Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Functional analysis of the N-FLAG-tagged Orai1 channel. A, thapsigargin (TG)-induced [Ca2+]i rises in Orai1-expressing HEK293 cells. Left, average time course of Ca2+ responses induced by 2 μm TG in Orai1 (gray triangles)-, N-FLAG-tagged Orai1 (black circles)-, and control vector (white circles)-transfected HEK293 cells (n = 27–28). The perfusion solution was first changed to 0.5 mm EGTA containing Ca2+-free HBS (EGTA), and 2 μm TG was applied to the cells in the absence of extracellular Ca2+. Eleven minutes after the application of TG, 2 mm Ca2+ was added to the extracellular solution. Right, maximum [Ca2+]i rises (Δ[Ca2+]i) induced by TG in EGTA and those after re-addition of 2 mm external Ca2+. B, TG-induced [Ca2+]i rises in Orai1 and STIM1-coexpressing HEK293 cells. Left, average time course of Ca2+ responses induced by 2 μm TG in Orai1 plus STIM1 (gray triangles)- and N-FLAG-tagged Orai1 plus STIM1 (black circles)-cotransfected HEK293 cells and control vector (white circles)-transfected HEK293 cells (n = 25–41). Right, Δ[Ca2+]i induced by TG in EGTA and those after re-addition of 2 mm external Ca2+. C, activation of CRAC current induced by 40 μm IP3 in Orai1- and STIM1-coexpressing cells. Left, representative current-voltage relationships in Orai1 plus STIM1 (a)- and N-FLAG-tagged Orai1 plus STIM1 (b)-cotransfected HEK293 cells and control vector (c)-transfected HEK293 cells. Data represent leak-subtracted current densities (pA/picofarad) evoked by 50-ms voltage ramps from -100 to +100 mV. Right, average CRAC current densities at -80 mV in Orai1 plus STIM1 (n = 11), N-FLAG-tagged Orai1 plus STIM1 (n = 9) and control (n = 9) cells. **, p < 0.01; and ***, p < 0.001.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Functional Assay, Expressing, Control, Plasmid Preparation, Transfection, Activation Assay

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Purification of Orai1 protein from transiently expressed HEK293 cells. N-terminally FLAG-tagged Orai1 was solubilized using DDM, then purified with immunoaffinity chromatography followed by SEC. A, SDS-PAGE of aliquots at each step in anti-FLAG affinity chromatography visualized by silver staining, and B, Western blotting using anti-FLAG antibody. Orai1 proteins are demonstrated as glycosylated (indicated as Orai1*, 44 kDa) and unglycosylated (Orai1, 36 kDa) forms. C, SEC profile using Superdex 200 column. The Orai1-rich eluates from the immunoaffinity column were concentrated using the Microcon YM-100 filter unit and analyzed by Superdex 200 SEC. Orai1 was mainly eluted in a peak at 1.04 ml elution (indicated by an arrow). Another peak at 1.96 ml contains FLAG-peptide. D, SDS-PAGE analysis of the SEC fractions, visualized by silver staining. Intensity of the bands of Orai1 in the gel corresponds well to the absorbance in SEC. The peak fraction at 1.04 ml (arrow) was used for electron microscopic analysis.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Purification, Chromatography, SDS Page, Affinity Chromatography, Silver Staining, Western Blot

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Chemical cross-linking, native PAGE and Stokes radius of Orai1. A, chemical cross-linking of Orai1. Purified proteins with (right panel) or without (left panel) PNGase F treatment were reacted with glutaraldehyde (GA) at indicated concentrations and separated by SDS-PAGE followed by silver staining. Left, the bands of Orai1 at 36 kDa and 44 kDa were shifted upward to ∼200 kDa, suggesting glycosylated tetramer formation (44 × 4 = 176 kDa). Right, the deglycosylated band at 36 kDa was also shifted upward by the cross-linking to ∼170 kDa. B, native gel electrophoresis of Orai1. Purified proteins were separated in native gel and visualized by silver staining. The Orai1 proteins were detected as a broad band at 520 kDa, and shifted downward to 380 kDa after PNGaseF treatment. This much larger estimation of the molecular weight may suggest the swollen structure of the Orai1 molecule. C, Stokes radius (Rs) of purified Orai1 calculated from SEC. From the calibration curve obtained from the standards, the Rs of glycosylated and deglycosylated Orai1 were calculated to be 82.7 ± 1.1Å(n = 5) and 75.0 ± 1.0Å(n = 3), respectively. Standard proteins are thyroglobulin (Rs, 85 Å), ferritin (Rs, 61 Å), catalase (Rs, 52 Å), and aldolase (Rs, 48 Å).

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Clear Native PAGE, Purification, SDS Page, Silver Staining, Nucleic Acid Electrophoresis, Molecular Weight

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Electron microscopy of negatively stained Orai1. A, Orai1 particles were observed as variously shaped but uniformly sized projections of the molecule. For statistical analysis, 3681 particles were picked up by a combination of the auto-accumulation method (39) and the three-layer neural network (NN) auto-picking system (36, 37). B, assignment of the cytoplasmic domain of Orai1. Gallery of negatively stained complexes between N-FLAG Orai1 and anti-FLAG antibodies (panels 1–4) and between C-FLAG Orai1 and anti-FLAG antibodies (panels 5 and 6). Both the cytoplasmic N and C termini were assigned at the smaller end of the Orai1 molecule. The binding of multiple antibodies to a single Orai1 molecule is frequently observed (panels 3 and 4). Gallery of complex between N-FLAG Orai1 and Fab-gold (panels 7 and 8). Fab fragments of anti-FLAG antibodies are conjugated with colloidal gold, and then mixed with purified Orai1. The gold conjugate binds to similar positions as indicated in panels 1–4. C, assignment of the extracellular glycan moiety of Orai1. Gallery of complex between N-FLAG Orai1 and wheat germ agglutinin-gold (panels 1–4). The gold conjugate binds to the larger end of the Orai1 molecule. Contours are shown below each image. Scale bars represent 100 Å.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Electron Microscopy, Staining, Binding Assay, Purification, Glycoproteomics

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Three-dimensional reconstruction of Orai1. A, raw images of Orai1 with different Euler angles (row 1) are compared with their corresponding class averages (row 2), the surface representations of the three-dimensional reconstruction (row 3) and the reprojections from the reconstruction (row 4) along the corresponding Euler directions. The Euler angle (α, β, γ) is denoted below each column. Protein is displayed in bright shades. Scale bar represents 100 Å. B, plot of the Euler (β, γ) angles of 70 adopted class averages. Orai1 molecules are almost randomly oriented on the carbon surface. C, FSC function indicates a resolution limit of 21 Å by the FSC > 0.5 criterion.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques:

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Structural features of Orai1. A, surface representations of Orai1 viewed from four different Euler angles (α, β, γ): 1 (0, 180, -45), 2 (0, 0, -45), 3 (0, 90, -45), and 4 (0, 90, 0). The molecular mass enclosed by the isosurface is 210 kDa, corresponding to 149% of the tetrameric Orai1 protein. Protein is displayed in bright shades. Two blue lines, ∼30 Å apart in panels 3 and 4, indicate the putative position of the lipid bilayer. A red arrowhead in panel 4 indicates one of inverted-V-shaped orifices in the cytoplasmic domain. B, horizontal sections parallel to the membrane plane. Sections at 7.7-Å intervals through the molecule are indicated by numbers 1–20 on the left side of panel A3. The internal structure of the Orai1 molecule is sparse. Scale bar represents 100 Å.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Membrane

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Predicted topology of Orai1 and a docking model with STIM1. A, schematic diagram of N-FLAG Orai1. Orai1 is a highly glycosylated protein with glycan moiety of ∼8 kDa per subunit. The Orai1 polypeptide has a cytosolic N terminus, four TM segments (yellow columns: S1–S4), and a cytosolic C terminus with a putative coiled-coil sequence (green box) involved in binding with STIM1. The FLAG tag (red box) was introduced in the N terminus. B, illustration of putative docking between Orai1 and STIM1. The three-dimensional reconstruction in this study suggests that the cytoplasmic domain of Orai1 is long enough for direct association with STIM1. STIM1 contains a coiled-coil motif (green box) in the cytoplasm, a single TM segment (yellow column), a sterile α motif domain (purple box), and an EF-hand motif (blue crescent) in the ER lumen.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Glycoproteomics, Sequencing, Binding Assay, FLAG-tag, Sterility