interaction with Orai1 and STIM1 are conserved between the two isoforms (Figure 4.2), CRACR2A-a has an additional proline-rich domain (PRD) and a Rab GTPase domain in its C terminus and is enriched in lymphoid organs. CRACR2A-a is unique because it clearly distinguishes itself from small Rab GTPases (∼20 kDa) due to its large size (∼85 kDa) and presence of multiple functional domains [34,35]. Interestingly, the Rab GTPase domain establishes the localization of CRACR2A-a in a GTP-/GDP-binding and prenylation-dependent manner. GTP-bound and prenylated CRACR2A-a localizes within vesicles close to the trans-Golgi network, whereas GDP-bound or unprenylated CRACR2A-a is cytosolic and rapidly degraded. Prenylation of CRACR2A-a involves geranylgeranylation at an unconventional site (CCx, x; any amino acid) in the C terminus. Upon TCR stimulation, CRACR2A-a translocates into the immunological synapse via interaction of its PRD with Vav1, a proximal TCR signaling molecule, to activate SOCE and the Ca²⁺-NFAT and the JNK signaling pathways. CRACR2A-a also translocates into the ER-PM junctions via vesicle trafficking after passive ER Ca²⁺ store depletion and recovers SOCE in Jurkat T cells depleted of both the isoforms, similar to CRACR2A-c. Because CRACR2A-a retains the Orai-STIM interaction domain, one can assume that it supports SOCE by interacting with both Orai1 and STIM1, similar to CRACR2A-c. The molecular mechanism of translocation of CRACR2A-a and activation of SOCE upon passive store depletion remains to be investigated.

4.4. Store-Independent Regulation of Orai1 via Protein Interaction

Only a few cases of store-independent regulation of Orai1 have been identified so far. An isoform of the secretory pathway Ca²⁺ ATPase, SPCA2, was shown to enhance mammary tumor cell growth by raising [Ca²⁺]_i via a direct interaction with the N and C termini of Orai1 in a STIM1 and store-independent manner [41]. Another example of store-independent interaction is the one between STIM1 and arachidonate-regulated Ca²⁺ (ARC) channels, which are activated by low concentrations of arachidonic acid. While CRAC channels are formed of homomultimers of Orai1, ARC channels are heteromers of Orai1 and Orai3 monomers and are opened by a pool of STIM1 that constitutively resides in the PM [42,43]. Albarran et al. showed that in addition to the ER, SOCE-associated regulatory factor (SARAF) also localizes to the PM (see Section 4.6). Addition of the ARC channel agonist, arachidonic acid, increases the association of PM-resident SARAF with Orai1 to negatively regulate Ca²⁺ entry via the ARC channels [44]. Accordingly, knockdown of SARAF increases arachidonic acid-induced Ca²⁺ entry, while its overexpression decreases arachidonic acid-induced Ca²⁺ entry (see Chapter 11). These studies describe a novel and interesting aspect of Orai channel activation via store depletion-independent mechanisms, and future studies are needed to uncover other mechanisms regulating Orai and STIM function in a store-independent manner.

4.5. STIM1-Interacting Molecules at the ER-PM Junctions

Orai1 and STIM1 cluster at preexisting junctions of the ER and the PM, a space of 10–25 nm [45,46]. In excitable cells (e.g., muscle cells), proteins localized to the junctions between the PM and ER/sarcoplasmic reticulum (SR) membrane form a structural foundation for Ca²⁺ dynamics essential for excitation-contraction coupling [2,47]. Various biochemical screening approaches have identified junctophilins, mitsugumins, sarcalumenin, junctin, and junctate as components of these junctions [47–49]. Recent studies have shown that homologues and isoforms of these junctional proteins are also expressed in T cells. Srikanth et al. identified the EF-hand-containing protein, junctate, as an interactor of STIM1 [50]. Junctate localization defined the sites of accumulation of CRAC channel components since after store depletion, Orai1 and STIM1 accumulated at junctions that were already marked by junctate (Figure 4.1a). In a recent study, Woo et al. identified an important role of another junctional protein, JP4, in regulation of SOCE in T cells [51]. Junctophilin family consists of four genes, JP1, JP2, JP3, and JP4, that are expressed in a tissue-specific manner and are known to form ER-PM junctions in excitable cells including skeletal muscle, cardiac, and neuronal cells [48,52]. Junctophilins contain eight repeats of the membrane occupation and recognition nexus motifs that bind to phospholipids in the N terminus and a C-terminal ER membrane-spanning TM [48,53]. Depletion of JP4 inhibited STIM1 recruitment into the ER-PM junctions and significantly decreased SOCE. Biochemical analyses showed a direct interaction of JP4 cytoplasmic domain with CC1 and CC2 regions of STIM1. JP4 was also shown to interact with the N-terminal cytoplasmic region of junctate. Therefore, this study demonstrates that junctate-JP4 complex is an important component of the ER-PM junctions in T cells that synergistically recruits STIM1 into these junctions by protein interaction (Figure 4.1a). When overexpressed, STIM1 alone is sufficient to establish the ER-PM junctions using its C-terminal polylysine regions. However, in a physiological condition when the concentration of STIM1 is low or STIM1 has a defect in its phospholipid-binding capacity (e.g., low [PIP₂] in the PM), interaction of STIM1 with the junctate-JP4 complex can be important for efficient assembly of a functional CRAC channel complex at the junctions. This junctional protein complex also acts as a determinant of the site of Ca²⁺ entry because it is preassembled at the regions where Orai1 and STIM1 accumulate after store depletion.

Two studies identified transmembrane protein 110 (TMEM110 or STIM-activating enhancer) as a positive regulator of Ca²⁺ influx by the Orai1 and STIM1 complex using biotin-labeled protein purification and a genome-wide RNAi screen, respectively [54,55]. TMEM110 is a multipass ER-resident protein with its N and C termini facing the cytoplasm. Jiang et al. showed that TMEM110 physically interacted with the CC1 region of STIM1 and induced its active conformation to interact with Orai1 [54] (Figure 4.1a). The CC1 region of STIM1 contains an acidic amino acid motif that binds to the Orai1-interacting CAD/SOAR fragment, blocking its interaction with Orai1 in an autoinhibitory manner. Interaction of TMEM110 with the CC1 region of STIM1 facilitated the release of this autoinhibition in STIM1. Furthermore, this study also showed that depletion of TMEM110 had a modest influence on the frequency of the ER-PM junctions with 8%–12% decrease in cortical ER (ER that is in close proximity to the PM). Thus, the observed phenotype of more than 60% reduction in SOCE in TMEM110 KO cells was predominantly ascribed to its direct interaction with STIM1 to relieve its autoinhibition. The molecular mechanism proposed in this study is different from the one reported by Hogan and colleagues, who showed that siRNA-mediated depletion of TMEM110 significantly reduced the density of ER-PM junctions by >60% in HeLa/HEK293 cells, both under resting conditions and after store depletion. Importantly, artificial expansion of the junctions by overexpression of a yeast junctional protein lst2, which is unlikely to affect STIM1 autoinhibition, significantly rescued STIM1 translocation and SOCE. Therefore, this study concluded that TMEM110 is important for maintenance of the ER-PM junctions involved in SOCE in resting conditions and for dynamic remodeling of these junctions after store depletion. Although the molecular mechanism proposed in each study is different, the common conclusion from both studies is that TMEM110 is localized at the ER-PM junctions and is essential for translocation of STIM1, and thereby for SOCE.

In another study, extended synaptotagmin proteins (E-Syts) were shown to play a critical role in the formation of ER-PM junctions in HeLa cells [56]. Using siRNA-mediated depletion of all three E-Syt proteins, the authors showed a >50% reduction in the density of ER-PM junctions; however, E-Syts-dependent junctions were completely dispensable for SOCE. The authors did observe a significant decrease in accumulation of Orai1 and STIM1 at the ER-PM junctions; however, this decrease in accumulation did not significantly affect SOCE. Collectively, these studies suggest that specialized proteins like TMEM110, junctate, or JP4 may play a significant role in the formation and/or dynamic regulation of junctions specifically involved in SOCE, which are likely to be distinct from those involved in other functions of ER-PM junctions including PIP₂ replenishment and lipid transfer.

4.6. Modulators of STIM1 Function

An ER-resident protein SARAF was identified as an interacting partner of STIM1 that facilitates the Ca²⁺-dependent slow inactivation of CRAC channels [57]. By screening a cDNA overexpression library, Palty et al. identified SARAF as a candidate that reduces mitochondrial Ca²⁺ accumulation. It turned out that this overexpression strategy was useful specially to identify inhibitors of Orai1 and STIM1 function because siRNA-based screens are excellent to pick up positive regulators, but not inhibitory proteins. SARAF plays multiple roles in regulation of basal and ER [Ca²⁺] as well as SOCE. Human SARAF encodes a 339-amino acid protein containing a single transmembrane segment (aa 173–195) with its N terminus facing the ER lumen (aa 1–172) and its C terminus facing the cytoplasm (aa 196–339) (Figure 4.1a). It shares similarities with STIM1 in global domain structure together with the presence of positively charged residues, which may interact with the PM phospholipids and a serine/proline-rich domain in its C-terminal end. Depletion of SARAF increased intracellular Ca²⁺ concentration and enhanced SOCE after store depletion, whereas its overexpression showed an opposite effect. Therefore, SARAF plays a negative role in SOCE with different modes of action: first, it interacts with the inactive form of STIM1 in the resting condition to stabilize its inactive state in the ER; second, it translocates to the ER-PM junctions together with STIM1 to induce CDI of Orai1 channels; and finally it also facilitates dissociation of clustered STIM1 proteins. More detailed structure-function studies identified a C-terminal inhibitory domain (CTID, aa 448–530) within STIM1 that regulates SARAF-STIM1 interaction [58]. STIM1 CTID is located at the C-terminal region of the Orai1-interacting CAD/SOAR domain, and interestingly, deletion of CTID from full-length STIM1 resulted in constitutively active Orai1 channels. CTID does not bind to SARAF directly but mediates the interaction of SARAF with the CAD/SOAR region. Therefore, this study highlights the important role of STIM1 and SARAF in CDI of Orai1, which is necessary to avoid excessive Ca²⁺ entry and its ensuing outcomes including cell death.

4.7. STIM1 as a Regulator for Non-CRAC Channel-Related Functions

Two independent studies have shown an interaction between STIM proteins and Ca_v1.2 [59,60]. STIM1 interacted with Ca_v1.2 and inhibited the channel activity in a short term and blocked its surface expression in a long term [59,60]. Both studies showed a direct interaction between the CAD/SOAR domain of STIM1 and the C terminus of the Ca_v1.2 channel. These results provide an interesting scenario where the same region of STIM1 plays exactly opposite roles in regulation of CRAC channels and Ca_v1.2 channels. This must be a useful strategy in excitable cells where timing and activities of Ca_v1.2 and SOC channels can be simultaneously regulated. However, in T cells, the activation of CRAC channels and Ca_v1 channels occurs simultaneously; hence, it would be interesting to understand how STIM1 interacts with both these channels at the same time. Recently, several reports have shown a positive role of Ca_v1 family of Ca²⁺ channels in activation, homeostatic proliferation, and cytokine production by CD4+ and CD8+ T cells [61–64]. It would be interesting to examine the activity of Ca_v1 channels in T cells isolated from mice lacking the expression of STIM proteins. In addition to CRAC and Ca_v channels, STIM1 also interacts with and activates transient receptor potential type C (TRPC) channels via an electrostatic interaction between dilysine motif in its polybasic tail and a conserved diaspartate motif in TRPC1 (and other TRPC channels) [65–69]. One report shows expression of TRPC3 among all the TRP channels in human T cells; however, the authors did not observe any significant defect in T cell activation, SOCE, or proliferation in the presence of physiological amounts of extracellular [Ca²⁺] [70]. Only under Ca²⁺-limiting conditions, with an extracellular [Ca²⁺] of ∼30–50 μM, the authors observed very mild reduction in TCR stimulation-induced SOCE and proliferation of human T cells. Hence, the role of TRPC channels in T cell activation and proliferation awaits further detailed investigation.

STIM1 negatively regulates plasma membrane Ca²⁺ ATPase (PMCA) pump directly or indirectly via a novel 10-transmembrane segment-containing protein, POST (partner of STIM1, TMEM20). Ritchie et al. showed that STIM1 interacts with PMCA and inhibits its Ca²⁺ clearance function, while Quintana et al. suggested that Ca²⁺ buffering by mitochondria in the immunological synapse inhibits PMCA activation [71,72]. In addition, while POST expression did not affect CRAC currents, POST negatively regulated PMCA activity. These studies suggest that both stimulation of CRAC channels and inhibition of PMCA activity at the immunological synapse may be important for generation of sustained, local Ca²⁺ entry required for NFAT activation. As discussed earlier, STIM1 also functions in a completely store-independent manner. STIM1 in the PM interacts with and activates ARC channels independent of Ca²⁺ stores as previously described [73]. Another function of STIM1, which is independent from store depletion, is growth of microtubules. When a growing microtubule tip comes across the ER membrane, STIM1 bound to a microtubule tip-binding protein, EB1, and pulled out a new ER tubule through the “tip attachment complex” mechanism [74,75]. This interaction of STIM1 with EB1 does not affect SOCE and does not require store depletion [74]. Together, STIM1 plays an important role not only in gating of CRAC channels but also in the function of other Ca²⁺ transport proteins including Ca_v, TRPC, PMCA, or ARC channels as well as SOCE-independent functions such as growth of microtubule tips [74,75].

4.8. Methods Used to Identify Interacting Partners of Orai1 and STIM1

The history of studying Orai1 and STIM1 began with the identification of the SOCE mechanism and measurement of CRAC currents by electrophysiological methods of whole-cell patch-clamp recording by depleting Ca²⁺ stores with Ca²⁺-chelating reagents or a blocker for the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump, thapsigargin [76] (see Chapter 1). The CRAC channels were later shown to be present in multiple immune cell types including T cells and mast cells, identified by their unique biophysical properties; however, the molecular components remained unknown for more than two decades. Orai1 and STIM1 were identified by siRNA-mediated functional screens when the discovery of siRNA allowed large-scale loss-of-function studies. Limited RNAi screens in Drosophila and HeLa cells identified STIM1 [14–16], and genome-wide RNAi screens in Drosophila cells identified Drosophila Orai first, which led to revealing its human homologues; among them, Orai1 as a pore subunit of the CRAC channels [10–13]. Two types of readout were used for these screens: direct measurement of SOCE by Ca²⁺ indicators (e.g., Fluo-4) and the use of translocation of GFP-fused NFAT into the nucleus as a readout for functional SOCE pathway. The benefits of using SOCE measurement as readout include a high chance of picking up direct components and identification of potential regulators of SOCE. Shortcomings can be potentially high false positives due to difficulties in plate handling for live-cell Ca²⁺ measurement, and a less robust signal-to-noise ratio between resting and stimulated conditions. On the contrary, NFAT translocation assay as readout yielded robust results due to the ease of plate handling after fixing the cells and very high signal-to-noise ratio that resulted in <30 candidates from a whole genome-wide screen. The shortcomings include the absolute necessity for a secondary screen using Ca²⁺ measurement because it is possible that the depletion of non-SOCE-involved components of the NFAT pathway (e.g., calcineurin) can influence its nuclear translocation.

After identification of Orai1 and STIM1, numerous interacting partners have been identified from protein purifications using affinity tags (e.g., FLAG) [33], biotin-labeling techniques [54], plasmon resonance assays [77], and targeted or genome-scale high-throughput screens [55]. Functions of interacting partners of Orai1 and STIM1 in SOCE were validated by single-cell Ca²⁺ measurement as well as electrophysiological tools of CRAC current measurements when they act on channel gating or inactivation. In addition, SOCE levels can be measured by flow cytometry using ratiometric Ca²⁺ indicators such as Indo-1 to measure responses from a large population of cells. For example, one can label a mixture of cells with various cell type-specific markers (e.g., CD4+ T cells, CD8+ T cells, and B cells) and measure SOCE from all of them simultaneously. Later, during data analysis, one can specifically examine the cell type of their choice based on the expression of various surface markers. However, it is technically difficult to accurately determine the basal and ER Ca²⁺ levels using this method. Single-cell ratiometric Ca²⁺ imaging using microscopy is the method of choice to accurately measure basal, ER [Ca²⁺], as well as SOCE. The physiological outcomes of Ca²⁺ signaling can be measured by checking nuclear translocation or dephosphorylation of NFATc2, or induction of NFATc1 expression [78,79]. To determine the roles of these associating molecules in translocation, clustering, and dissociation of Orai1 and STIM1, high-resolution confocal and total internal reflection fluorescence (TIRF) microscopy imaging are being commonly used. TIRF imaging is a very useful tool to determine the rate of association and dissociation of Orai1 and STIM1 at the ER-PM junctions and monitor if interacting partners alter this rate or translocate together with Orai/STIM. The shortcomings of these imaging techniques are an extensive use of overexpression system because these tools require fluorescently labeled Orai1/STIM1 and interacting molecules. It is difficult to uncover roles of these regulators in SOCE, when Orai1 and STIM1 are overexpressed, because overexpression of Orai1 and STIM1 are necessary and sufficient to restore CRAC currents in various cell types. The regulators are likely to play an important role in modulating the function of Orai/STIM when their expression is limiting, as observed in physiological conditions. These problems can be partly resolved by selecting cells with mild expression of Orai1 and STIM1 for imaging or using stable cell lines, which express proteins at much lower levels than transient transfection, by generation of mutants defective in dominant functions (e.g., STIM1ΔK) [50], and also by comparing phenotypes with loss-of-function studies using more physiological experimental settings without overexpression, for example, checking the effect on endogenous Orai1 or STIM1.

To determine protein interactions of regulators with Orai1 and STIM1, GST pulldown and immunoprecipitation analyses have been widely used to establish a foundation for other techniques. When combined with mutational analyses within specific domains, it can provide useful information about the site of interaction between various molecules and unravel the working mechanism of regulators. These are common and conventional methods that have been used to determine protein interactions for many decades and are still widely used due to their straightforwardness, sensitivity, and reproducibility. The shortcoming of these techniques is that they are very laborious, and it is difficult to quantify the results from multiple independent experiments (e.g., biological replicates). In addition, standardization of these biochemical techniques often requires trial and error with different kinds and concentrations of detergents, concentrations for monovalent or divalent cations, incubation duration, and the amount of proteins. Negative control proteins play a very important role here, because both Orai1 and STIM1 contain transmembrane segments. Appropriate controls/mutants will be important to validate authentic protein interactions. These techniques can be combined with cell-based assays such as FRET or biomolecular fluorescence complementation [80,81]. These techniques allow validating protein interactions in intact cells, but this is only possible when detailed topology of candidate molecules is clearly known for appropriate design of experiments. Therefore, these methods cannot be the best choice for any primary or secondary screening to identify novel proteins with unknown topology. In summary, the usage of these biochemical and imaging techniques to determine protein interactions has tremendously advanced our understanding of the mechanism of CRAC channel regulation, and they have proven to be very powerful tools, especially when combined with relevant functional assays.

4.9. Conclusions and Perspectives

The activation mechanism of CRAC channels provides a new paradigm of channel activation that solely depends on protein interactions. The studies on conformational changes of STIM1 from the folded inactive state to the open active state provide a fundamental insight into the mechanism of how signal is transmitted from sensing ER Ca²⁺ depletion to activation of Orai1. Identification of Orai1 and STIM1 as components of the CRAC channels has allowed the uncovering the ubiquitous role of SOCE in multiple cell types including immune cells, platelets, keratinocytes, osteoblasts, cardiac myocytes, skeletal muscle cells, and even neuronal cells. Some regulators of Orai1 and STIM1 also show tissue-selective expression patterns, which may determine the unique properties of CRAC channels in T cells. For example, CRACR2A and junctophilin-4 are highly abundant in T cells to selectively support CRAC channel activities [34,51]. While we mostly focus on protein interactions in regulation of Orai1 and STIM1 functions here, second messengers such as cAMP, the phospholipid PIP₂, or reactive oxygen species are also able to regulate Orai1 and STIM1 functions. Furthermore, posttranslational mechanisms such as phosphorylation and glycosylation covered elsewhere [7] also play an important role in regulation of Orai1 and STIM1 functions and should be considered as topics for future investigations.

Ion channels are considered excellent drug targets because many of them are localized at the cell surface providing a relatively easily accessible target for the drug. There is a notion that Ca²⁺ signaling is broadly involved in most cellular activities because Ca²⁺ is a ubiquitous second messenger for many signaling pathways. However, numerous studies argue that cells have an amazing capability to distinguish minor differences in the agonists, amplitude, timing, duration, oscillation frequency, and location (e.g., microdomain) of Ca²⁺ signaling and to generate unique downstream responses [82,83] (see Chapter 5). Cell type-specific Ca²⁺ response can also be mediated by unique expression of regulatory proteins as discussed earlier. The next challenging question is to develop better therapy for immune and inflammatory disorders by modulation of Ca²⁺ signaling based on these studies, and the core of such trials is to understand the accurate composition of Orai1-STIM1 complex with its auxiliary proteins, specifically formed in immune cells.

Acknowledgments

This work was supported by the National Institutes of Health grants AI-083432 and AI-109059 (Y.G.) and a scientist development grant from the American Heart Association, 12SDG12040188 (S.S.).

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Jin Seok Woo

Department of Physiology

University of California

Los Angeles, California

Sonal Srikanth

Department of Physiology

University of California

Los Angeles, California

Yousang Gwack

Department of Physiology

University of California

Los Angeles, California