Abstract
STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca2+ sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca2+ channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.
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Introduction
Calcium signals controlling a vast array of cellular functions are generated through the operation of channels that either release internally stored Ca2+ or allow external Ca2+ entry. The endoplasmic reticulum (ER) membrane-spanning STIM proteins are key mediators and coordinators of these Ca2+ signalling events1. Triggered by sensing decreased Ca2+ stored within the ER, STIM1 undergoes an intricate activation process and translocates into ER–PM junctions where it couples with and gates the highly Ca2+-selective family of Orai channels in the PM1,2. The entering Ca2+ sustains Ca2+ oscillations, maintains Ca2+ homoeostasis, and provides crucial long-term Ca2+ signals controlling gene expression and cellular growth1,3. Understanding the molecular basis of STIM-mediated control over Orai channel function has important clinical implications in autoimmunity, inflammation, vascular diseases and cancer4,5,6.
Much new information on the structure and function of STIM and Orai proteins has come to light1,2,4,7. Molecular understanding of the luminal amino-terminal Ca2+-sensing domain of STIM1 has provided much information on the initial triggering of STIM1 as Ca2+ stored within the ER is depleted during Ca2+ signalling events8,9. The cytoplasmic carboxy-terminal domain that includes the Orai-interacting site has also been structurally scrutinized. A small active fragment known as the STIM-Orai-activating region (SOAR)10 or CRAC activation domain (CAD)11 was identified within this cytoplasmic domain of STIM1 as the minimal sequence necessary for interaction with and activation of Orai channels. Recent crystallographic analysis of SOAR from human STIM1 reveals a well-organized tetra-helical arrangement stabilized within a dimeric configuration of two SOAR peptides12 compatible with biochemical evidence that STIM1 is a dimer in its resting state12,13,14,15. Crystallographic data has also elucidated the Orai channel architecture, analysis of Drosophila Orai revealing a hexameric arrangement of channel subunits each of which comprises four membrane-spanning helices16. These analyses support strong evidence17,18,19,20 that the N-terminal helices of each of the six subunits, form the channel selectivity filter and line the central Orai channel pore, surrounded by the other membrane helices. The cytoplasmic ends of the N- and C-terminal Orai membrane helices extend into the cytosol and are in close proximity with each other16,21. Both the N- and C-terminal membrane helices in Orai1 are shown to be regions of interaction with STIM1 (refs 11, 22), and both are clearly necessary for STIM1-induced channel activation22,23,24,25,26,27,28. However, the molecular site within the STIM1 molecule that interacts with Orai channels and how it induces channel gating has remained unresolved.
Similar in structure and also widely expressed, the little-studied STIM2 protein differs subtly from STIM1 in its N-terminal domain, affecting luminal Ca2+-sensitivity29 and self-activation9,30,31. Thus, small differences in residues forming the canonical Ca2+-sensing EF-hand domain may explain the greater sensitivity of STIM2 to changes in ER luminal Ca2+ (refs 29, 32, 33, 34). In addition, differences in the rates of unfolding of the luminal N-terminal domain may underlie distinct rates of activation of STIM1 and STIM2 in response to store-depletion8,9,30,31,32. Significantly, it was noted also that the efficacy of coupling between STIM2 and Orai1 was considerably reduced compared with that of STIM1, and that STIM2 might function as a competitive inhibitor of STIM1-mediated Ca2+ entry33,35,36.
We sought to determine whether the differential Orai1-coupling efficacy of STIM1 and STIM2 might be related to structural differences in their Orai-interacting domains. While the corresponding SOAR sequence in STIM2 is highly conserved, we reveal that it has a profoundly diminished interaction with and ability to gate Orai1 channels. We narrow this distinction in Orai1 activation to a small sequence in SOAR within which substitution of a single phenylalanine in STIM1 with leucine in STIM2 confers a severe decrease in Orai1 channel-gating efficacy. This amino acid is strategically positioned at the structural apex of the SOAR domain. Remarkably, modification of this single phenylalanine within the full-length STIM1 protein with the dimensionally similar but polar histidine sidechain, prevents both Orai channel binding and gating, revealing the pivotal role of this residue in both interaction with and activation of Orai. The results not only pinpoint a crucial STIM-Orai-coupling locus, but also reveal the basis of a physiologically important distinction between STIM1 and STIM2. While STIM1 is a full Orai1-agonist, leucine-replacement of this critical residue in STIM2 endows it with partial agonist properties, likely critical for limiting Orai activation stemming from the enhanced sensitivity of STIM2 to store-depletion.
Results
SOAR1 and SOAR2 are functionally distinct
In addition to small differences in the luminal N-terminal Ca2+-sensing domains in STIM1 and STIM2 (refs 9, 29, 30, 31), distinctions between STIM1 and STIM2 in their apparent efficacy of Orai channel-coupling33,35,36 prompted us to functionally compare the SOAR fragments from STIM1 (SOAR1) and STIM2 (SOAR2) (Supplementary Fig. 1). Despite sharing >80% amino acid identity, expressed equally in stable Orai1-CFP-expressing HEK293 cells (HEK-Orai1 cells), their coupling to Orai1 channels was remarkably different (Fig. 1). In fura-2-loaded cells with replete Ca2+ stores, constitutive Ca2+ entry after transient (5 min) exposure to Ca2+-free medium was assessed by Ca2+ readdition. Expressed YFP-SOAR1 caused robust constitutive Ca2+ entry rapidly blocked by 50 μM 2-APB (Fig. 1a,c) the well-described store-operated Ca2+ entry (SOCE) modifier37,38. In contrast, YFP-SOAR2 induced only small constitutive Ca2+ entry that was rapidly and transiently enhanced with 2-APB (Fig. 1b,c), despite equal expression levels (Fig. 1d,e). Although YFP-SOAR1 was avidly attached to Orai1 at the PM (Fig. 1f) consistent with earlier observations39,40, little YFP-SOAR2 was PM-associated in HEK-Orai1 cells (Fig. 1g, left). YFP-SOAR1 remained attached even after 2-APB treatment (Fig. 1f) despite blocked Ca2+ entry; in contrast, 2-APB caused a rapid association of SOAR2 with the PM (Fig. 1g). Förster resonance energy transfer (FRET) analysis revealed that the resting FRET level between YFP-SOAR1 and Orai1-CFP was 2.5-fold higher than that between YFP-SOAR2 and Orai1-CFP (Fig. 1h). Upon 2-APB addition to YFP-SOAR1-expressing cells, the FRET level increased relatively little compared with that observed for YFP-SOAR2-expressing cells (Fig. 1i). Indeed, the relative fractional FRET change for YFP-SOAR2 was some fivefold higher than YFP-SOAR1 (Fig. 1j). Thus, despite having similar sequence and predicted secondary structure (Fig. 2), SOAR1 and SOAR2 differ with respect to functional coupling with the Orai1 channel.
SOAR domains determine distinct STIM1 and STIM2 functions
The stark differences between SOAR1 and SOAR2 were unexpected. We considered whether such different functions were still observed using the whole C-terminal domains from STIM1 and STIM2 in which the SOAR components operate within the larger C-terminal molecular environment (Supplementary Fig. 1). Expression of the soluble cytoplasmic C-terminal domain of STIM1 (STIM1ct) reveals it to be a somewhat poor activator of Orai1 (refs 12, 26, 41, 42, 43). This likely results from an internal folding of the STIM1ct domain that may occlude the SOAR domain. A group of four acidic amino acids within the 318–322 sequence of STIMct (EEELE) is important in mediating this internal occlusion of STIM1ct12,26, these residues lying within a coiled-coil sequence (amino acids 310–337) described as an ‘inhibitory helix’ within STIM1ct12. Using HEK293 cells stably expressing CFP-Orai1 (ref. 39) (HEK-Orai1 cells), we expressed STIM1ct in which these four glutamates were replaced with alanines (AAALA; STIMct-4EA-YFP). Extracellular Ca2+ add-back experiments as above revealed robust Ca2+ entry mediated in cells expressing STIMct-4EA-YFP (Supplementary Fig. 2a). Addition of 50 μM 2-APB caused a brief activation followed by large inhibition of the Ca2+ entry, consistent with its actions on STIM1-mediated SOCE in several cell types38. In contrast, expression of STIM2ct in which the equivalent region containing three glutamates (409–413; EQELE) were mutated to alanines (AQALA; STIM2ct-3EA-YFP) resulted in a much smaller level of constitutive Ca2+ entry, and the addition of 50 μM 2-APB caused a rapid, large and transient activation of Ca2+ (Supplementary Fig. 2b). Expression of both constructs was similar as measured by fluorescence intensity of transfected cells (Fig. 2c). Fluorescence imaging of the YFP-tagged proteins revealed that much STIM1ct-4EA was associated with the PM whereas there was little PM-association of STIM2ct-3EA (Fig. 2d,e).
Considering that the large C-terminal variable regions within the STIM1ct and STIM2ct (Supplementary Fig. 1) might contribute to differences in function, we deleted the entire variable regions from STIM1ct and STIM2ct and expressed the shortened STIM1ctΔV-4EA-YFP (234–505) and STIM2ctΔV-3EA-YFP (325–595) constructs. Despite elimination of these variable regions, the distinct activation of Orai channels by the two C-terminal constructs was almost exactly the same (Fig. 3a,b). STIM1ctΔV-4EA-YFP expression resulted in robust constitutive activation blocked by 2-APB, whereas identical expression (Fig. 3c,d) of STIM2ctΔV-3EA-YFP gave virtually no constitutive Ca2+ entry but was rapidly activated by 2-APB. As with the larger whole C-terminal fragments, the STIM1ctΔV-4EA-YFP molecule was clearly visible at the plasma membrane while the STIM2ctΔV-3EA-YFP was largely cytosolic (Fig. 3e,f). FRET analysis of the interaction with Orai revealed that the STIM2ctΔV-3EA-YFP had a much lower basal FRET value than the STIM1ctΔV-4EA-YFP molecule (Fig. 3g). Upon addition of 2-APB, the STIM1-Orai1 FRET value increased by more than twofold for STIM2ctΔV-3EA-YFP whereas the value for the STIM1 construct changed by only ~20% with 2-APB (Fig. 3h). Clearly, the STIM2ct construct was again much poorer at interacting with and activating Orai1. These results reveal that intrinsic differences in the SOAR domains, mediate distinct functions of the entire C-terminal domains of the two STIM proteins.
SOAR1/SOAR2 chimeras reveal the crucial Orai-activating site
The distinct functions of the SOAR domains not only revealed fundamental differences in Orai coupling between STIM1 and STIM2, they also provided a valuable strategy to identify the molecular site on STIM proteins mediating Orai channel activation. The recent human SOAR1 crystal structure reveals a dimeric configuration12 compatible with biochemical evidence that resting STIM1 is a dimer12,15. Each SOAR1 monomer comprises a group of four α-helices12. We constructed SOAR1/SOAR2 chimera in which we exchanged each of these α-helical sequences (Sα1-Sα4) (Fig. 2). Compared with wild-type-SOAR1 and SOAR2 (Fig. 4a,b), a chimera in which the Sα1 helix in SOAR1 was replaced with Sα1 from SOAR2 (SOAR-2111; Fig. 4c), behaved like SOAR2 giving low constitutive Ca2+ entry that was activated by 2-APB. Its largely cytoplasmic location and low basal FRET activated by 2-APB were also similar to SOAR2. The reciprocal chimera, SOAR-1222 in which Sα1 from SOAR1 was substituted within SOAR2, took on an interesting mixed phenotype (Fig. 4d). Like SOAR2, the constitutive Ca2+ entry mediated by SOAR-1222 was low and sharply activated by 2-APB. However, unlike SOAR2, SOAR-1222 was tightly PM-associated and FRET between Orai1-CFP and YFP-SOAR-1222 was high and changed little with 2-APB, resembling SOAR1. The constitutive Ca2+ entry (summary in Fig. 4g), resting FRET (summary in Fig. 4h), and cytosolic distribution, indicate that the Sα1 helix confers SOAR1 with the ability to strongly bind Orai1, whereas at least part or all of the remaining α-helices from SOAR1 contribute to its greater efficacy of channel activation.
We next considered the role of the two small intervening α-helices, Sα2 and Sα3, within SOAR. The 4-amino-acid Sα3 helix is identical within SOAR1 and SOAR2. However, the 6-amino-acid Sα2 helix has one amino-acid difference; phenylalanine (F394) in SOAR1 is a leucine residue in SOAR2 (L485). Hence, we constructed the YFP-SOAR-1211 chimera simply by making the F394L point mutation in SOAR1. This single substitution in SOAR1 led to a profoundly different phenotype when expressed in HEK-Orai1 cells. Compared with SOAR1, the SOAR-1211 chimera (Fig. 4e,g) induced much lower constitutive Ca2+ entry that was enhanced with 2-APB, much like SOAR2. Yet, SOAR-1211 was mostly PM-associated and FRET between YFP-SOAR-1211 and Orai1-CFP was high and barely altered by 2-APB (Fig. 4e,h), similar to that for SOAR1. In the reciprocal experiment, we made the L485F point mutation in SOAR2 to create the SOAR-2122 chimera. Although its expression was lower than SOAR2, its phenotype appeared similar to SOAR2 with respect to low constitutive Ca2+ entry activated by 2-APB, mostly cytoplasmic expression, and relatively low FRET enhanced by 2-APB (Fig. 4f–h). Thus, the diminished Orai1 binding conferred on SOAR-2122 by virtue of its STIM2 Sα1 helix, was dominant in preventing its activation of Orai1.
We also substituted the Sα4 helices between SOAR1 and SOAR2 and expressed these chimeras in stable HEK-Orai1 cells. However, the Sα4 substitutions (SOAR-1112 and SOAR-2221) did not change SOAR1 or SOAR2 function (Fig. 5). Thus, like SOAR1, SOAR-1112 expression led to large Ca2+ entry that was blocked by 2-APB (Fig. 5a,c,e,f). Also like SOAR1, SOAR-1112 was PM-associated, and FRET between SOAR-1112 and Orai1-CFP was high and relatively unchanged by 2-APB. Conversely, SOAR-2221 behaved in Ca2+ imaging, cytoplasmic distribution, and FRET, much like SOAR2 (Fig. 5b–f). Thus, the Sα4 helices in SOAR1 and SOAR2 function similarly and do not contribute to the distinct characteristics of these proteins.
STIM activation of Orai is tuned by a single amino acid
The results from the Sα2 chimera reveal that the single phenylalanine-394 residue in the Sα2 helix of SOAR1 is a crucial locus for the induction of Orai1 channel opening. Considering SOAR exposure and function within STIM1 is a complex and highly controlled process1, we asked whether the F394 residue plays a similar role in the full-length STIM protein environment. Certainly, SOAR1 and SOAR2 function is preserved within their expressed whole C-terminal domains (Fig. 3, Supplementary Fig. 2). Important was to examine the role of F394 within the intact STIM1 protein. Compared with STIM1-wt, equal expression of the F394L mutation in HEK-Orai1 cells resulted in substantially reduced SOCE (Figs 6a,b and 7a). Diminishing the side-chain size of the hydrophobic F394 residue even further to alanine (F394A), decreased the Ca2+ entry yet further. Remarkably, mutation of the phenylalanine to histidine (F394H) to introduce polarity while preserving side-chain size, completely abolished SOCE. These STIM1 mutants did not change ionomycin-induced Ca2+ release and hence did not alter either size or releasability of Ca2+ stores. With all STIM1 mutants, 2-APB addition after Ca2+ readdition rapidly induced Ca2+ entry, indicating Orai1 channels were activatable even though store-depletion was not sufficient for activation. Using identically transfected HEK-Orai1 cells but without depleting stores, no Ca2+ entry was observed upon Ca2+ readdition, nor was there any 2-APB induced Ca2+ entry (Fig. 6c). Thus, 2-APB overcomes the deficit in Orai1-coupling but still requires STIM1 activation by store-depletion. Patch-clamp measurements revealed that the Ca2+ entry in response to each STIM1 mutant was mediated by authentic CRAC current (Figs 6d–h and 7c). 2-APB enhanced or restored CRAC current in each case (Figs 6i and 7d).
These results reveal the strategic significance of the STIM1-F394 residue in Orai1 channel activation. Analysis of FRET between STIM1-YFP and Orai1-CFP reveals this amino acid also has a critical role in coupling to Orai1 (Figs 6j and 7b). The F394L mutation had only a slight effect on store-depletion-mediated FRET between STIM1-YFP and Orai1-CFP. However, STIM1-Orai1 FRET was greatly decreased with the STIM1-F394A mutant and almost abrogated with the STIM1-F394H mutation. In both cases, 2-APB restored the STIM1-Orai1 FRET almost to the maximal levels seen with wild-type STIM1. These FRET changes were entirely dependent on store-depletion, no FRET changes occurring without ionomycin treatment, even upon 2-APB addition (Fig. 6k). The role of F394 in Orai1 binding was clearly observable in STIM1-YFP/Orai1-CFP localization experiments (Fig. 6l–n). Thus, we observed that, although the STIM1-F394H mutation still underwent store-depletion-induced puncta formation, it barely altered Orai1 distribution (Figs 6l,m and 7e,f). However, 2-APB restored the STIM1-Orai1 interaction and Orai1 now appeared within puncta (Fig. 6n). Thus, the modest F394H mutation profoundly alters the STIM1-Orai1 interaction in addition to entirely eliminating Orai1 gating. Importantly, STIM1-Orai1 antibody immunoprecipitation studies reveal the F394 residue in STIM1 critically determines the interaction between STIM1 and Orai1 (Fig. 8). When expressed in normal HEK293 cells, the F394A and F394H STIM1 mutants functionally repressed endogenous SOCE (Supplementary Fig. 4). Indeed, the F394H mutant in particular functioned as a powerful dominant negative.
Discussion
The unexpected distinction we reveal between the active sites of STIM1 and STIM2 not only has major physiological implications, but has allowed us to pinpoint the crucial Orai-coupling locus in STIM proteins. From the SOAR1 crystal structure, F394 is strategically located at the SOAR1 apex12 with high accessibility to Orai channels (Fig. 9a). Homology modelling of SOAR2 predicts it has an almost identical structure to SOAR1 (Fig. 9a,b). SOAR1 is known to interact with both the N- and C-termini of Orai1 channels and binding across both termini is predicted to mediate channel opening11. SOAR1 strongly interacts with the C-terminal cytoplasmic extension of the fourth membrane-spanning helix of Orai1 (M4-extension)11,12,22,23,24,25,26,28. The positively charged region within the Sα1 helix of SOAR1 (382–387; KIKKKR; Fig. 2b) may contribute to this strong Orai1 binding perhaps through electrostatic interactions with the Orai1 C-terminus12,25,26. Our SOAR chimera results reveal the Sα1 helix is critical for Orai1 binding. The STIM1-Sα1 helix confers strong Orai1 binding when inserted into SOAR2, whereas the STIM2-Sα1 helix prevents Orai1 binding when placed in SOAR1 (Fig. 4). Since the positively charged residues are identical in STIM1 and STIM2 (Fig. 2b), the substantial difference in binding must be conferred by other amino acids.
SOAR1 undergoes a second weaker interaction with the 73–85 amino-acid N-terminal segment of Orai1 (refs 11, 22, 23, 24, 44). This Orai1 region is a helical extension of the M1 pore-forming transmembrane helix shown by crystallography to protrude into the cytosol16. The interaction of SOAR with this sequence is likely key to Orai1 channel gating11,21,22,23,24,27,28. Our chimera experiments reveal that the Sα2 helix is crucial for Orai1 channel gating (Fig. 4). Substitution of STIM2-Sα2 within SOAR1 confers poor channel gating properties even though the chimera still avidly associates with Orai1 by virtue of its STIM1-Sα1. The single amino acid difference between STIM1-Sα2 (F394) and STIM2-Sα2 (L485) reveals the pivotal role of this residue in the Orai-activation site of STIM proteins. The F394L mutation in STIM1 substantially reduces Orai1 channel activation (Fig. 6); conversely, the L485F mutation in STIM2 enhances its constitutive activation of Orai1 (Supplementary Fig. 5). The relatively conservative STIM1-F394L mutation affects channel gating (Fig. 6) consistent with STIM2 behaving as a partial agonist of Orai1 (Fig. 9c,d). The more severe STIM1-F394A substitution affects both Orai1 activation and binding, operating as a still weaker channel agonist (Fig. 9e). Significantly, the STIM1-F394H mutation is unable to activate or bind to Orai1 and is a complete non-agonist of Orai1 (Fig. 9f). That such a modest amino-acid substitution (F394H) would exact such profoundly altered function is remarkable. Likely, F394 undergoes a strategically important hydrophobic interaction with Orai1 that is prevented by the introduction of charge within the dimensionally similar histidine ring. Surprisingly, this single substitution also overrides the strong Orai1 interaction mediated by the Sα1 helix. This suggests that the apical regions of both Sα1 and Sα2 may function as a single tight Orai-interacting unit, agreeing with the recent suggestion by McNally et al.22 that the close proximity of the M1 and M4 cytoplasmic extensions in Orai1 (ref. 16) creates a single binding pocket for SOAR. We suggest exposed hydrophobic residues on the Orai1 M1-extension may constitute part of this binding site (Supplementary Fig. 6).
Thus, in addition to defining a crucial Orai-interacting site, the results provide a new molecular understanding of the physiological distinctions between STIM1 and STIM2. The functionally pivotal Sα2 phenylalanine is replaced in STIM2 by a leucine reducing its gating efficacy. In addition, the closely associated Sα1 region has lowered binding efficacy in STIM2. However, since STIM2 has subtly lower affinity for Ca2+ than STIM1 (ref. 29) and is on the ‘cusp’ of activation at resting ER Ca2+ levels29,35, there will be more STIM2 than STIM1 in junctions under resting or partially ER-depleted conditions. Thus, under this condition, activated STIM2 in ER–PM junctions would out-compete STIM1. In earlier studies, we showed that STIM2 over expression suppresses endogenous SOCE35,36, a result that is explained by the poor efficacy of STIM2 on the Orai1 channel. The combination of increased sensitivity to Ca2+ store-depletion and decreased Orai channel efficacy likely contributes to the different roles of STIM1 and STIM2 in receptor-induced Ca2+ signal generation45,46. Moreover, the lower target channel efficacy of STIM2 is likely important in preventing uncontrolled Ca2+ entry due to the near-constitutive activation of STIM2. Certainly, the competitive interaction between STIM1 and STIM2 may have special importance in those tissues with prominent STIM2 expression, including neural, cardiac, smooth muscle, pancreatic and dendritic cells1,47,48.
Methods
DNA Constructs
STIMct C-terminally YFP-labelled constructs were inserted within the pIRESneo3 plasmid (Clontech). SOAR and SOAR chimera constructs were inserted within the pEYFP-c1 plasmid (Clontech) between XhoI/EcoRI sites to create N-terminally-labelled SOAR derivatives. STIM2ct-3EA-YFP was derived from STIM2ct-YFP41 utilizing the Quikchange II site directed mutagenesis kit (Agilent Technologies). STIM1ct-4EA-YFP was mutated from the previously described STIM1ct-YFP construct41. STIM2ct-ΔV-3EA-YFP was generated by deletion of the C-terminal 238 amino acids (596–833 on STIM2) on STIM2ct-3EA-YFP by PCR. STIM1ct-ΔV-4EA-YFP was generated by deletion of the C-terminal 180 amino acids (506–685 on STIM1) on STIM1ct-4EA-YFP. Constructs not generated on site were made by (Mutagenex, NJ).
Cell culture and transfection
Stable HEK293 expressing Orai1-CFP were derived and maintained in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 10% fetal bovine serum, penicillin and streptomycin (Gemini Bioproducts, CA) (37 °C; 5% CO2) in G418 (100 μg ml−1)36,41. All plasmid transfections were undertaken by electroporation using the Bio-Rad Gene Pulser Xcell system in OPTI-MEM medium41. DNA constructs were introduced by electroporation at 180 V, 25 ms in 4 mm cuvettes (Molecular BioProducts). Transfected cells were cultured on coverslips (25 mm) in OPTI-MEM with 5% FBS and measurements undertaken 14–48 h after transfection39.
Cytosolic Ca2+ measurements
Intracellular Ca2+ levels were measured by ratiometric imaging using fura-2. Loading of fura-2 and imaging of fura-2 fluorescence utilized loading and fluorescence solutions comprising: 107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl2, 11.5 mM glucose, 20 mM Hepes-NaOH, pH 7.2 (ref. 40). During dye-loading, cells were treated with 2 μM fura-2/AM for 30 min at room temperature, then transferred to fura-2-free solution for a further 30 min. Ratio fluorescence imaging was measured utilizing the Leica DMI 6000B fluorescence microscope controlled by Slidebook 5.0 software (Intelligent Imaging Innovations; Denver, CO). Consecutive excitation at 340 nm (F340) and 380 nm light (F380) was undertaken every 2 s; emission fluorescence was collected at 505 nm. Intracellular Ca2+ levels are shown as F340/F380 ratios obtained from groups of 5–35 single cells on coverslips. All Ca2+ imaging experiments were undertaken at room temperature and representative traces of at least three independent repeats are shown as mean±s.e.m.
Förster resonance energy transfer measurements
To determine FRET signals between stably expressed Orai1-CFP and transiently expressed YFP-tagged STIM fragments, we used the Leica DMI 6000B fluorescence microscope equipped with CFP (438Ex/483Em), YFP (500Ex/542Em), FRETraw (438Ex/542Em) filters. Images captured with the CFP, YFP and FRET filters (FCFP, FYFP and Fraw, respectively) were collected every 20 s at room temperature using a × 40 oil objective (N.A.1.35; Leica) and processed using Slidebook 5.0 software (Intelligent Imaging Innovations). Three-channel corrected FRET was calculated using the following formula: FRETc=Fraw−Fd/Dd*FCFP−Fa/Da*FYFP where FRETc represents the corrected energy transfer, Fd/Dd represents measured bleed-through of CFP through the YFP filter (0.447), and Fa/Da is measured bleed-through of YFP through the CFP filter (0.049). Although the CFP signal was constant in Orai1-CFP stable cells, there was some variation in transiently expressed YFP levels. To compensate for these variations in YFP expression, the above FRETc values were normalized against acceptor fluorescence (FYFP) to generate ‘N-FRET’ signals. Thus, N-FRET=FRETc/FYFP. In some figures, the relative changes in N-FRET were compared with resting N-FRET (N-FRETrest) levels. This is denoted as ΔN-FRET/N-FRETrest, in which ΔN-FRET=N-FRET—N-FRETrest. Average FRET measurements shown are typical of at least three independent analyses ±s.e.m.
Enhanced Fluorescence Image Analysis
Enhanced images of the cellular distribution of fluorescently-tagged STIM and Orai constructs were obtained from stacks of 10–20 3-D Z-axis image-planes collected at 1 μm steps. The constrained iterative deconvolution function of the Slidebook 5.0 software was used to analyse image stacks and derive enhanced deconvolved images with minimized fluorescence contamination from out-of focus planes. STIM and Orai images shown were typical of at least three independent experiments.
Analysis of STIM and Orai Fluorescence Intensity in Puncta
Levels of STIM1 and Orai1 within punctal regions were quantified by comparing YFP or CFP fluorescence in punctal regions, and in corresponding adjacent control regions. Average wild-type or mutant STIM1-YFP fluorescence within puncta (YFPpuncta) and adjacent control areas (YFPcontrol) were exported using the ‘ratio/timelapse-data’ function in Slidebook. The ratio of YFPpuncta/YFPcontrol was used to define the relative enrichment of STIM1 in puncta after ionomycin. Levels of Orai1-CFP were measured in the same superimposed punctal and adjacent control regions selected from the STIM-YFP images, to give CFPpuncta/CFPcontrol. The relative changes (Δ) are shown compared with resting levels before ionomycin application. Analyses shown are from six cells taken from three independent experiments, with 20–30 punctal and control regions selected from each cell.
Electrophysiological measurements
Conventional whole-cell recordings in HEK-Orai1 cells transiently expressing STIM1 or its mutants were performed39. After establishing whole-cell configuration, a 50 ms voltage step (−100 mV) from a holding potential of 0 mV, followed by a voltage ramp spanning from −100 to +100 mV in 50 ms was delivered every 2 s. The intracellular solution contained: 135 mM Cs aspartate, 8 mM MgCl2, 8 mM BAPTA, and 10 mM Cs-HEPES (pH 7.2). The extracellular solutions contained: 130 mM NaCl, 4.5 mM KCl, 20 mM CaCl2, 10 mM TEA-Cl, 10 mM D-glucose, and 5 mM Na-HEPES (pH 7.4). A 10 mV junction potential compensation was applied to correct the liquid junction potential between pipette solutions relative to extracellular solution. All whole-cell currents were filtered at 3.3 kHz and sampled at 10 kHz.
Western analyses
Cells were lysed in cell lysis buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% IGEPAL CA‐630, and 100 μM phenylmethylsulfonyl fluoride and 5% protease inhibitor cocktail (Sigma-Aldrich). Lysis took place on ice for 30 min, followed by centrifugation at 14,000 g, 4 °C for 10 min. Supernatants were quantified using Bio-Rad DC protein assay kits. Protein extracts (20 μg per lane of each) were resolved on 8% SDS-polyacrylamide gels and electroblotted onto Bio-Rad Immun-Blot PVDF membranes. After transfer, the PVDF membranes were blocked in Tris-buffered saline-Tween 20 (TBST; containing 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% BSA (Sigma-Aldrich) for 1 h at room temperature, then incubated with Rabbit GFP primary antibody (dilution 1:1000) overnight at 4 °C. Membranes were washed two times (7 min) in TBST and incubated with secondary antibody (Anti-Rabbit immunoglobulin G (H+L) from KPL, Inc.) and Precision Protein StrepTactin-horseradish perioxidase conjugate (Bio-Rad) for 30 min. Subsequently, membranes were washed three times (5 min) in TBST. Peroxidase activity was examined with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) using the manufacturer’s protocols; the resulting fluorescence was collected using an Alpha Inotech MultiImage II system.
Immunoprecipitation
Cells were treated as indicated in the legend and lysed in chilled lysis buffer containing 150 mM NaCl, 10 mM Tris–HCl (pH 7.4), 1% Igepal Ca-360, and one tablet of complete protease inhibitors (Santa Cruz, sc-29131) per 25 ml. For 2-APB treatment, lysis buffer was supplemented with 50 μM 2-APB. Cell lysates were centrifuged (14,000 g, 10 min at 4 °C). After quantification with Bio-Rad DC protein assay kits, 500 μg cell lysate samples (2 μg μl−1) were incubated with protein G agarose (Invitrogen, 15920-010) for 10 min at 4 °C. Input samples were prepared from preincubated cell lysates (whole-cell lysate), the remaining lysates were immunoprecipitated with sheep anti-STIM1 antibody (2 μl per sample) by rotation for 2 h at 4 °C, followed by an additional 1 hr incubation with protein G agarose. Sheep anti-STIM1 antibody was purified from a polyclonal anti-STIM1 serum39. The precipitates were washed three times with lysis buffer and eluted in gel-loading buffer by heating at 100 °C for 10 min, and resolved in 4–12% NuPAGE Bis-Tris gels. Proteins of interest were immunoblotted using anti-GFP (1 μg ml−1, Santa Cruz, sc-9996), anti-Orai1 (1 μg ml−1, Sigma, O8264), or anti-GAPDH (1:1000, Proteintech, 60004-1-Ig) antibody. To minimize immunoglobulin G contamination from primary antibodies used in immunoprecipitation, clean-blot IP detection reagent (horseradish perioxidase; Thermo Scientific, 21232) was applied. Peroxidase activity was visualized using the ECL kit (Thermo Scientific, SuperSignal West Femto Chemiluminescent Substrate) with the FluorChem HD2 imaging system from Alpha Innotech. Original membrane scans are provided in Supplementary Fig. 3.
Homology modelling
The SOAR2 domain of the STIM2 protein was modelled as a dimer using Biological Assembly Modeler (BAM, version 2.0) which allows simultaneous modelling of both chains based on a single template ( http://dunbrack.fccc.edu/BAM; Maxim V. Shapovalov, Qiang Wang, Qifang Xu, and Roland L. Dunbrack, Jr.) (PDB code 3TEQ). Modelling also utilized MolIDE software49. Side-chain rotamers for the dimer were optimized with a backbone dependent library and the SCWRL 4 software50,51. Superpositioning of the SOAR2 model with the SOAR1 structure was undertaken using the Chimera software52 that was also used to prepare figures.
Statistical analyses
Analysis of statistical significance was performed using unpaired Student’s t-test with GraphPad Prism 5 software. Results are expressed as means±s.e.m., **P<0.01; ***P<0.001.
Additional information
How to cite this article: Wang, X. et al. Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site. Nat. Commun. 5:3183 doi:10.1038/ncomms4183 (2014).
References
Soboloff, J., Rothberg, B. S., Madesh, M. & Gill, D. L. STIM proteins: dynamic calcium signal transducers. Nat. Rev. Mol. Cell Biol. 13, 549–565 (2012).
McNally, B. A. & Prakriya, M. Permeation, selectivity and gating in store-operated CRAC channels. J. Physiol. 590, 4179–4191 (2012).
Kar, P., Nelson, C. & Parekh, A. B. CRAC channels drive digital activation and provide analog control and synergy to Ca2+-dependent gene regulation. Curr. Biol. 22, 242–247 (2012).
Lewis, R. S. Store-operated calcium channels: new perspectives on mechanism and function. Cold Spring Harb. Perspect. Biol. 3, 1–24 (2011).
Feske, S., Skolnik, E. Y. & Prakriya, M. Ion channels and transporters in lymphocyte function and immunity. Nat. Rev. Immunol. 12, 532–547 (2012).
Shaw, P. J. & Feske, S. Regulation of lymphocyte function by ORAI and STIM proteins in infection and autoimmunity. J. Physiol. 590, 4157–4167 (2012).
Hogan, P. G., Lewis, R. S. & Rao, A. Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28, 491–533 (2010).
Stathopulos, P. B., Zheng, L., Li, G. Y., Plevin, M. J. & Ikura, M. Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry. Cell 135, 110–122 (2008).
Zheng, L. et al. Auto-inhibitory role of the EF-SAM domain of STIM proteins in store-operated calcium entry. Proc. Natl Acad. Sci. USA 108, 1337–1342 (2011).
Yuan, J. P. et al. SOAR and the polybasic STIM1 domains gate and regulate Orai channels. Nat. Cell Biol. 11, 337–343 (2009).
Park, C. Y. et al. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136, 876–890 (2009).
Yang, X., Jin, H., Cai, X., Li, S. & Shen, Y. Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1). Proc. Natl Acad. Sci. USA 109, 5657–5662 (2012).
Penna, A. et al. The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 456, 116–120 (2008).
Muik, M. et al. A cytosolic homomerization and a modulatory domain within STIM1 C-terminus determine coupling to ORAI1 channels. J. Biol. Chem. 284, 8421–8426 (2009).
Covington, E. D., Wu, M. M. & Lewis, R. S. Essential role for the CRAC activation domain in store-dependent oligomerization of STIM1. Mol. Biol. Cell 21, 1897–1907 (2010).
Hou, X., Pedi, L., Diver, M. M. & Long, S. B. Crystal structure of the calcium release-activated calcium channel Orai. Science 338, 1308–1313 (2012).
McNally, B. A., Yamashita, M., Engh, A. & Prakriya, M. Structural determinants of ion permeation in CRAC channels. Proc. Natl Acad. Sci. USA 106, 22516–22521 (2009).
Zhou, Y., Ramachandran, S., Oh-Hora, M., Rao, A. & Hogan, P. G. Pore architecture of the ORAI1 store-operated calcium channel. Proc. Natl Acad. Sci. USA 107, 4896–4901 (2010).
Zhang, S. L. et al. Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91. Proc. Natl Acad. Sci. USA 108, 17838–17843 (2011).
McNally, B. A., Somasundaram, A., Yamashita, M. & Prakriya, M. Gated regulation of CRAC channel ion selectivity by STIM1. Nature 482, 241–245 (2012).
Rothberg, B. S., Wang, Y. & Gill, D. L. Orai channel pore properties and gating by STIM: implications from the Orai crystal structure. Sci. Signal 6, pe9 (2013).
McNally, B. A., Somasundaram, A., Jairaman, A., Yamashita, M. & Prakriya, M. The C- and N-terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels. J. Physiol. 591, 2833–2850 (2013).
Li, Z. et al. Mapping the interacting domains of STIM1 and Orai1 in CRAC channel activation. J. Biol. Chem. 282, 29448–29456 (2007).
Muik, M. et al. Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J. Biol. Chem. 283, 8014–8022 (2008).
Calloway, N., Holowka, D. & Baird, B. A basic sequence in STIM1 promotes Ca2+ influx by interacting with the C-terminal acidic coiled coil of Orai1. Biochemistry 49, 1067–1071 (2010).
Korzeniowski, M. K., Manjarres, I. M., Varnai, P. & Balla, T. Activation of STIM1-Orai1 involves an intramolecular switching mechanism. Sci. Signal 3, ra82 (2010).
Lis, A., Zierler, S., Peinelt, C., Fleig, A. & Penner, R. A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating. J. Gen. Physiol. 136, 673–686 (2010).
Zheng, H. et al. Differential roles of the C and N termini of Orai1 protein in interacting with stromal interaction molecule 1 (STIM1) for Ca2+ release-activated Ca2+ (CRAC) channel activation. J. Biol. Chem. 288, 11263–11272 (2013).
Brandman, O., Liou, J., Park, W. S. & Meyer, T. STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell 131, 1327–1339 (2007).
Stathopulos, P. B., Zheng, L. & Ikura, M. Stromal interaction molecule (STIM)1 and STIM2 EF-SAM regions exhibit distinct unfolding and oligomerization kinetics. J. Biol. Chem. 284, 728–732 (2009).
Zhou, Y. et al. The short N-terminal domains of STIM1 and STIM2 control the activation kinetics of Orai1 channels. J. Biol. Chem. 284, 19164–19168 (2009).
Zheng, L., Stathopulos, P. B., Li, G. Y. & Ikura, M. Biophysical characterization of the EF-hand and SAM domain containing Ca2+ sensory region of STIM1 and STIM2. Biochem. Biophys. Res. Commun. 369, 240–246 (2008).
Bird, G. S. et al. STIM1 is a Calcium Sensor Specialized for Digital Signaling. Curr. Biol. 19, 1724–1729 (2009).
Thiel, M., Lis, A. & Penner, R. STIM2 drives Ca2+ oscillations through store-operated Ca2+ entry caused by mild store depletion. J. Physiol. 591, 1433–1445 (2013).
Soboloff, J. et al. Orai1 and STIM reconstitute store-operated calcium channel function. J. Biol. Chem. 281, 20661–20665 (2006).
Soboloff, J. et al. STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ Entry. Curr. Biol. 16, 1465–1470 (2006).
Prakriya, M. & Lewis, R. S. Potentiation and inhibition of Ca2+ release-activated Ca2+ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP3 receptors. J. Physiol. 536, 3–19 (2001).
Ma, H. T., Venkatachalam, K., Parys, J. B. & Gill, D. L. Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes. J. Biol. Chem. 277, 6915–6922 (2002).
Wang, Y. et al. The calcium store-sensor, STIM1, reciprocally controls Orai and CaV1.2 channels. Science 330, 105–109 (2010).
Mancarella, S. et al. Hypoxia-induced acidosis uncouples the STIM-Orai calcium signaling complex. J. Biol. Chem. 286, 44788–44798 (2011).
Wang, Y. et al. STIM protein coupling in the activation of Orai channels. Proc. Natl Acad. Sci. USA 106, 7391–7396 (2009).
Muik, M. et al. STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation. EMBO J. 30, 1678–1689 (2011).
Kim, J. Y. & Muallem, S. Unlocking SOAR releases STIM. EMBO J. 30, 1673–1675 (2011).
Zhou, Y. et al. STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat. Struct. Mol. Biol. 17, 112–116 (2010).
Oh-Hora, M. et al. Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance. Nat. Immunol. 9, 432–443 (2008).
Kar, P., Bakowski, D., Di Capite, J., Nelson, C. & Parekh, A. B. Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression. Proc. Natl Acad. Sci. USA 109, 6969–6974 (2012).
Williams, R. T. et al. Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins. Biochem. J. 357, 673–685 (2001).
Lopez, E., Salido, G. M., Rosado, J. A. & Berna-Erro, A. Unraveling STIM2 function. J. Physiol. Biochem. 68, 619–633 (2012).
Wang, Q., Canutescu, A. A. & Dunbrack, R. L. Jr SCWRL and MolIDE: computer programs for side-chain conformation prediction and homology modeling. Nat. Protoc. 3, 1832–1847 (2008).
Shapovalov, M. V. & Dunbrack, R. L. Jr A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density estimates and regressions. Structure 19, 844–858 (2011).
Krivov, G. G., Shapovalov, M. V. & Dunbrack, R. L. Jr Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77, 778–795 (2009).
Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Calloway, N., Vig, M., Kinet, J. P., Holowka, D. & Baird, B. Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions. Mol. Biol. Cell 20, 389–399 (2009).
Acknowledgements
This work was supported by NIH Grant AI058173 (to D.L.G.), NIH Grant HL105066 (to S.M.), and a Novartis Institutes for Biomedical Research Fellowship (Y.W.).
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X.W., Y.W. and D.L.G. conceived the project, designed the experiments and wrote the manuscript. X.W., Y.W. and Y.Z. performed the experiments and analysed the data. M.D.A. undertook homology modelling and structural analysis. B.S.R. provided valuable structural insight. J.S. and B.S.R. helped write the manuscript. E.H. and S.M. were involved in data discussions.
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This work was supported in part by a research fellowship from Novartis Institutes for Biomedical Research.
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Wang, X., Wang, Y., Zhou, Y. et al. Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site. Nat Commun 5, 3183 (2014). https://doi.org/10.1038/ncomms4183
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DOI: https://doi.org/10.1038/ncomms4183
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