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Store-operated Ca2+ entry through the plasma membrane Ca2+ release–activated Ca2+ (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca2+ flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.
Ca2+ influx through the CRAC channel in mammalian T cells and mast cells is essential for transcriptional responses and other effector responses to physiological stimuli1–4. Gating of the CRAC channel is a classical instance of store-operated Ca2+ entry, where an initial release of Ca2+ from internal cellular stores, by depleting the stores, triggers sustained Ca2+ signaling due to opening of plasma membrane Ca2+ channels5–7. RNA interference (RNAi) screens and determination of the genetic basis of a human severe combined immunodeficiency (SCID) syndrome have identified two proteins required for CRAC channel function: STIM1, a protein anchored in the endoplasmic reticulum (ER) that senses depletion of ER Ca2+ stores8–10, and ORAI1, a pore subunit of a plasma-membrane Ca2+ channel that is gated directly or indirectly by STIM1 (refs. 11–16).
The early steps of STIM1-ORAI1 signaling have been worked out in studies using engineered fluorescent STIM proteins. STIM1 senses a reduction in the ER luminal Ca2+ concentration, when Ca2+ dissociates from its luminal EF hand 1, a canonical helix-loop-helix Ca2+-binding motif9,10. Dissociation of Ca2+ leads to oligomerization of STIM1, followed by a local redistribution within the ER by which STIM1 becomes enriched at sites of ER–plasma membrane apposition, termed “puncta”9,10,17–22. Subsequently, STIM1 recruits ORAI1 to ER–plasma membrane contacts, where Ca2+ enters the cell through the opened ORAI1 channels23–27. Structural and biochemical studies with recombinant ER-luminal portions of STIM1 and STIM2 have illuminated the molecular mechanism by which STIM proteins sense Ca2+ changes in the ER lumen28,29.
Despite these insights, it remains unclear whether STIM1 directly gates ORAI1 channels. Overexpressed ORAI1 seems to be part of a larger channel complex30, and RNAi screens have identified other proteins that contribute measurably to store-operated Ca2+ entry12,13 (S. Sharma, P.G.H. and A.R., unpublished data), leaving open the possibility that proteins in addition to STIM and ORAI have a direct role in channel opening. The observation that overexpression of STIM with ORAI is sufficient for large store-operated Ca2+ currents12,18,31,32 has been taken as an indication that STIM by itself can gate ORAI. However, the cells used for expression of STIM and ORAI normally possess a store-operated Ca2+ entry pathway and may supply other proteins necessary to gate the overexpressed ORAI channels.
To dissect the essential steps in STIM1-ORAI1 signaling, we have expressed ORAI1 in the yeast Saccharomyces cerevisiae and isolated membrane vesicles containing functional ORAI1 channels. The yeast cells do not use the classical store-operated Ca2+ entry pathway that has been defined in mammalian cells—S. cerevisiae has no appreciable reservoir of Ca2+ in the ER33, does not possess orthologs of the ER Ca2+-ATPase (ATP2A1–ATP2A3) or IP3 receptor (ITPR1–ITPR3)34,35 and has no STIM or ORAI homologs—and therefore they are not likely to contribute proteins dedicated to this pathway. Here we report that recombinant STIM1 cytoplasmic fragments gate the ORAI1 channel in vitro.
We set out to express properly assembled recombinant ORAI1 channels in the yeast S. cerevisiae. S. cerevisiae strains carrying the temperature-sensitive sec6-4 mutation have a defect in fusion of vesicles trafficking from the Golgi compartment to the plasma membrane at the restrictive temperature, 37 °C, and newly synthesized plasma-membrane proteins accumulate in vesicles in the cell cytoplasm36,37 (Fig. 1a, above). The isolated vesicles have been used in flux assays to investigate transport mediated by several plasma-membrane proteins38–41, and they seemed particularly suited to our planned experiments because recombinant ORAI1 would be oriented with its cytoplasmic face accessible for interaction with recombinant STIM1 (Fig. 1a, above). Myc-tagged human ORAI1 expressed in a sec6-4 strain of S. cerevisiae seemed to be correctly targeted to the plasma membrane at the permissive temperature, 25 °C, as indicated by a circumferential pattern of immunocytochemical staining of the Myc tag in most cells, and was retained in the cell interior at the restrictive temperature, 37 °C (Fig. 1a, below). Secretory vesicles isolated from cells that had been incubated at the restrictive temperature contained Myc-ORAI1 as demonstrated by western blotting (Fig. 1b).
To investigate the interaction by which STIM1 gates ORAI1 channels, we have focused on the cytoplasmic region of STIM1 (STIM1CT), which is sufficient in cells to trigger activation of ORAI1 (refs. 26,42–45). Sequence alignments of human STIM1 with its vertebrate orthologs show that the region of pronounced conservation ends around residue 531 of human STIM1; additional alignments with insect Stim proteins and with STIM2 show a shorter region of conservation, ending around residue 498 (Fig. 1c and Supplementary Fig. 1). On the premise that interaction with either ORAI itself or other proteins of the ORAI channel complex is a basic function of STIM proteins that will be reflected in sequence conservation, we expressed and purified STIM1 C-terminal proteins truncated at residue 498, at residue 531 and at other sites suggested by sequence conservation. The anchoring of STIM1 in the ER and the measured ER–plasma membrane distance, 17 ± 10 nm (ref. 19), render it unlikely that the initial part of the STIM1 coiled coil interacts directly with ORAI1, but we have retained the entire coiled coil in our constructs because of its possible role in proper STIM1 multimer assembly. The STIM1 proteins used in this study support store-operated Ca2+ influx when expressed in mammalian cells (Supplementary Data and Supplementary Figs. 2 and 3).
Analysis of recombinant STIM1CT by size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) showed that the freshly prepared protein is dimeric under our conditions in vitro (Fig. 1d). STIM1233–498 also formed dimers; STIM1233–463 was a heterogeneous mixture of trimers and smaller material, tetramers and large aggregates (Fig. 1e). STIM1CT and its fragments eluted earlier than expected for compact globular proteins of comparable mass, consistent with an elongated shape, and in particular consistent with the predicted STIM1 coiled coil. CD spectroscopy of STIM1CT, STIM1233–498 and STIM1233–463 indicated α-helical contents of 49%, 54% and 57%, respectively (Supplementary Fig. 4). The α-helical contents and the [θ]222/[θ]208 ratios, ~1, of the latter fragments provide strong experimental support for the predicted coiled coil.
We used a membrane-flotation assay to evaluate binding of these recombinant STIM1 proteins to ORAI1. For these experiments, we expressed ORAI165–301, an N-terminally truncated ORAI1 protein that forms functional Ca2+ channels in mammalian cells (Supplementary Fig. 5), in the yeast Pichia pastoris. We prepared microsomal membranes from P. pastoris expressing ORAI165–301, layered these membranes at the bottom of a discontinuous sucrose gradient and centrifuged them. After centrifugation, the recombinant ORAI1 was recovered near the top of the gradient in the fraction visually identified as containing membranes (Fig. 2a), as expected for an integral membrane protein. STIM1CT centrifuged together with control membranes from yeast not expressing ORAI1 remained at the bottom of the gradient, as expected for a soluble protein. However, when we mixed STIM1CT with membranes containing ORAI1 and then centrifuged them, a substantial fraction of STIM1CT rose with the membranes into the upper part of the gradient, demonstrating its interaction with ORAI1. STIM1233–498 and all the longer STIM1 fragments tested in this assay also clearly bound ORAI1, whereas STIM1233–463 did not bind detectably.
Recruitment of ORAI1 to puncta by full-length STIM1 depends on a direct or indirect interaction of STIM1 with the C-terminal cytoplasmic tail of ORAI1 (refs. 26,46). Therefore, we next examined the direct interaction of recombinant STIM1 proteins with a bacterially expressed fusion protein, GST-ORAI1CT, containing the cytoplasmic tail of ORAI1, residues 259–301. STIM1CT and the other STIM1 fragments, except for STIM1233–463, bound to GST-ORAI1CT immobilized on resin (Fig. 2b). Binding was dependent on recognition of ORAI1CT, as there was no binding to GST alone. Because we visualized all input and bound proteins on the gel with Coomassie Brilliant Blue staining and detected no other proteins, the current experiment provides unambiguous proof of a direct protein-protein interaction between STIM1CT and ORAI1CT.
A conserved region of ORAI1 immediately preceding, and extending into, ORAI1 transmembrane segment 1 is implicated in channel opening11,46–48, and GFP-ORAI148–91 expressed in mammalian cells co-immunoprecipitates with STIM1342–448 (ref. 47). Here again, the protein-protein interaction is direct, because purified recombinant STIM1CT bound to purified GST-ORAI165–87 (Fig. 2c). This experiment required a large amount of input protein, and the fraction of input STIM1 retained by immobilized GST-ORAI1 peptide was small, indicating that the interaction is weaker than the STIM1CT-ORAI1CT interaction.
We next asked whether STIM1-ORAI1 protein-protein interactions are sufficient to open the ORAI1 Ca2+ channel. We obtained vesicles from sec6-4 S. cerevisiae expressing ORAI1 and incubated them under conditions in which Fura-2 was the principal Ca2+ buffer in the extravesicular solution, to monitor Ca2+ efflux49. Treatment with the Ca2+ ionophore ionomycin increased the prominence of the peak of the Fura-2 excitation spectrum near 340 nm (Ca2+–dye complex) relative to the peak near 365 nm (free dye), indicating release of Ca2+ to the extravesicular solution (Fig. 3a). Addition of STIM1CT or STIM1233–498 also elicited efflux of Ca2+ from vesicles with ORAI1, whereas addition of STIM1233–463 had little effect (Fig. 3a). The effects of STIM1CT and STIM1233–498 required ORAI1, because neither STIM1 fragment was effective in releasing Ca2+ from control vesicles lacking ORAI1 (Fig. 3a).
In complementary measurements, we monitored vesicular Ca2+ directly by coexpressing ORAI1 with the Ca2+ sensor D3cpV or D4cpV (ref. 50) fused to the α-mating–factor secretion signal to target the sensor to the vesicles. We isolated vesicles that had incorporated ORAI1 from sec6-4 yeast incubated at the restrictive temperature, and we diluted the vesicles into assay buffer. The intravesicular sensor gave a stable FRET signal at ~528 nm due to the internal Ca2+, indicating that the vesicles were not allowing Ca2+ to leak out. Treatment with ionomycin reduced the FRET signal at ~528 nm and increased the donor signal at ~475 nm, corresponding to depletion of vesicular Ca2+ (Fig. 3b). Addition of either STIM1CT or STIM1233–498 similarly triggered substantial loss of vesicular Ca2+ (Fig. 3b). For control vesicles lacking ORAI1, only ionomycin was effective in releasing Ca2+ (Fig. 3b).
We sought confirmation that Ca2+ release from the vesicles was due to gating of the ORAI1 channel. Two mutant ORAI1 proteins that do not support Ca2+ influx in mammalian cells—ORAI1 R91W, which cannot be activated by STIM1 (ref. 11), and ORAI1 E106Q, which is disabled in the ion-conducting pore15—were expressed individually in the sec6-4 strain for control experiments. Although each mutant ORAI1 protein was present in isolated vesicles at levels comparable to those of wild-type ORAI1 (Fig. 1b), STIM1CT did not elicit Ca2+ release from vesicles containing either mutant protein (Fig. 4). We conclude that the in vitro assay reflects STIM1-dependent gating of ORAI1 and Ca2+ flux through the ORAI1 channel pore.
We have taken advantage of the absence of STIM-ORAI signaling in the yeast S. cerevisiae to show that recombinant STIM1 gates ORAI1 directly, without assistance from other proteins that have a dedicated role in the mammalian store-operated Ca2+ entry pathway. It seems certain from RNAi studies that additional proteins in mammalian cells can modulate Ca2+ influx through ORAI1 channels, but these proteins are not essential to channel function.
Our work implies that native STIM1 in cells interacts directly with ORAI1 across the ~17-nm distance19 that separates the ER and plasma membrane. The store-operated channels formed by ORAI165–301 (Supplementary Fig. 5) and ORAI174–301 (ref. 46) have short cytoplasmic portions that cannot span this distance. Rather, as we have shown here, STIM1 forms a coiled coil of sufficient length to position the central region of the STIM1 cytoplasmic domain near the cytoplasmic face of the plasma membrane. The latter finding complements evidence that the region of STIM1 encompassing residues 344–442 causes constitutive activation of ORAI1 channels when expressed in mammalian cells47,51–53.
Major questions remain about how specific STIM1-ORAI1 interactions participate in the gating conformational change, and about the conformational change itself. Our development of in vitro functional assays, with defined protein reagents, to probe the STIM1-ORAI1 interaction and Ca2+ flux through the ORAI1 channel is an essential step toward the rigorous biochemical characterization of STIM1-ORAI1 gating.
Methods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/.
We are grateful to H. Li for guidance on protein expression and purification. We thank R. Rao for S. cerevisiae strain NY17 and for advice on membrane protein expression in that sec6-4 strain, R.Y. Tsien for plasmids encoding the calcium sensors D3cpV and D4cpV, J. Cregg for advice on protein expression in P. pastoris, the Department of Neurobiology, Harvard Medical School, for use of their spectrofluorimeter and T. Rapoport for access to SEC-MALLS instrumentation. This work was supported by US National Institutes of Health grants AI40127, GM075256 and AI084167 (to A.R. and P.G.H.) and by an Irvington Fellowship from the Cancer Research Institute and a Postdoctoral Fellowship from the Leukemia and Lymphoma Society (to Y.Z.).
Note: Supplementary information is available on the Nature Structural & Molecular Biology website.
AUTHOR CONTRIBUTIONSP.G.H. set overall goals for the project and coordinated the work; Y.Z., P.M., A.R. and P.G.H. designed experiments and wrote the manuscript; Y.Z. prepared and characterized the STIM1 reagents, measured STIM-ORAI interactions and conducted the assays using mammalian cells; P.M. prepared and characterized sec6-4 ORAI1 vesicles; Y.Z. and P.M. conducted the in vitro Ca2+ flux assays; D.M. and H.T.K. developed the P. pastoris membrane-flotation assay; M.O., with Y.Z., carried out reconstitution and Ca2+-imaging experiments using STIM1−/− T cells; J.Z., with Y.Z., carried out SEC-MALLS analyses; Y.H., with Y.Z., contributed confocal microscopy; A.S. helped P.M. with construction of S. cerevisiae expression plasmids.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/nsmb/.
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