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.

Summary Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGL) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer’s disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo and whole transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

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.

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Stim and Orai proteins comprise the molecular machinery of Ca 2+ release-activated Ca 2+ (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca 2+ -store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca 2+ current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca 2+ -permeable conductance that was relatively resistant to block by Gd 3+. The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.store-operated Ca 2+ entry | gating mechanism | Ca 2+

Ca 2+ release-activated Ca 2+ (CRAC) channels, located in the plasma membrane, are opened upon release of Ca 2+ from intracellular stores, permitting Ca 2+ entry and sustained [Ca 2+ ] i signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca 2+ influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca 2+ ions into cells.