Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized 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-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca 2+ influx and Ca 2+ release-activated Ca 2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) 1,2 and Orai 3-5 . Stim probably functions as a sensor of luminal Ca 2+ content and triggers activation of CRAC channels in the surface membrane after Ca 2+ store depletion 1,6 . Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca 2+ influx and CRAC channel activity in patient T cells 3 . The overexpression of Stim and Orai together markedly increases CRAC current 5,[7][8][9] . However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca 2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca 2+ -selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other chargeneutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd 3+ . These results indicate that Orai itself forms the Ca 2+ -selectivity filter of the CRAC channel.Orai and Orai1 possess four hydrophobic stretches that are predicted to span the membrane. On the basis of strategies used previously for several different ion channels, we made point mutations to investigate the most conserved loop between putative transmembrane segments ( Supplementary Fig. 1a) and examined properties of ion selectivity, current-voltage rectification and block that are intimately associated with a pore-forming subunit. Wild-type or mutant Orai proteins were overexpressed together with wild-type Stim in S2 cells; messenger RNA and protein expression were verified by RT-PCR and western blotting (Fig. 1a and Correspondence and requests for materials should be addressed to M.D.C. (mcahalan@uci.edu). * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Supplementary Fig. 1b). In addition, by co-immunoprecipitation of the epitope-tagged wildtype proteins, we evaluated whether Stim and Orai are associated with each other before and after Ca 2+ store depletion. When cells were cultured without stimulation, only limited...

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca 2؉ influx and Ca 2؉ release-activated Ca 2؉ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca 2؉ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca 2؉ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca 2؉ pump sarco-͞ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca 2؉ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.capacitative calcium entry (CCE) ͉ genome-wide screen ͉ CRAC channel ͉ RNA interference ͉ store-operated calcium (SOC) influx P atch-clamp experiments have identified the biophysical characteristics of Ca 2ϩ release-activated Ca 2ϩ (CRAC) channels in lymphocytes and other human cell types (1, 2). Despite the acknowledged functional importance of storeoperated Ca 2ϩ (SOC) influx in cell biology (2) and of CRAC channels for immune cell activation (3), the intrinsic channel components and signaling pathways that lead to channel activation remain unidentified. In previous work (4), we demonstrated that SOC influx in S2 cells occurs through a channel that shares biophysical properties with CRAC channels in human T lymphocytes. In a medium-throughput RNA interference (RNAi) screen targeting 170 candidate genes in S2 cells, we discovered an essential conserved role of Stim and the mammalian homolog STIM1 in SOC influx and CRAC channel activity (5). STIM1 and STIM2 also were identified in an independently performed screen of HeLa cells by using the Drosophila enzyme Dicer to generate small interfering RNA species from dsRNA (6). Drosophila Stim and the mammalian homolog STIM1 appear to play dual roles in the CRAC channel activation sequence, sensing the luminal Ca 2ϩ store content through an EF hand motif and trafficking from an endoplasmic reticulum (ER)-like localization to the plasma membrane to trigger CRAC channel activity (6-8). However, as single-pass transmembrane proteins, Stim and its mammalian homolog STIM1 are unlikely to form the CRAC channel itself. To search systematically for additional components of the CRAC channel, and to analyze the signaling network and other required factors th...

Ca2+ release-activated Ca2+ (CRAC) channels underlie sustained Ca2+ signaling in lymphocytes and numerous other cells following Ca2+ liberation from the endoplasmic reticulum (ER). RNAi screening approaches identified two proteins, Stim1, 2 and Orai3-5, that together form the molecular basis for CRAC channel activity6, 7. Stim senses depletion of the ER Ca2+ store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane (PM)1, 8, 9, and Orai embodies the pore of the PM calcium channel10-12. A close interaction between Stim and Orai, identified by co-immunoprecipitation12 and by Förster resonance energy transfer13, is involved in opening the Ca2+ channel formed by Orai subunits. Most ion channels are multimers of poreforming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the PM. Here we show by biochemical analysis after cross-linking in cell lysates and in intact cells, and by non-denaturing gel electrophoresis without cross-linking that Orai is predominantly a dimer in the PM under resting conditions. Moreover, single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent again with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the C-terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded predominantly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C-terminus of Stim thus induces Orai dimers to dimerize, forming a tetramer that constitutes the Ca2+-selective pore. This represents a novel mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution toward the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signaling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. Based on these findings we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

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