Abstract-Recent breakthroughs in the store-operated calcium (Ca 2ϩ ) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca 2ϩ sensor and Orai1 as the pore forming subunit of the highly Ca 2ϩ -selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca 2ϩ release-activated Ca 2ϩ current (I CRAC ), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na ϩ currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I CRAC in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I CRAC . Ectopic expression of Stim1 in ECs increased their I CRAC to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway. ATPase inhibitors such as thapsigargin activates SOCE. 1,2 Under physiological conditions SOCE is initiated by inositol 1,4,5 triphosphate (IP 3 )-mediated depletion of ER Ca 2ϩ in response to a plethora of stimuli acting through phospholipase C-coupled receptors. The best characterized SOC current is the Ca 2ϩ release-activated Ca 2ϩ current (I CRAC ), first recorded in rat basophilic leukemia (RBL) mast cells, 3 and later described in other cell types. 4 SOCE is necessary for the replenishment of ER Ca 2ϩ content and is a key regulator of many Ca 2ϩ -dependent physiological processes. 4 Recently, high-throughput RNA silencing (siRNA) screens by several laboratories have identified 2 molecules, Stim1 and Orai1, as key components of the I CRAC pathway in mast cells, lymphocytes, and HEK293 cells. 5-8 On ER Ca 2ϩ depletion, Ca 2ϩ -sensing Stim1 proteins translocate to close proximity of the plasma membrane, where they aggregate into multiple puncta. Strikingly, Orai1 molecules also translocate to the same Stim1-containing structures on store depletion, where they open to mediate Ca 2ϩ influx. 9 -12 In endothelial cells (ECs), SOCE in response to passive store depletion was reported for several EC ...

The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.

Breast cancer is the most widespread cancer in women accounting for almost a third of all new cancer cases among women in Western countries(1, 2). Whereas most breast cancers express hormone receptors, primarily estrogen receptors (ER), 2 and depend on these hormones for their growth, an estimated 15-20% of all cases harbor the triple-negative (estrogen receptor/progesterone receptor/epidermal growth factor receptor 2-negative) phenotype (3, 4). Although breast cancer tumors are widely heterogeneous, the presence or absence of estrogen receptors on breast tumor cells represents one of the main criteria used for prognosis and for choice of hormonal and chemotherapeutic drugs.Store-operated calcium (Ca 2ϩ ) entry (SOCE) is a ubiquitous pathway necessary for refilling internal Ca 2ϩ stores and for signaling downstream to the nucleus(5-9). SOCE have been implicated in many cell functions such as proliferation, migration, and differentiation (5, 10 -12). However, a thorough characterization of the SOCE pathway in estrogen receptor-positive (ER ϩ ) and negative (ER Ϫ ) breast cancer cells is so far missing. Upon store depletion, the Ca 2ϩ sensor STIM1, that resides in the endoplasmic reticulum, oligomerizes and translocates to subplasmalemmal puncta (13,14) where it activate Orai1 channels located in the plasma membrane that mediate highly Ca 2ϩ -selective currents(15-17). Mammals possess three Orai proteins (Orai1/2/3) (15); Orai1 and Orai3 are highly expressed in similar tissues including liver, lymphoid organs, skin, and skeletal muscle (18,19). However, Orai2 is mainly found in lung, brain, spleen, and kidney (20, 21). Orai1 proteins were shown to encode the archetypical SOCE current called Ca 2ϩ release-activated Ca 2ϩ current (I CRAC )(15, 22); I CRAC was first recorded by Hoth and Penner in RBL mast cells(23). Since their discovery three years ago, Orai1, along with STIM1, were shown to be the predominant contributors to SOCE in many cell types, including T cells, B cells, mast cells, platelets, endothelial cells, smooth muscle cells, microglia, human embryonic kidney cells (HEK293), hepatocytes, and oocytes (10, 11, 15, 18, 19, 22, 24 -28). However, the role of Orai2 and Orai3 in mediating native SOCE pathways and CRAC currents remain unknown. Here we show the first evidence for I CRAC in breast cancer cells and for a native SOCE/I CRAC pathway mediated by Orai3. We demonstrate that the SOCE pathway is mediated by STIM1/2 and Orai3 proteins in ER ϩ breast cancer cells whereas the canonical STIM1/Orai1 encodes the SOCE pathway in ER Ϫ breast cancer cells. MATERIALS AND METHODSReagents-Thapsigargin, 2-APB, and nimodipine were purchased from Calbiochem, Fura-2AM was from Molecular Probes and GdCl 3 was from Acros Organics. Cs ϩ BAPTA and Pluronic F-127 were from Invitrogen. siRNAs were purchased from Dharmacon (see supplemental Table S2 for sequences). All the cell lines were bought from ATCC. All primers were synthesized by Integrated DNA Technologies. The transfection kit (VCA-1003) was from Lonza. All other ch...

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3 H]aspartate efflux, and VRAC-mediated Cl -currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl -currents. The expression of candidate Cl -channels was confirmed by RT-PCR. The maxi Cl -channel (p-VDAC) blocker Gd 3+ , the ClC-2 inhibitor Cd 2+ , and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Clchannels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl -currents. The phorbol ester PDBu, which blocks ClC-3-mediated Clcurrents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.

The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel Orai1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+ selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotrieneC4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of breast, prostate and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA), and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.