The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress.

Cell culture

The glutamate-resistant HT22R cell line was generated from the parental mouse hippocampal cell line HT22 as previously described. 19 Both cells lines, HT22S and HT22R, were cultured in DMEM high glucose (PAA Laboratories, Pasching, Austria ) supplemented with 5% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U per ml penicillin and 100 μg per ml streptomycin (Gibco, Darmstadt, Germany) in a humidified incubator with 5% CO 2 and 95% air.

Cell survival assays

HT22 cells were seeded in 96-well plates at a density of 5000 cells/well and 24 h later subjected to cell death experiments. siRNA-transfected cells were seeded 24 h post transfection. Pharmacological agents were added together with, or at specified time periods after, induction of cell death by the indicated drugs. Cell viability was quantified 16–24 h after stress onset with the cell titer blue (CTB) reagent (Promega, Madison, WI, USA). Alternatively, in some experiments, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used. 19 For CTB fluorescence, emission was measured at 590 nm after excitation at 562 nm using a GENios Pro microplate reader (Tecan, Männedorf, Switzerland); for the MTT-assay, absorption was measured at 570 nm. Calcium live-cell imaging Fura2 Ca 2+ imaging experiments were performed on a BD Pathway 855 High Content Imaging System (BD Biosciences, Heidelberg, Germany). The cells were seeded in 96-well imaging plate (BD Bioscience) the day before the experiment in a density of 5000 cells/well and loaded with 5 μℳ Fura2-AM (Molecular Probes, Darmstadt, Germany) in HBSS for 30 min before the experiment. Measurement was performed with excitation at 340 and 380 nm for ratiometric analysis and pictures were taken with a delay of 5 s. For comparison of SOCE in HT22S and HT22R, cells were seeded on 12 mm coverslips at a density of 100 000/well, loaded with Fura2-AM and placed into a flow chamber. The measurement was performed using an Olympus IX81 fluorescence microscope with the cell^R imaging software. Images were acquired at 340 and 380 nm excitation and the ratio was calculated every 5 s for every single cell. After 1 min of baseline recording, ER-calcium stores were depleted in EGTA buffer (Ca 2+ -free HBSS supplemented with 0.5 mℳ EGTA, 20 mℳ HEPES, 1 mℳ MgCl 2 and 1 g per l Glucose) with 2 μ ℳ thapsigargin for 6 min and subsequently Ca 2+ was readded by changing the buffer to HBSS.

Electrophysiology

We used whole-cell patch clamping to measure currents through ion channels of single cells. Whole-cell recordings were performed using an Axopatch 200B patch clamp amplifier (Axon Instruments, Sunnyvale, CA, USA) and digitized with Digidata 1332 (Axon Instruments). For SOC channels recordings the pipette solution contained (in mℳ) 120 CsCl, 5 Bapta-Na, 30 Cs-HEPES pH 7.3, 1.5 MgCl 2 , 4 Na 2 ATP, 0.4 Na 2 GTP and 1.6 CaCl 2 (pCa 7.0). Extracellular solution contained (in mℳ) 140 NMDG-Asp, 10 BaCl 2 , 10 Cs-HEPES, pH 7.3. During recording the currents were sampled at 5 kHz and filtered digitally at 500 Hz, the pClamp9 software (Axon Instruments) was used for data acquisition and analysis. In all whole-cell experiments the holding potential was −40 mV, periodically (once every 5 s) the membrane potential was stepped to −100 mV (for 30 ms) and a 200 ms voltage ramp to +100 mV was applied. Currents were evoked by application of 1 μ ℳ thapsigargin (Sigma, Munich, Germany) in external solution, which was added to the bath perfusion. The traces recorded before current activation were used as templates for leak subtraction. Whole-cell currents were normalized to the cell capacitance. The mean value of cell capacitance was 19 pF±5 (total number of experiments n =20).

Quantitative RT-PCR Whole-cellular

RNA was isolated using ZR RNA MiniPrep Kit (Zymo, Irvine, CA, USA) and transcribed into cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). RT-PCR analysis was performed on a 7500 Fast cycler (Applied Biosystems) with FAST BLUE qPCR MasterMix (Eurogentec, Cologne, Germany) running the 7500 standard program. Primers and Probes for STIM1 and 2, ORAI1 and TRPM7 were designed by the universal probe library assay design center (Roche, Mannheim, Germany) and the hypoxanthine-phosphoribosyltransferase gene ( HPRT ) served as an endogenous control with individually designed primers and probe purchased from MWG (Ebersberg, Germany). siRNA transfections Flexitube siRNAs against ORAI1, STIM1, STIM2 and TRPM7 were purchased from Qiagen (Hilden, Germany) (no.SI00972251, no.SI00972272, no.SI1435623, no.SI1435637, no.SI01435665, no.SI01435672, no.SI02694727 and no.SI02742663) and transfected into cells with Lipofectamine RNAiMAX (Invitrogen, Darmstadt, Germany) when cells were grown to 70–80% confluence in six-well plates. Successful knockdown was verified by RT-PCR or immunoblot.

Immunoblotting

Cells were lysed in ice-cold RIPA buffer (Thermo Fisher Scientific) containing mini complete protease inhibitor cocktail (Roche) and centrifuged for 30 min at 16 000 g. The supernatants were separated on 8–16% polyacrylamide gels (Thermo Fisher Scientific), transferred onto nitrocellulose membranes with the iBlot System (Invitrogen, Darmstadt, Germany) and blocked in 3% nonfat dry milk in phosphate-buffered saline containing 0.5% Tween-20 (PBS-T) for 1 h at room temperature before overnight incubation with primary antibodies against STIM1 (BD Biosciences, no.610955 1 : 250), STIM2 (Cell Signaling, Danvers, MA, USA, no.4917 1 : 1000), ORAI1, Alomone Jerusalem, Israel, no.ACC-062 1 : 500), Actin (Millipore, Billerica, MA, USA, no.MAB1501 1 : 5000) or GAPDH (Cell Signaling, no.2118 1 : 5000) followed by α -mouse (respectively rabbit) IgG (Fc) infrared fluorescence−conjugated secondary antibody (Licor, Lincoln, NE, USA, 1 : 30 000). The membranes were scanned for infrared fluorescence at 680 or 800 nm using the Odyssey system (Licor) and the signal was analyzed quantitatively with the image-processing software ImageJ ( http://rsbweb.nih.gov/ij/ ).

Long-term calcium live-cell imaging

GCaMP5 was a kind gift of Douglas S. Kim (Howard Hughes Medical Institute, Ashburn) and subcloned with EcoRI and NotI into the IRES-RFP-containing vector PB531A-1 (System Biosciences, Mountain View, CA, USA). Cells were transfected with GCaMP5-IRES-RFP together with two different siRNAs against ORAI1 or non-targeting control-siRNA with Lipofectamine 2000 (Invitrogen) in six-well plates and 24 h later transferred to 96-well imaging plates (BD Biosciences) at a density of 5000 cells/well. For calcium imaging, phenol red containing medium was replaced by colorless DMEM (PAA Laboratories, Pasching, Austria). Two hours before the onset of measurement, 25 mℳ glutamate was added to the cells, control wells were supplemented with vehicle and the plate was incubated in the BD Pathway 855 at 37 °C with 5% CO2 and 95% air. For pCPT-cGMP-induced Ca 2+ influx, measurement was started immediately after addition of 2.5 mℳ pCPT-cGMP. Cytosolic calcium was monitored by GCaMP5 and normalized to RFP fluorescence; pictures were taken every 15 min. For analysis of glutamate-induced Ca 2+ peaks, the maximal ratio was divided by the average ratio and a threshold was defined by the mean of control-siRNA transfected cells. Every cell above that threshold was counted as Ca 2+ peak positive. For analysis of pCPT-cGMP-induced Ca 2+ influx, the slope of Ca 2+ increase was calculated over the first 2 h of the measurement.

Statistical analysis

Data were analyzed as mean±S.E.M. and the statistical significance assessed using two-tailed t -tests or analysis of variance (ANOVA) with Tukey's Multiple Comparison Test as indicated.