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.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

Role of STIM1 in the surface expression of SARAF

The store-operated Ca²⁺ entry-associated regulatory factor (SARAF), a protein expressed both in the endoplasmic reticulum and the plasma membrane, has been presented as a STIM1-interacting protein with the ability to modulate intracellular Ca²⁺ homeostasis. SARAF negatively modulates store-operated Ca²⁺ entry (SOCE) by preventing STIM1 spontaneous activation and regulating STIM1-Orai1 complex formation. In addition, SARAF is a negative regulator of Ca²⁺ entry through the arachidonate-regulated Ca²⁺ (ARC) channels. Here we explored the possible role of the surface expression of SARAF on the location of STIM1 in the plasma membrane. In NG115-401L cells, lacking a detectable expression of native STIM1, transfection with pHluorin-STIM1, which is able to translocate to the cell surface, enhances the plasma membrane location of SARAF as compared to cells transfected with YFP-STIM1, lacking the ability to translocate to the cell surface. These findings suggest that the surface location of SARAF is dependent on the expression of STIM1 in the plasma membrane.     

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.     

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

The inactivation domain of STIM1 (ID_STIM: amino acids 470–491) has been described as necessary for Ca²⁺-dependent inactivation (CDI) of Ca²⁺ release–activated Ca²⁺ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within ID_STIM that control the extent of CDI and examine functional interactions of ID_STIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three ID_STIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling ID_STIM by a triple alanine substitution for these three residues (“STIM1 3A”) or by truncation of the entire domain (STIM1_1–469) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM1_1–469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca²⁺ conductance, factors that determine local Ca²⁺ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between ID_STIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling ID_STIM or W76 were not additive, demonstrating that ID_STIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling ID_STIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking ID_STIM generated approximately two thirds of the WT extent of CDI but those with ID_STIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although ID_STIM is not required as a Ca²⁺ sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.     

The N-terminal domain of Orai3 determines selectivity for activation of the store-independent ARC channel by arachidonic acid

Although highly selective Ca²⁺ entry pathways play a critical role in agonist-activated Ca²⁺ signals in non-excitable cells, only with the recent discovery of the Orai proteins have the first insights into the molecular nature of these pathways been possible. To date, just two such highly Ca²⁺-selective “Orai channels” have been identified in native cells—the storeoperated CRAC channels and the store-independent, arachidonic acid-activated ARC channels. Studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 subunits, whilst a heteropentamer of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore. Importantly, this inclusion of Orai3 subunits in the ARC channel structure has been shown to play a specific role in determining the selectivity of these channels for activation by arachidonic acid. Using an approach based on the expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. We show that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain of Orai3 as the region specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete single Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is exclusively activated by arachidonic acid.Key words: STIM1, Orai1, Orai3, calcium channel, calcium entry, arachidonic acid     

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⁺.     

Selective activation of distinct Orai channels by STIM1

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Recent progress on STIM1 domains controlling Orai activation

Ca²⁺ entry in non-excitable cells is mainly carried by store-operated channels among which the CRAC channel is best characterized. Its two limiting molecular components are represented by the Ca²⁺ sensor protein STIM1 located in the endoplasmic reticulum and Orai1 in the plasma membrane. STIM1 senses a decrease of the Ca²⁺ content in internal stores and triggers its accumulation into puncta like structures resulting in coupling to as well as activation of Orai1 channels. The STIM1–Orai coupling process is determined by an interaction via their C-termini. This review highlights recent developments on domains particularly within the cytosolic part of STIM1 that govern this interaction.     

Ubiquilin 1 interacts with Orai1 to regulate calcium mobilization

Store-operated calcium entry (SOCE) channels composed of Stim and Orai proteins play a critical role in diverse biological processes. Upon endoplasmic reticulum (ER)-mediated calcium (Ca²⁺) depletion, Stim proteins oligomerize with Orai to initiate Ca²⁺ influx across the plasma membrane. The ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of ubiquilin 1 are involved in the degradation of presenilin and polyglutamine proteins. Through screening of Orai1 interaction partner(s) that might have an effect on SOCE, ubiquilin 1 was identified as a target of Orai1. However, the UBL and UBA domains of ubiquilin 1 were dispensable for this interaction. Additionally, ubiquilin 1 and Orai1 colocalized in the cytosolic compartment. Ubiquilin 1 increased the ubiquitination of Orai1, resulting in the formation of a high-molecular-weight form. MG132, a proteasome inhibitor, failed to block the degradation of Orai1, whereas bafilomycin A, a lysosome inhibitor, prevented Orai1 degradation. Confocal microscopy studies demonstrated that a fraction of Orai1 colocalized with ubiquilin 1 and the autophagosomal marker LC3. Because Orai1 is a constituent of SOCE, we determined the effect of ubiquilin 1 on Orai1-mediated Ca²⁺ influx. As we expected, intracellular Ca²⁺ mobilization, a process normally potentiated by Orai1, was downregulated by ubiquilin 1. Taken together, these findings suggest that ubiquilin 1 downregulates intracellular Ca²⁺ mobilization and its downstream signaling by promoting the ubiquitination and lysosomal degradation of Orai1.     

Orai1 and STIM1 mediate SOCE and contribute to apoptotic resistance of pancreatic adenocarcinoma

The store-operated calcium channels (SOCs) represent one of the major calcium-entry pathways in non-excitable cells. SOCs and in particular their major components ORAI1 and STIM1 have been shown to be implicated in a number of physiological and pathological processes such as apoptosis, proliferation and invasion. Here we demonstrate that ORAI1 and STIM1 mediate store-operated calcium entry (SOCE) in pancreatic adenocarcinoma cell lines. We show that both ORAI1 and STIM1 play pro-survival anti-apoptotic role in pancreatic adenocarcinoma cell lines, as siRNA-mediated knockdown of ORAI1 and/or STIM1 increases apoptosis induced by chemotherapy drugs 5-fluorouracil (5-FU) or gemcitabine. We also demonstrate that both 5-FU and gemcitabine treatments increase SOCE in Panc1 pancreatic adenocarcinoma cell line via upregulation of ORAI1 and STIM1. Altogether our results reveal the novel calcium-dependent mechanism of action of the chemotherapy drugs 5-FU and gemcitabine and emphasize the anti-apoptotic role of ORAI1 and STIM1 in pancreatic adenocarcinoma cells. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

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.     

The extracellular domain determines the kinetics of desensitization in acid-sensitive ion channel 1

The acid-sensitive ion channel 1 (ASIC1alpha or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that has the broadest distribution in the nervous system. Hallmarks of ASIC1alpha are gating by external protons and rapid desensitization. In sensory neurons ASIC1 may constitute a nociceptor for pain induced by local acidification, whereas in central neurons it may modulate synaptic activity. To gain insight into the functional roles of ASIC1, we cloned and examined the properties of the evolutionarily distant species toadfish (Opsanus tau), approximately 420-million year divergent from mammals. Analysis of the protein sequence from fish ASIC1 revealed 76% amino acid identity with the rat orthologue. The regions of highest conservation are the second transmembrane domain and the ectodomain, whereas the amino and carboxyl termini and first transmembrane domain are poorly conserved. At the functional level, fish ASIC1 is gated by external protons with a half-maximal activation at pHo 5.6 and a half-maximal inactivation at pHo 7.30. The fish differs from the rat channel on having a 25-fold faster rate of desensitization. Functional studies of chimeras made from rat and fish ASIC1 indicate that the extracellular domain specifically, a cluster of three residues, confers the faster desensitization rate to the fish ASIC1.     

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.     

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.