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...

Pulmonary vascular medial hypertrophy caused by excessive pulmonary artery smooth muscle cell (PASMC) proliferation is a major cause for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased Ca 2؉ influx is an important stimulus for PASMC proliferation. Transient receptor potential (TRP) channel genes encode Ca 2؉ channels that are responsible for Ca 2؉ entry during cell proliferation. Normal human PASMC expressed multiple canonical TRP (TRPC) isoforms; TRPC6 was highly expressed and TRPC3 was minimally expressed. The protein expression of TRPC6 in normal PASMC closely correlated with the expression of Ki67, suggesting that TRPC6 expression is involved in the transition of PASMC from quiescent phase to mitosis. In lung tissues and PASMC from IPAH patients, the mRNA and protein expression of TRPC3 and -6 were much higher than in those from normotensive or secondary pulmonary hypertension patients. Inhibition of TRPC6 expression with TRPC6 small interfering RNA markedly attenuated IPAH-PASMC proliferation. These results demonstrate that expression of TRPC channels correlates with the progression of the cell cycle in PASMC. TRPC channel overexpression may be partially responsible for the increased PASMC proliferation and pulmonary vascular medial hypertrophy in IPAH patients.I diopathic pulmonary arterial hypertension (IPAH) is a fatal disease that causes right heart failure and death. The elevated pulmonary vascular resistance (PVR) and arterial pressure in IPAH patients result mainly from pulmonary vasoconstriction, vascular remodeling, and in situ thrombosis (1). A central aspect of pulmonary vascular remodeling is medial hypertrophy caused by sustained pulmonary vasoconstriction (2-4), excessive pulmonary artery smooth muscle cell (PASMC) proliferation (5), and inhibited PASMC apoptosis (6, 7), resulting in a narrowed vascular lumen and increased PVR. Although its etiology remains unclear, elevated levels of circulating mitogens, dysfunction or down-regulation of receptors and ion channels, upregulation of transporters, and heightened activity of elastases and glycoproteins have been implicated in IPAH (5,6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21). High levels of [Ca 2ϩ ] cyt and sufficient levels of Ca 2ϩ in the SR are required for vascular smooth muscle cell proliferation (22,25,39). Because they regulate SR and cytoplasmic Ca 2ϩ , CCE and SOC may play significant roles in regulating cell proliferation (28,29). This study tested the hypothesis that canonical TRP (TRPC...

. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol 284: C316-C330, 2003; 10.1152/ajpcell.00125.2002 entry (CCE) through store-operated Ca 2ϩ (SOC) channels plays an important role in returning Ca 2ϩ to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ]cyt). A rise in [Ca 2ϩ ]cyt and sufficient Ca 2ϩ in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5-48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca 2ϩ from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca 2ϩ ]cyt, facilitates return of Ca 2ϩ to the SR, and enhances PASMC growth. store-operated cation channels; pulmonary hypertension; vascular remodeling; platelet-derived growth factor PLATELET-DERIVED GROWTH FACTOR (PDGF) is an important autocrine and paracrine mitogen for vascular smooth muscle cells, mediating hyperplasia, hypertrophy, endoreduplication, and migration, and for pulmonary vascular remodeling (3,4,58,60,71). As a tyrosine kinase-coupled receptor agonist, PDGF is not only itself sufficient to initiate DNA synthesis and mitosis, but it is also a stimulus for its own expression (56) and synthesis of other mitogens such as endothelin-1 (ET-1) and heparin-binding epidermal growth factor in vascular smooth muscle cells (4). High levels of PDGF have been implicated in the blood and lung tissues of patients with primary and secondary pulmonary hypertension, suggesting a critical role of PDGF in the elevated pulmonary vascular resistance and pulmonary arterial pressure in these patients. Indeed, the mitogenic effect of PDGF on pulmonary artery smooth muscle cells (PASMC) has been demonstrated to contribute to the progression of pulmonary vascular wall remodeling in patients with pulmonary hypertension (3,26,58,60,64).Ionized Ca 2ϩ in the cytoplasm, intracellular organelles, and nucleus is a critical signal transduction element in many cell types (5,57,61,62). An increase in cytoplasmic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) is a major trigger for smooth muscle contraction (57, 62) and an important stimulus for smooth muscle cell growth (6-8, 43). Removal (or chelation) of ext...