STIM1 is a Ca sensor that activates CRAC channels and migrates from the Ca store to the plasma membrane

As the sole Ca entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca signalling andmany other cellular processes. A calcium-releaseactivated calcium (CRAC) channel in T lymphocytes is the bestcharacterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-handmutants of Stim or STIM1 activates CRACchannels constitutivelywithout changingCa store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulumlike sites to the plasma membrane upon depletion of the Ca store. We propose that STIM1 functions as the missing link between Ca store depletion and SOC influx, serving as a Ca sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels. We previously characterized a SOC current in Drosophila S2 cells with biophysical properties similar to CRAC channels in human T cells. More recently, Stim was identified in an RNA-mediated interference (RNAi)-based screen with the SERCA (sarcoplasmic/ endoplasmic reticulum Ca2þATPase) pump inhibitor thapsigargin (TG) to evoke SOC influx in Drosophila S2 cells. Uniquely among the 170 candidate genes that were screened, including all trp-related genes, RNAi-mediated suppression of Stim inhibited the Ca2þ influx evoked by TG. By single-cell imaging of cytosolic Ca2þ concentration ([Ca]i) and patch clamp analysis, we confirmed a functional requirement for Stim, and for the human homologue STIM1, to mediate CRAC channel activity in S2 cells and in Jurkat T cells, respectively. Drosophila Stim and mammalian STIM1 (collectively referred to here as Stim1) are modular type I transmembrane proteins with an EF-hand motif near the amino terminus located in the lumen of the endoplasmic reticulum (ER) or outside the cell (Supplementary Fig. 1). Because Stim1 does not resemble any known ion channel, the presence of the EF-hand motif and its localization indicated that Stim1 might function as a sensor of the ER Ca2þ store. According to this proposal, Ca2þ binding to the EFhand domain of Stim1 within the lumen of the Ca2þ store would keep CRAC channels in the plasma membrane closed. To test this possibility, full-length STIM1 and Stim cDNA were cloned from RBL cells and S2 cells and placed into appropriate expression vectors. Mutants in the EF-hand region were prepared, two for STIM1 and four for Stim, on residues known to be critical for Ca2þ binding. Overexpression of wild-type (WT) or mutant Stim1 after transient transfection was confirmed by western blotting (Supplementary Fig. 2). In single-cell Ca2þ imaging experiments, overexpression of WT Stim1 produced no significant difference in resting [Ca]i, TG-independent Ca 2þ influx or TG-evoked store release compared with control blank-transfected cells (Jurkat cells are shown in Fig. 1, S2 cells in Supplementary Fig. 3). A modest increase in TG-dependent (store-operated) Ca2þ influx was seen in Jurkat T cells (Fig. 1b) but not in S2 cells, consistent with the hypothesis that Stim1 by itself is not a functional CRAC channel. In contrast, Jurkat or S2 cells transfected with the EF-hand mutants bore two severe phenotypes: resting [Ca]i and TG-independent Ca 2þ influx were increased from about 50 nM to more than 200 nM on average. Histograms of resting [Ca]i show that expression of EF-hand mutants increased resting [Ca]i to more than 600 nM in many individual cells (Supplementary Fig. 4). The Ca2þ release transient, obtained by adding TG in zero-Ca2þ solution, was not changed (Supplementary Fig. 5), showing that the Ca2þ store content was not affected. To test whether the high values of resting [Ca]i and enhanced TG-independent Ca2þ influx were caused by constitutively opened CRAC channels, 2-aminoethyldiphenyl borate, SKF96365 and Gd3þ were applied as pharmacological tools that block CRAC channels in Jurkat cells and in S2 cells. Each of these agents inhibited TG-evoked SOC influx in control cells and, at the same concentrations, all three inhibited both the high resting Ca2þ concentration and the enhanced TG-independent Ca2þ influx in Jurkat and S2 cells transfected with EF-hand mutants (Fig. 1g–i, and Supplementary Fig. 3c). These results demonstrate that CRAC channels in Jurkat or S2 cells are constitutively opened by overexpression of Stim1 EF-hand mutants. In addition to affecting resting [Ca]i, expression of Stim EFhandmutants arrested the growth of S2 cells, whereas overexpressing WT Stim had a small effect comparedwith that in S2 cells undergoing blank transfection (Supplementary Fig. 6). Most S2 cells overexpressing Stim EF-hand mutants were stained by annexin V, an early marker for the exposure of phosphatidylserine that occurs during apoptosis. The growth arrest and apoptosis were probably triggered by abnormally high resting [Ca]i (refs 19–21). A similar but milder growth defect in Jurkat cells overexpressing STIM1 EF-handmutants was also observed. How does STIM1 regulate the activity of CRAC channels? The subcellular localization of STIM1 was examined by LETTERS