A Novel Native Store-operated Calcium Channel Encoded by Orai3

Store-operated calcium (Ca2+) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca2+ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca2+ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca2+-selective Ca2+ release-activated Ca2+ current (ICRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca2+ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and ICRAC in estrogen receptor-positive (ER+) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER−) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER+ breast cancer cells represent the first example where the native SOCE pathway and ICRAC are mediated by Orai3. Future studies implicating Orai3 in ER+ breast cancer progression might establish Orai3 as a selective target in therapy of ER+ breast tumors.

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)(6)(7)(8)(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 2ϩselective currents (15)(16)(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.
Real-time PCR-Cultured cells were grown to ϳ80% confluence before RNA extraction. Total RNA was extracted using a Qiagen RNeasy Mini kit following the manufacturer's protocol. cDNA was made from 1 g of RNA reverse transcribed using oligo(dT) primers (Invitrogen, Carlsbad, CA) and SuperScript III reverse transcriptase (Invitrogen). Real-time PCR analysis was performed using a Bio-Rad iCycler and iCycler iQ Optical System Software (Bio-Rad). PCR reactions were performed using Bio-Rad iQ SYBR Green Supermix. The PCR protocol started with 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 30 s at 54.3°C, and 45 s at 72°C. Quantification was measured as sample fluorescence crossed a predetermined threshold value that was just above the background. Expressions of STIM and Orai were compared with those of the housekeeping gene GAPDH and were measured using comparative threshold cycle values. The sense and antisense primers targeting human GAPDH, STIM, and Orai isoforms are described in supplemental Table S1.
Cell Transfections-Sets of either three or four different siRNAs per target gene were initially assessed for their ability to reduce mRNA levels using quantitative PCR (qPCR) as described above. siRNA sequences that induced most significant decrease in their target mRNA were used in Western blotting to confirm protein knockdown (described below). All transfections in MDA-MB231 and MCF 7 were done using Nucleofector device II (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions i.e. using program X-013 and P-020 for MDA-MB231 and MCF7, respectively. As a marker of cell transfection, 0.5 g of green fluorescent protein (GFP) was co-transfected with siRNA for identification of successfully transfected cells during experiments. As a control, we used a non-targeting sequence (Control siRNA). Sequences of all siRNAs used in the study are listed in supplemental Table S2. Cells were transfected with 20 g of siRNA of choice per ϳ1 ϫ 10 6 cells, seeded on either round glass coverslips (for Ca 2ϩ imaging and patch-clamp studies) or plates (for Western blotting) and were assayed between 72 and 96 h post-transfection.
Ca 2ϩ Measurements-Ca 2ϩ measurements were performed as described previously (29 -31). Briefly, coverslips with attached cells were mounted in a Teflon chamber and incubated at 37°C for 40 -60 min in culture medium containing 4 M Fura-2/AM for all cell lines expect MCF 10A and 184A1 where 3 l of Pluronic F127 was added along with 4 M Fura-2/AM in the loading solution. Cells were then washed (3 times) and bathed in HEPES-buffered saline solution (140 mM NaCl, 1.13 mM MgCl 2 , 4.7 mM KCl, 2 mM CaCl 2 , 10 mM D-glucose, and 10 mM HEPES, adjusted to pH 7.4 with NaOH) for at least 10 min before Ca 2ϩ measurements were made. For Ca 2ϩ measurements, fluorescence images of several cells were recorded and analyzed with a digital fluorescence imaging system (InCyt Im2; FIGURE 2. Western blot analysis of STIM1, Orai1, and Orai3 expression in breast cancer cells. A, all 10 breast cancer cells lines were lysed, and 50 g of proteins each were loaded in the same gels. After transfer, membranes were probed with specific antibodies against either STIM1, Orai1, or Orai3 followed by the appropriate secondary antibody coupled to peroxidase as described under "Materials and Methods," and proteins bands were visualized using the ECL kit. B, blots obtained from 3-4 independent experiments were analyzed using Image J software and densitometric ratios to corresponding actin were calculated. All densitometric values were then normalized to those of the normal cell line MCF10A. Intracellular Imaging Inc., Cincinnati, OH). Fura-2 fluorescence at an emission wavelength of 510 nm was induced by exciting Fura-2 alternately at 340 and 380 nm. The 340/ 380 ratio images were obtained on a pixel-by-pixel basis. All experiments were conducted at room temperature. All figures depicting Ca 2ϩ imaging traces are an average from several cells from one coverslip and are representative of several independent recordings.
Whole Cell Patch Clamp Electrophysiology-Patch clamp electrophysiology was essentially conducted as described previously (10,11 . Upon obtaining G⍀ seal and break-in, recordings were made from cells with Ͻ15 M⍀ series resistance. Cells were maintained at 0 mV holding potential during experiments and subjected to voltage ramps from ϩ80 to Ϫ140 mV lasting 250 ms every 3 s. Reverse ramps were designed to inhibit voltage-gated channels. 3 M nimodipine was added to bath solution to generally stabilize membrane patches and reach better seals. The ground silver-silver chloride electrode was connected to the bath  through an agar bridge. Pulses of divalent-free (DVF) solutions were delivered focally to cells with least possible pressure to minimize accidental seal damage, which could lead to leak currents contaminating recordings.
Statistical Analysis-Data are expressed as means Ϯ S.E., and statistical analysis using one way ANOVA was done with Origin software (OriginLab, Northampton, MA). Differences are considered significant when p values are Ͻ 0.05. The p values of Ͻ0.05, Ͻ0.01, and Ͻ0.001 are represented as *, **, and ***, respectively.

RESULTS
SOCE Sensitivity to Pharmacological Blockers in ER ϩ and ER Ϫ Breast Cell Lines-Native SOCE in many cell types has been shown to be inhibited by low concentrations of lanthanides (Gd 3ϩ ; 1-10 M) and by the drug 2-aminoethoxydiphenyl borate (2-APB; 30 -50 M) (5,32). Previous studies comparing the functions of Orai1/2/3 when ectopically co-expressed individually with STIM1 in HEK293 cells showed that while 2-APB inhibits Orai1 and Orai2 it has the ability to activate Orai3 and to alter their ion selectivity by presumably increasing their pore size (33)(34)(35)(36). We undertook a thorough analysis of the pharmacological features of thapsigargin-activated SOCE in 5 different ER ϩ and 5 ER Ϫ breast cell lines (two "normal" breast epithelial cells and 3 cancerous ones) in response to 5 M Gd 3ϩ and 30 M 2-APB using Fura2 imaging. Cell lines were picked in an unbiased manner; the ER ϩ cell lines were: MCF7, BT474, ZR751, T47D, and HCC1500. The ER Ϫ cell lines were: MCF10A and 184A1 normal breast epithelial cells and the cancerous cell lines MDA-MB231, BT20, and HCC1937. Our results show that 5 M Gd 3ϩ inhibits SOCE in both ER ϩ and ER Ϫ cell lines (Fig. 1, A, C, E). However, while 30 M 2-APB essentially abrogated SOCE in ER Ϫ cell lines (Fig. 1,  D and F) the effect of that concentration of 2-APB on ER ϩ cell lines was more complex involving a transient and robust potentiation followed by sustained levels of Ca 2ϩ entry (Fig. 1B). One exception was the cell line T47D where SOCE was equally potentiated by 2-APB, followed by partially inhibited yet sustained Ca 2ϩ entry. There was small and transient potentiation of SOCE by 2-APB in the ER Ϫ cell lines MDA-MB231 and BT20, which is typical of Orai1(11, 37). However, the more robust and sustained potentiation of SOCE by 2-APB in ER ϩ cell lines clearly indicated the presence of a calcium entry channel unique to ER ϩ cells with features similar to those previously described for 2-APB-mediated Ca 2ϩ entry in HEK293 cells ectopically expressing human Orai3 (33)(34)(35)(36). Furthermore, analysis of the extent of SOCE in ER ϩ and ER Ϫ cell lines consistently showed that SOCE is significantly more robust in the ER Ϫ breast cancer cell lines (MDA-MB231, BT20, and HCC1937) compared with the ER ϩ cell lines or the ER Ϫ normal breast epithelial cells (MCF10A, 184A1) (Fig. 1G). Statistical analysis on the extent of Ca 2ϩ release induced by thapsigargin in all 10 cell lines is also represented in Fig. 1G. Western blot analysis of STIM1, Orai1, and Orai3 proteins in all 10 cell lines revealed a significantly higher expression of Orai3 in ER ϩ cell lines while the expression levels of STIM1 and Orai1 were slightly lower compared with the ER Ϫ cell lines ( Fig. 2A), suggesting that in ER ϩ cell lines the balance of STIM1/Orai interaction might be tipped toward STIM1/Orai3. Statistical analysis of the ratio of protein band densitometry to actin normalized to the protein levels of MCF10A from 3-4 independent experiments is shown in Fig. 2B.
For the rest of this study, we focused on two representative and widely studied cell lines, the ER ϩ MCF7 and the ER Ϫ MDA-MB231. Given the robust 2-APB potentiation of thapsigargin-activated SOCE in MCF7 cells, we reasoned that Orai3 might be contributing to SOCE in ER ϩ cells. While 2-APB is a nonspecific drug that has been shown to activate TRPV channels, it does so at concentrations that are 10-fold higher than those used in our study (300 M; (38 -41)). Nevertheless, to rule out contributions from TRPV channels to 2-APB-mediated potentiation of SOCE in ER ϩ cells, we conducted additional experiments in MCF7 cells depicted in Fig. 1H. In these experiments, pre-incubation of MCF7 cells with low concentrations of Gd 3ϩ (1-10 M), known to specifically inhibit Orai channels, was used to block SOCE, followed by thapsigargin stimulation using the standard Ca 2ϩ off/Ca 2ϩ on protocol and subsequent addition of 2-APB. At these low concentrations, Gd 3ϩ does not inhibit TRPV channels (concentrations as high as 500 M Gd 3ϩ were reported to induce a slow developing block of TRPV4 (42)). Fig. 1H shows that the concentration-dependent inhibition of SOCE by Gd 3ϩ directly correlated with the reduction of 2-APB-mediated potentiation, strongly arguing that 2-APB action in MCF7 cells is likely mediated by Orai3 channels.
Electrophysiological Recordings of I CRAC in MDA-MB231 ER Ϫ and MCF7 ER ϩ Breast Cancer Cells-To substantiate these pharmacological data and provide confidence for the involvement of Orai3 channels in mediating SOCE in ER ϩ cells, we conducted whole cell patch clamp electrophysiological recordings in MDA-MB231 ER Ϫ and MCF7 ER ϩ cells. To char- acterize the current mediating SOCE in MDA-MB231 and MCF7 cells, we used the standard method for measuring I CRAC in whole-cell mode with intracellular dialysis of high concentrations of the pH-independent, fast Ca 2ϩ chelator BAPTA, as described previously (10,11,33). Upon break-in with a pipette solution containing 12 mM BAPTA, a relatively small inwardly rectifying I CRAC -like current with very positive reversal potential developed in the presence of external Ca 2ϩ (10 mM) in MDA-MB231 and MCF7 (Fig. 3A-D; 1.05 Ϯ 0.25 pA/pF in MDA-MB231 versus 0.57 Ϯ 0.1 pA/pF in MCF7 cells, at Ϫ140 mV; n ϭ 3). Fig. 3, B and D show the current-voltage relationship (I/V) where currents recorded immediately after break-in were subtracted from fully developed currents revealing inward rectification with very positive reversal potential. To better study these I CRAC -like currents, we conducted subsequent current measurement using divalent-free (DVF) bath conditions to amplify these currents, as described previously (43,44). Wholecell current measurements were performed using reverse voltage ramps to minimize potential contributions from voltagegated channels. Upon break-in in the MDA-MB231 cell line monovalent I CRAC currents were revealed in divalent free conditions that were inwardly rectifying with positive reversal potential similar to those described for Orai1-mediated I CRAC in different cell types (Fig. 3E; 5.9 Ϯ 1.08 pA/pF at Ϫ140 mV, n ϭ 11). These currents were essentially abrogated with 5 M Gd 3ϩ and 50 M 2-APB (Fig. 3, E, I; also see supplemental  (45). These currents displayed the peculiar phenomenon of rapid time-dependent inactivation of inward Na ϩ currents (called depotentiation) in DVF solutions and a very positive reversal potential of ϳϩ50mV in DVF which corresponds to a P Cs /P Na permeability ratio of ϳ 0.1, all characteristics of I CRAC (5) (Fig.  3, E and F). After Gd 3ϩ addition, a small Gd 3ϩ -insensitive current remained presumably corresponding to a background current. After Gd 3ϩ washoff, only a portion of I CRAC was restored (Fig. 3E).
Upon break-in in MCF7, we also observed currents in divalent-free conditions typical of monovalent I CRAC that were inwardly rectifying with positive reversal potential (Fig. 3, G, H;  4.57 Ϯ 1.03 pA/pF at Ϫ140 mV; n ϭ 11). These currents observed in MCF7 also showed depotentiation in DVF solutions, although to a much lesser extent than MDA-MB231 (also see Fig. 3C) consistent with the properties described for ectopically expressed Orai3 channels (33), and a very positive reversal potential of ϳϩ50mV (Fig. 3H). These currents were reversibly inhibited by 5 M Gd 3ϩ (supplemental Fig. S2). However, upon 50 M 2-APB addition, we observed a robust potentiation of these membrane currents by over 3-fold (Fig. 3, G, H, J). Importantly, the currents activated by 2-APB in MCF7 cells showed large outward currents at potentials greater than 0 mV with a left shift of reversal potential that is indicative of change in ion selectivity and Cs ϩ permeation (Fig. 3H), features that are con- sistent with those of 2-APB causing pore enlargement of ectopically expressed Orai3 channels that were recently reported by four independent groups (33)(34)(35)(36). Similar results of 2-APB actions on MDA-MB231 and MCF7 cells were obtained using regular voltage ramps from Ϫ120 mV to ϩ60 mV lasting for short durations (150 ms; supplemental Fig. S1).
We next tested whether 2-APB can cause Ca 2ϩ entry and membrane currents in MCF7 and MDA-MB231 cells, in the absence of store depletion. Fig. 4A shows a Fura2 experiment where cells were incubated in HBSS containing 2 mM Ca 2ϩ and stimulated with 2-APB. In this case, 2-APB caused a small but significant increase in intracellular Ca 2ϩ in MCF7 cells with no apparent effect on MDA-MB231 cells (Fig. 4A). We conducted whole cell patch clamp recordings using a pipette solution where Ca 2ϩ was buffered to 100 nM using the chelator BAPTA to avoid store depletion (buffered 100 nM Ca 2ϩ was achieved using the software Maxchelator). Using DVF protocols we found that while 2-APB failed to generate a membrane current in MDA-MB231 cells (Fig. 4, B and C), it caused significant current in MCF7 cells consistent with the Fura2 data (Fig. 4, D  and E). These 2-APB-activated currents in MCF7 cells were substantially smaller than the currents generated in the same cells when 2-APB was added after store depletion (e.g. see Fig. 3, G and J). Also note the I/V curve (Fig. 4E) that is consistent with the modification of Orai3 pore properties by 2-APB.

SOCE and I CRAC Are
Mediated by Orai1 and Orai3 in MDA-MB231 and MCF7 Cells, Respectively-Because the specificity of 2-APB is questionable despite the low concentrations used in our study (30 -50 M), we conducted molecular knockdown on all the 5 potential SOCE proteins to unequivocally determine the molecular identity of SOCE in ER Ϫ and ER ϩ breast cancer cells. Systematic gene silencing using short interfering RNA (siRNA) against STIM and Orai isoforms was performed to establish the molecular nature of SOCE in MCF7 and MDA-MB231. The siRNA sequences used are listed in the online supplement, some of which have been used previously for successful protein knockdown in human endothelial cells (10). Fig. 5A shows that these siRNA sequences were efficient in causing robust reduction in mRNA expression of their respective target genes (Fig. 5A). Western blots analysis using anti-STIM1, anti-Orai1 and anti-Orai3 antibodies confirmed knockdown of these proteins in MDA-MB231 and MCF7 72 h after transfection with specific siRNA (Fig. 5B  STIM2, Orai2 and Orai3 had no significant effect on SOCE in these cells (Fig. 6). Statistical analysis of all Ca 2ϩ imaging data using target-specific and control siRNA in MDA-MB231 are shown in Fig. 6F. Similar results were obtained in MDA-MB231 cells with a second set of siRNA/shRNA sequences directed against STIM1, Orai1, and Orai3 (supplemental Fig. S3).
Interestingly, protein knockdown studies revealed that SOCE in MCF7 was mediated by STIM1 and Orai3 independently of Orai1 and Orai2 (83.2% Ϯ 3.9 inhibition for siSTIM1 and 67% Ϯ 3.4 for siOrai3); STIM2 knockdown had a modest yet statistically significant effect (21.8% Ϯ 5.9 inhibition for siSTIM2) on SOCE in MCF7 cells (Fig. 7). Statistical analysis of all Ca 2ϩ imaging data using target-specific and control siRNA in MCF7 are shown in Fig. 7F. Similar results were obtained in MCF7 cells with a second set of siRNA/shRNA sequences directed against STIM1, Orai1 and Orai3 (supplemental Fig. S4). Furthermore, Orai3 knockdown was accompanied by strong inhibition of 2-APB-mediated SOCE potentiation in MCF7 cells, further indicating that Orai3 is the major SOCE channel in MCF7 cells (supplemental Fig. S5). Whole-cell patch clamp recordings demonstrated that Orai1 knockdown significantly inhibited store depletion-activated I CRAC membrane currents measured in DVF conditions in MDA-MB231 (54.16% Ϯ 6.55 inhibition, n ϭ 6; Fig. 8, A-C) while Orai3 knockdown inhibited monovalent I CRAC currents in MCF7 cells (58.84% Ϯ 7.04 inhibition, n ϭ 4; Fig. 8, D-F). Current/voltage relationships of monovalent I CRAC currents in MDA-MB231 and MCF7 cells with control siRNA and siRNA against Orai isoforms (Fig. 8, B and E) as well as statistical analysis (Fig. 8, C and F) are shown.

DISCUSSION
During the past few decades, increased understanding of the molecular heterogeneity of breast cancers has led to significant improvements in therapeutic modalities of this disease. In over two-third of breast cancers, ER signaling is a key instigator of tumor growth, and inhibition of this important pathway using ER modulators such as tamoxifen has shown a clear therapeutic benefit (3). However, most of breast cancer therapies currently available are effective only in a proportion of ER ϩ breast cancers and new targets are constantly needed. The newly discovered STIM/Orai pathway is known to control many physiological and pathophysiological functions, but its expression and role in cancer is only starting to emerge. Only recently, STIM1 and Orai1 were shown to play an important role in breast cancer cell migration and metastasis using the MDA-MB231 ER Ϫ cell model (46). Ritchie et al. demonstrated that STIM1 gene expression in primary Wilms tumors is reciprocally regulated by Wilms tumor suppressor 1 (WT1) and early growth response 1 (EGR1) (47). The expression and role of STIM/Orai isoforms in ER ϩ breast cancer progression is currently unknown. Our data clearly implicate a selective involvement of Orai3 in the ER ϩ breast cancer cell line MCF7, one of the most recognized and widely studied cell models for ER ϩ breast cancer. The four other ER ϩ cell types used in this study were picked randomly based solely on their positivity for the ER; according to 2-APB pharmacology and Orai3 protein expression, all these ER ϩ cell lines (BT474, ZR75-1, HCC1500, and T47D) likely use Orai3 as their predominant SOCE pathway. However, the ER Ϫ cell lines use the canonical STIM1/Orai1 as their main SOCE pathway, like all cell types from a number of tissues analyzed so far (10, 11, 15, 18, 19, 22, 24 -28).
Whereas ion channels in general and calcium channels in particular represent attractive targets for drug therapy of human disease, to this day no channel molecule has been exploited for either therapy or diagnosis of breast cancer (48,49). Clearly, many important questions need to be resolved by future studies before Orai3 could represent such a target. Namely, what is the precise role of Orai3 in ER ϩ breast cancer growth, migration and apoptotic/anti-apoptotic pathways? What is the connection between the presence of ER and Orai3? What are the molecular mechanisms that control the functional switch of Orai isoforms in breast cancer? What are the growth factors that mediate their function through Orai3 in ER ϩ breast cancer cells? The answer to these questions might establish Orai3 as a target for therapy of ER ϩ breast cancers. Our study showing a unique involvement of Orai3 in the SOCE pathway in ER ϩ cells represents a first step toward this important goal.