Selective activation of distinct Orai channels by STIM1

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Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation

STIM1 and Orai1 are essential components of Ca(2+) release-activated Ca(2+) channels (CRACs). After endoplasmic reticulum Ca(2+) store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and Orai1 in protein clustering, migration, interaction, and, ultimately, opening CRAC channels. Here we demonstrate that the coiled-coil domain in the C terminus of STIM1 is crucial for its aggregation. Amino acids 425-671 of STIM1, which contain a serine-proline-rich region, are important for the correct targeting of the STIM1 cluster to the cell periphery after calcium store depletion. The polycationic region in the C-terminal tail of STIM1 also helps STIM1 targeting but is not essential for CRAC channel activation. The cytoplasmic C terminus but not the N terminus of Orai1 is required for its interaction with STIM1. We further identify a highly conserved region in the N terminus of Orai1 (amino acids 74-90) that is necessary for CRAC channel opening. Finally, we show that the transmembrane domain of Orai1 participates in Orai1-Orai1 interactions.     

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

Orai proteins contribute to Ca²⁺ entry into cells through both store-dependent, Ca²⁺ release–activated Ca²⁺ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca²⁺ (ARC) and leukotriene C₄ (LTC₄)-regulated Ca²⁺ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC₄-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC₄- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC₄ or AA, with LTC₄ being more potent. Although PM-STIM1 was required for current activation by LTC₄ and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.     

STIM proteins,Orai1, and gene expression

Cytoplasmic Ca²⁺ is an universal intracellular messenger that activates cellular responses over a broad temporal range, from neurotransmitter release to cell growth and proliferation.^1,2 Inherent to the use of the multifarious Ca²⁺ signal is the question of specificity: how can some Ca²⁺-dependent responses be activated in a cell and not others? A rise in cytoplasmic Ca²⁺ can evoke a response either by binding directly to the target (as occurs with certain Ca²⁺-activated K⁺ and Cl⁻ channels, for example) or through recruitment of intermediary proteins, such as calmodulin and troponin C. A substantial body of evidence has now established that Ca²⁺-binding proteins differ both in their affinities for Ca²⁺ and in their on- and off-rates for Ca²⁺ binding/unbinding. Furthermore, different Ca²⁺-binding proteins often occupy distinct locations within the cell. Therefore, the size, kinetics and spatial profile of a cytoplasmic Ca²⁺ signal are all important in determining which Ca²⁺-dependent response will be activated, when and for how long.³     

Orai1 and STIM reconstitute store-operated calcium channel function

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.     

Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits

The Ca²⁺ release-activated Ca²⁺ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca²⁺ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an “all-or-none” fashion but undergoes a graded process via binding of different numbers of STIM1.     

Functional interactions among STIM1, Orai1 and TRPC1 on the activation of SOCs in HL-7702 cells

STIM1, Orai1 and TRPC1 are all reported to be important for store-operated Ca²⁺ entry (SOCE) in diverse cells. However, there is no evidence for the functional interaction of the three proteins in SOCE in human liver cells. The objective of this study is to determine whether they are involved in SOCE in normal human liver cells. Liposomal transfection method was used to increase expression levels of the three proteins in HL-7702 cells, a normal human liver cell line. Western blot and single cell RT–PCR were applied to evaluate transfection effectiveness. Changes in store-operated current (I_SOC) and SOCE were investigated using whole-cell patch-clamp recording and calcium imaging. I_SOC is detected in HL-7702 cells and it is inhibited either by 2-Aminoethoxydiphenyl borate (2-APB) or La³⁺. Overexpression of STIM1 or Orai1 alone did not induce any change in I_SOC. TRPC1-transfection, however, caused approximate 2.5-fold increase in I_SOC. A large increase (>10-fold) in I_SOC emerged when both STIM1 and Orai1 were co-transfected into HL-7702 cells. Co-overexpression of STIM1 + TRPC1 also caused >10-fold increase in I_SOC, and addition of Orai1 did not cause any further increase. In HL-7702 cells, TRPC1 and Orai1 take part in SOCE independently of each other. Functional interactions of STIM1 and Orai1 or TRPC1 contribute to I_SOC activation.     

STIM and Orai in platelet function

Physiological platelet activation and thrombus formation are essential to stop bleeding in case of vascular injury, whereas inadequate triggering of the same process in diseased vessels can lead to fatal thromboembolism and tissue ischemia of vital organs. A central step in platelet activation is agonist-induced elevation of the intracellular Ca²⁺ concentration. This happens on the one hand through the release of Ca²⁺ from intracellular stores and on the other hand through Ca²⁺ influx from the extracellular space. In platelets, the major Ca²⁺ influx pathway is the so-called store operated Ca²⁺ entry (SOCE), induced by store depletion. Studies in the last five years discovered the molecular background of platelet SOCE. Stromal interaction molecule 1 (STIM1) and Orai1, two so far unknown molecules, got in the focus of research. STIM1 was found to be the Ca²⁺ sensor in the endoplasmic reticulum (ER) membrane, whereas Orai1 was identified as the major store operated Ca²⁺ (SOC) channel in the plasma membrane. These two molecules and their role in platelet function and thrombus formation are the topic of the present review with a special focus on apoptosis and apoptosis-like processes in platelet physiology.     

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding

Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca²⁺ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca²⁺-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.     

Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca²⁺ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca²⁺ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca²⁺ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P₂ depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca²⁺ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I_CRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P₂ is a likely determinant of Orai1 channel activity.Store-operated Ca²⁺ entry (SOCE)³ is a ubiquitous Ca²⁺ entry pathway that is regulated by the Ca²⁺ content of the endoplasmic reticulum (ER) (1). SOCE has been identified as the major route of Ca²⁺ entry during activation of cells of the immune system such as T cells and mast cells (2, 3), and it is also present and functionally important in other cells such as platelets (4) and developing myotubes (5). The long awaited mechanism of how the ER luminal Ca²⁺ content is sensed and the information transferred to the plasma membrane (PM) has been clarified recently after identification of the ER Ca²⁺ sensor proteins STIM1 and -2 (6, 7) and the PM Ca²⁺ channels Orai1, -2, and -3 (8–10). According to current views, a decrease in the ER Ca²⁺ concentration is sensed by the luminal EF-hand of the single-transmembrane STIM proteins causing their multimerization. This oligomerization occurs in the tubular ER, where it promotes the interaction of the cytoplasmic C termini of STIM with PM components and association with the PM-localized Orai channels, causing both their clustering and activation in the PM (reviewed recently in Refs. 11–13). Analysis of the interacting domains within the STIM1 and Orai1 proteins suggests that the cytoplasmic domain of STIM1 is necessary and sufficient to activate Orai1 (14), whereas the latter requires its C-terminal membrane-adjacent cytoplasmic tail to be fully activated by the STIM proteins (15, 16). Both STIM1 and -2 contain a polybasic segment in their C termini, and such regions are often responsible for the PM localization of proteins (mostly of the small GTP-binding protein class) via interaction with anionic phospholipids such as phosphatidylserine or PtdIns(4,5)P₂ (17). However, the role of this domain in STIM1 function(s) remains controversial. Deletion of the polybasic tail is reported to prevent PM association but not clustering of STIM1 upon ER store depletion (18). In other studies, truncated STIM1 lacking the polybasic domain shows only slightly altered activation (15) or inactivation (19) kinetics without major defects in supporting Orai1-mediated Ca²⁺ influx. The most recent studies identify the minimal Orai1 activation domain in STIM1 (20, 21) and find that the polybasic domain is not essential for this function but makes electrostatic interaction with classical transient receptor potential channels (22).PM phosphoinositides have been widely reported as regulators of the activity of several ion channels and transporters (23). However, only a few studies have addressed the inositide requirement of SOCE and none specifically that of the Orai1-mediated Ca²⁺ entry process. Sensitivity of SOCE to phosphatidylinositol 3-kinases (PI3K) inhibitors has been reported, but this required concentrations that suggested inhibition of targets other than PI3Ks, possibly myosin light chain kinase or the type-III PI4Ks (4, 24–26). Here we have described studies addressing the role of PM phosphoinositides in STIM1 movements as well as in Orai1 channel gating. Our results show that phosphoinositides do not have a major role in the prominent reorganization of STIM1 after Ca²⁺ store depletion but suggest a function of PtdIns4P rather than PtdIns(4,5)P₂ in supporting the Orai1-mediated Ca²⁺ entry process.     

The Short N-terminal Domains of STIM1 and STIM2 Control the Activation Kinetics of Orai1 Channels

STIM1 and STIM2 are dynamic transmembrane endoplasmic reticulum Ca²⁺ sensors, coupling directly to activate plasma membrane Orai Ca²⁺ entry channels. Despite extensive sequence homology, the STIM proteins are functionally distinct. We reveal that the short variable N-terminal random coil sequences of STIM1 and STIM2 confer profoundly different activation properties. Using Orai1-expressing HEK293 cells, chimeric replacement of the 43-amino-acid STIM1 N terminus with that of STIM2 attenuates Orai1-mediated Ca²⁺ entry and drastically slows store-induced Orai1 channel activation. Conversely, the 55-amino-acid STIM2 terminus substituted within STIM1 strikingly enhances both Orai1-mediated Ca²⁺ entry and constitutive coupling to activate Orai1 channels. Hence, STIM N termini are powerful coupling modifiers, functioning in STIM2 to “brake” the otherwise constitutive activation of Orai1 channels afforded by its high sensitivity to luminal Ca²⁺.The transmembrane ER⁴ proteins STIM1 and STIM2 function as sensors of Ca²⁺ within ER stores (1, 2). Depletion of luminal Ca²⁺ within the ER triggers aggregation and translocation of STIMs into junctions closely associated with the plasma membrane, where they activate the highly Ca²⁺-selective Orai family of store-operated channels (SOCs) via conformational coupling (3–8). Recent investigations of the cytoplasmic portion of STIM1 revealed that it alone is sufficient to activate Orai (9–12) via a short (∼100 amino acids) region centered around the second coiled-coil domain (see Fig. 1) (13–15). However, although activation of Orai1 is mediated entirely within the C-terminal portion of STIM, physiological control of STIM1 and STIM2 is exerted via their N-terminal ER-luminal Ca²⁺-sensing domains. The extent to which structural differences between these domains in STIM1 and STIM2 contribute to their distinct properties (16–19) remains poorly understood. Although STIM2 has the capacity to sense ER Ca²⁺ and activate SOCs (16, 17, 19), overexpressed STIM2 inhibits endogenous SOCs (18). Moreover, the kinetics of SOC activation by STIM2 are much slower than STIM1 (17). STIM2 was recently revealed to have a decreased Ca²⁺-sensing affinity when compared with STIM1 by virtue of three amino acid substitutions in the Ca²⁺-binding EF-hand domain (16). Although the lower affinity of the STIM2 EF-hand accounts for differences in the activation thresholds of STIM1 and STIM2 (16, 20, 21), it does not explain the slow kinetics of STIM2 nor its dominance over endogenous SOC activation. However, recent investigations reveal similar abilities of the cytosolic portions of STIM1 and STIM2 to activate Orai1 (12). Hence, although activation of Orai1 is mediated entirely within the C-terminal portion of STIM, physiological control of STIM1 and STIM2 is exerted via their N-terminal ER-luminal Ca²⁺-sensing domains.Open in a separate windowFIGURE 1.Schematic diagram depicting the domain structure of STIM1, STIM2, and STIM chimeras. The currently defined domains of STIM1 and STIM2 are depicted as canonical (cEF) and hidden (hEF) EF-hands, SAM domains, transmembrane domains (TM), coiled-coil structures, a proline-rich domain (P), and a polybasic tail (K). The sequences of the STIM1 and STIM2 N-terminal domains were aligned using the lalign program and depicted with red indicating identical amino acids and blue indicating similarity.The initial triggering events for STIM1 and STIM2 proteins involve the unfolding and aggregation of the N-terminal domains resulting from dissociation of Ca²⁺ from the luminal EF-hand Ca²⁺ binding domains (20–23). Recent evidence reveals that this unfolding is much slower for the N terminus of STIM2 than for STIM1 (21). Although most of the N termini of STIM1 and STIM2 are highly homologous, significant variability exists in the first 60 N-terminal amino acids upstream from the EF-hands, comprising a flexible random coil domain (21). Intriguingly, these upstream sequences appear to markedly modify the stability of the N-terminal domains of STIM1 and STIM2 (21). We reveal here that these sequences confer profound distinctions between STIM1 and STIM2 in their coupling to activate SOCs. In STIM2, this domain acts as a powerful “brake” to restrict constitutive activation of SOCs, occurring as a result of its high sensitivity to luminal Ca²⁺.     

Aggregation of STIM1 underneath the plasma membrane induces clustering of Orai1

STIM1 and Orai1 have recently been identified to be crucial in the regulation of store-operated Ca(2+) entry. However, it remains to be established how STIM1 couples store depletion to the functioning of Orai1 in the plasma membrane. Using quantitative measurement, we find little STIM1 on the surface membrane which is not increased by store depletion. We further demonstrate that Orai1 assembles into clusters that co-localize with STIM1 aggregations upon store depletion. The clustering of Orai1 is only seen when Oari1 are co-expressed with STIM1, but not when expressed alone. Moreover, ER retreat from cell periphery leads to mismatching of Orai1 and STIM1 puncta. Therefore, we propose that store depletion causes aggregation and translocation of STIM1 in close apposition to the plasma membrane, which in turn recruits Orai1 in the plasma membrane to the sites of STIM1 aggregates to assemble functional units of CRAC channels in a stoichiometric manner.     

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1

STIM1 and Orai1 represent the two molecular key components of the Ca²⁺ release-activated Ca²⁺ channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73–90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify “hot spot” residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca²⁺ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca²⁺ selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76–90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.