Reagents and antibodies
Fura 2-AM, Fura 4F-AM, and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA). Anti-FLAG antibody (F3165) from Sigma-Aldrich (St. Louis, MO) was used at 1:10,000 dilution. STIM1 was detected using monoclonal antibody (No. 610954, BD Biosciences, San Jose, CA) at 1:2,000 dilution. Anti-actin (sc-1616) and anti-Myc (sc-47694) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) (1:2,000 dilution). Polyclonal rabbit antibody for detection of CRACR2A was generated using purified human CRACR2A protein (Open Biosystems, Huntsville, AL) and used at 1:10,000 dilution.
Plasmids and cells
Full-length cDNAs of human Orai family and STIM1 were subcloned into pMSCV-CITE-eGFP-PGK-Puro16
. For generation of STIM1-YFP and STIM1-mCherry, cDNA was cloned into pEYFP-N1 or pmCherry-N1 plasmids (Clontech, Mountain View, CA). Orai1K85A/K87A
mutant was generated with an extracellular FLAG-tag in pMSCV-CITE-eGFP-PGK-Puro vector and with C-terminal GFP in pEGFP-N1 vector. Full-length cDNAs for human CRACR2A (clone ID 3531511) and mouse CRACR2B (clone ID 5256663) were purchased from Open Biosystems and subcloned into pMSCV-CITE-eGFP-PGK-Puro vector with a FLAG-tag. A plasmid encoding Myc-CRACR2A was generated by subcloning into pcDNA3.1mychis (Invitrogen). WT CRACR2A or CRACR2AEF2MUT
cDNA was subcloned into pEGFP-N1, pEGFP-C1 and pmCherry-N1 plasmids, respectively. CRACR2B cDNA was subcloned into pcDNA3.1mychis and pMSCV-CITE-eGFP-PGK-Puro vectors. Primers used are described in Suppl. Table 1
. HEK293, HeLa and Jurkat T cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA) center. HeLa O+S cells were generated by transducing with viruses encoding FLAG-tagged Orai1 and untagged STIM1 (1:3 ratio) together with hCD2544–45
followed by selection using hCD25 antibody-coated magnetic beads (Invitrogen).
Glycerol gradient analysis, affinity protein purification and mass spectrometry
2 × 1010
HeLa O+S cells were left untreated or treated with 1 µM thapsigargin for 10 min in PBS and cross-linked with 0.5 mM dithiobis succinimidyl propionate (DSP) for 1 h on ice, followed by quenching with 20 mM Tris-Cl, pH 7.5. Cells were lysed in cell lysis buffer (20 mM Tris-Cl, 2 mM EDTA, 100 mM NaCl, 10% glycerol, 0.5% Igepal CA-630, protease inhibitor cocktail [Roche]). Precleared lysates were applied onto a 20–50% glycerol cushion and centrifuged at 288,000g for 16 h. 56 fractions were collected from the top and analyzed by immunoblotting. Orai1 containing fractions were pooled and immunoprecipitated overnight at 4°C with anti-FLAG antibody agarose, washed five times in lysis buffer with high salt (200 mM NaCl), five times with low salt buffer (100 mM NaCl), and eluted with the FLAG-peptide (0.1 mg/ml). The eluates were run on a SDS-PAGE, and analyzed by silver staining or Colloidal Blue Staining Kit (Invitrogen) for mass spectrometry. In-gel digestion with trypsin, nanoLC-MSMS and automated database searching were performed essentially as described previously46
Immunoprecipitation and GST pulldown analysis
2 × 107 transfected HEK293 or HeLa cells were harvested in PBS and DSP cross-linked. Cells were lysed in lysis buffer (20 mM Tris-Cl, 2 mM EDTA, 100 mM NaCl, 10% glycerol, 0.5% Igepal CA-630, protease inhibitor cocktail [Roche]) and centrifuged at 100,000g for 1 h before preclearing with protein G-Sepharose. Lysates were immunoprecipitated overnight with anti-FLAG antibody agarose. Immunoprecipitates were washed in lysis buffer and analyzed by immunoblotting. For pulldown analysis, the GST-fused N terminus of Orai1, STIM1 fragments and 6× His-tagged full-length CRACR2A were incubated with glutathione sepharose 4B beads for 6 h in binding buffer (0.1% Igepal CA-630, 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, protease inhibitors, 10% glycerol, and 100 µg/ml of bovine serum albumin) with or without 2 mM CaCl2. After extensive washing, proteins bound to beads were analyzed by immunoblotting.
Purification of recombinant proteins from insect cells and E. coli
Full-length FLAG-tagged human Orai1 and STIM1 cDNAs were integrated into baculoviruses, expressed in Sf9 cells, and purified following manufacturer’s instructions (Bac-to-Bac Baculovirus Expression Systems, Invitrogen). Orai1, STIM1 fragments and full-length CRACR2A proteins were subcloned into pGEX4T-1 and pET21A plasmids. GST fusion protein expressing transformants were grown in liquid cultures and induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG, 0.2 mM) at 18°C overnight. Subsequently, cells were harvested and resuspended in lysis buffer (50 mM NaH2PO4, 500 mM NaCl, 10% glycerol, pH 8.0) containing protease inhibitors and 0.5% Triton X-100. The lysates were incubated with glutathione sepharose 4B beads for 2 h. The recombinant proteins were eluted using glutathione. For 6× His-tagged proteins, cells were harvested and resuspended in lysis buffer (50 mM NaH2PO4, 500 mM NaCl, 10% glycerol, 0.5% Triton X-100, and 10 mM Imidazole, pH 8.0). After sonication and clearing, the supernatant was incubated with Ni-NTA agarose (Qiagen). The recombinant proteins were eluted in buffer containing 250 mM imidazole.
siRNA/shRNA-mediated depletion and quantitative PCR
0.5 × 106
HEK293 cells were transfected with siRNAs (Dharmacon) using lipofectamine reagent. Cells were re-transfected after 24 h and harvested for RT- PCR or [Ca2+
measurement 3 days after transfection. Jurkat cells were electroporated with 100 nM of siRNA and used for experiments 3 days after transfection. The sequences of siRNAs are: hOrai1, UCACUGGUUAGCCAUAAGA; hCRACR2A, GCUCAGCAGCAGUUGGAAA; hCRACR2B, GAACAUGCAGAAAGAGAAA. Knockdown efficiency was quantified by RT-PCR analysis. Purified RNA was oligo(dT)-primed for first-strand cDNA synthesis (Superscript III kit; Invitrogen). Quantitative SYBR green real-time PCR was performed using an iCycler IQ5 (Bio-Rad). The cDNA levels were normalized to GAPDH. Sequences of the primers are mentioned in Suppl. Table 1
. For shRNA expression, the corresponding sense and antisense siRNA sequences were subcloned into pRetro-Super-puro (OligoEngine). For assessment of expression of CRACR2A and CRACR2B in various human tissues, cDNA made from commercially available purified total RNA (Applied Biosystems, Foster City, CA) was used for PCR with primers (Suppl. Table 1
Single-cell Ca2+ imaging, TIRF and confocal microscopy
T Cells were loaded at 1 × 106
with 1 µM Fura 2-AM for 30 min at 25°C and attached to poly-L-lysine–coated coverslips. Fibroblasts, HeLa and HEK293 cells were grown directly on UV-sterilized coverslips and loaded with 2 µM Fura 2-AM for 45 min. For SOCE measurements in , cells were loaded with 2 µM Fura 4F-AM for 45 min. Intracellular [Ca2+
measurements were performed using essentially the same methods as recently described47
. TIRF microscopy was performed using an Olympus IX2 illumination system mounted on an Olympus IX51 inverted microscope using recently described methods47
. For quantification of TIRF intensity across different cells, individual regions of interest were selected and data were analyzed as the ratio of fluorescence intensity at each time-point (F) to the fluorescence intensity at the start of the experiment (F0
). For confocal analysis, at 6 h after transfection, HEK293 cells were plated onto sterilized coverslips and maintained in complete DMEM for 18 h before imaging in Ringer’s solution containing (in mM): 155 NaCl, 4.5 KCl, 2 CaCl2
, 1 MgCl2
, 10 D-glucose, and 5 Na-HEPES (pH 7.4). For depletion of stores, cells were treated with 1 µM thapsigargin in Ca2+
-free Ringer’s solution (prepared by substitution of CaCl2
with 2 mM MgCl2
and 0.2 mM EGTA) for 5 min. eGFP and mCherry were excited simultaneously at 488 and 594 nm on a Leica SP2 AOBS inverted confocal microscope equipped with a PL APO 60×/NA 1.4 oil immersion objective. Fluorescence emission was collected at 615–700 nm (mCherry) and 510–570 nm (eGFP). Images were processed for enhancement of brightness or contrast using LCSlite (Leica) software.
T cell purification, cytokine measurement, Annexin V staining and immunocytochemistry
T cell purification, activation and transduction were carried out as previously described47
. Cells were harvested and used for measurement of Ca2+
entry or RNA extraction after 5–6 days of stimulation. Jurkat T cells were electroporated with 20 µg of plasmids. For intracellular cytokine staining, Jurkat T cells were stimulated with 10 nM phorbol 12-myristate 13-acetate (PMA) and 1 µM ionomycin for 16 h. Cells were fixed with 4% paraformaldehyde in PBS, and permeabilized in saponin buffer (PBS, 0.5% saponin, 1% bovine serum albumin, and 0.1% sodium azide). Cells were stained with Phycoerythrin-conjugated human IL-2 antibody (eBioscience) for 20 min, washed and analyzed. For apoptosis assay, transfected Jurkat cells were stained using Annexin V staining kit (BD Biosciences). Cells were examined on a FACSCalibur flow cytometer (BD Biosciences). Immunocytochemistry and flow cytometry on HEK293 cells expressing WT and mutant Orai1 constructs were performed as described47
Measurement of CRAC currents by whole cell patch clamp recording
CRAC current measurements were carried out as previously described47
Fluorescence protease protection (FPP) assays
FPP assays were performed using recently described protocol33
. Briefly, HEK293 cells expressing GFP-CRACR2A or CRACR2A-GFP on UV-sterilized coverslips were perfused with KHM buffer (110 mM potassium acetate, 20 mM HEPES, and 2 mM MgCl2
). For permeabilization, 75 µM of digitonin was used. Subsequently 50 µg ml−1
of proteinase K containing KHM buffer was perfused. GFP images were acquired at every 5-s interval after background subtraction. For each experiment, 50–100 individual HEK293 cells were analyzed using OriginPro (Originlab).
Detection of Ca2+ binding by using 45Ca2+ Overlay method
(1 mCi, 37 MBq) was purchased from NEN. The protocol was modified from a previously described method48
. Purified 6× His-fusion proteins and CaM (EMD Biosciences) were transferred to a nylon membrane after separating on a 12% SDS PAGE. The blot was extensively washed for 3 × 30 min with binding buffer (in mM: 60 KCl, 5 MgCl2
, and 10 imidazole-HCl, pH 6.8). The overlay assay was performed by incubating the blot with 8.8 µM 45
at 25°C for 1 h, followed by 3 × 5 min washes in dH2
O. After autoradiography, the blot was stained with Ponceau S solution for detection of proteins.
Database accession numbers
Genbank accession numbers for CRACR2A proteins are; Homo sapiens (NP_116069.1), Pan troglodytes (XP_001156341), Mus musculus (NP_001028636), Danio rerio (XP_001922837.1). Genbank accession numbers of CRACR2B proteins are: Mus musculus (NP_001020274), Homo sapiens (registered as GenBank accession number, GU952799).
Statistical analysis was carried out using Student’s t-test.