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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.041640

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A Stim1-dependent, noncapacitative Ca²⁺-entry pathway is activated by B-cell-receptor stimulation and depletion of Ca²⁺

¹ Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
² Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

View larger version (25K): [in this window] [in a new window]   Fig. 1. BCR-mediated La3+-resistant Ca2+ entry into DT40 cells. (A) WT DT40 cells were stimulated with 3 µg/ml anti-IgM in a nominally Ca2+-free medium, followed by the addition of 1.3 mM Ca2+ in the absence (black) or presence (red) of 0.3 µM La3+. Typical Ca2+ responses in single DT40 cells in the absence (black) and presence (red) of La3+ are shown. Fluorescence ratios (340 nm/380 nm) were normalized to the ratio obtained just before addition of Ca2+ (relative ratios). Horizontal bars indicate the presence of anti-IgM and Ca2+. Time points for the addition of reagents by exchanging medium are indicated by the arrows. The red arrow indicates the time of La3+ addition. (B) Normalized fluorescence ratios were collected at 5 second intervals from cells examined in the absence (black) or presence (red) of La3+. The gray trace indicates the normalized fluorescence ratio in the absence of La3+ and anti-IgM stimulation. Traces shown are the means ± s.e.m. of 187 and 204 cells in the absence or presence of La3+, respectively, and 237 unstimulated cells. Horizontal bars indicate the presence of anti-IgM and Ca2+. (C) WT cells were treated with 1 µM ThG in nominally Ca2+-free medium, followed by the addition of Ca2+ in the absence (black) or presence (red) of La3+. Traces are shown as the means ± s.e.m. of 125 and 191 cells in the absence or presence of La3+, respectively. Horizontal bars indicate the presence of ThG and Ca2+. (D) Anti-IgM (2 µg/ml) was added before the addition of Ca2+ in the presence La3+. The trace shown is the mean ± s.e.m. of 234 cells. Horizontal bars indicate the presence of ThG, anti-IgM, La3+ and Ca2+. (E,F) Effects of La3+ on BCR- or ThG-induced Ca2+ entry into WT (E) and IP3R-KO (F) DT40 cells. Maximal increases in fluorescence ratios following the addition of Ca2+ to cells treated with 0-5 µg/ml anti-IgM or 1 µM ThG in the absence (black columns) or presence (red columns) of La3+. Anti-IgM, ThG, La3+, and Ca2+ were applied at time points depicted in B or C. The total number of cells examined is shown above each column.

View larger version (19K): [in this window] [in a new window]   Fig. 2. BCR-mediated La3+-resistant Ca2+ entry after store depletion in WT and IP3R-KO DT40 cells. (A,B) WT (A) and IP3R-KO (B) DT40 cells were pretreated for 10 minutes with 1 µM ThG in nominally Ca2+-free medium, after which time La3+, Ca2+, and either 2 µg/ml anti-IgM (blue) or vehicle (gray) were added. Traces shown are the means ± s.e.m. of 191 and 202 cells in the absence or presence of anti-IgM in WT cells, and 173 and 198 cells in the absence or presence of anti-IgM in IP3R-KO cells, respectively. The horizontal bars in A and B indicate the presence of ThG, La3+ and Ca2+. Time points for the addition of reagents by exchanging medium are indicated by arrows. Anti-IgM was added at the time indicated by the blue arrow. (C) La3+-resistant Ca2+ entry in the presence of increasing anti-IgM stimulation of ThG-pretreated WT (black) and IP3R-KO (white) cells. Increases in fluorescence ratios at 10 minutes after the addition of various concentrations of anti-IgM (0 µg/ml to 5 µg/ml) to ThG-pretreated cells in the presence of La3+ and Ca2+. Data shown are means ± s.e.m. The total number of cells examined is shown above each column. **P<0.01.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Effects of genistein, U-73122, and staurosporine on BCR-mediated Ca2+ entry following ThG pretreatment. (A-D) WT DT40 cells were treated with ThG in nominally Ca2+-free medium. Subsequently, cells were treated with 50 µM genistein (A), 100 nM staurosporine (B), 2 µM U-73122 (C) or 2 µM U-73343 (D) followed by addition of La3+ and Ca2+ and stimulation with anti-IgM (blue) or vehicle (gray). Traces shown are the means ± s.e.m. of 221 and 201 cells in the absence or presence of anti-IgM in A; 177 and 185 cells in the absence or presence of anti-IgM in B; 180 and 166 cells in the absence or presence of anti-IgM in C; and 188 and 218 cells in the absence or presence of anti-IgM in D, respectively. The horizontal bars indicate the presence of inhibitors, ThG, La3+ and Ca2+. Time points for the addition of reagents by exchanging medium are indicated by the arrows. Anti-IgM was added at the time indicated by the blue arrow. (E) Effects of various inhibitors on BCR-mediated La3+-resistant Ca2+ entry in ThG-pretreated WT and IP3R-KO DT40 cells. Increases in fluorescence ratios of the ThG-pretreated cells were obtained 10 minutes after the addition of 2 µg/ml anti-IgM (black) or vehicle (white) in the presence of La3+, Ca2+, and each inhibitor. The total number of cells examined is shown above each column. **P<0.01.

View larger version (11K): [in this window] [in a new window]   Fig. 4. BCR-mediated Ca2+ entry in Stim1-KO cells. Stim1-KO cells were transfected with the YFP-Stim1 expression vector and Ca2+ responses in YFP-positive (black) and YFP-negative (red) cells were examined. (A) Cells were stimulated with 2 µg/ml anti-IgM in nominally Ca2+-free medium, followed by the addition of La3+ and Ca2+. (B,C) Cells were treated with ThG in nominally Ca2+-free medium, followed by the addition of La3+, Ca2+ and 2 µg/ml anti-IgM (B) or Ca2+ (C). Traces shown are the means ± s.e.m. of normalized fluorescence ratios from Stim1-KO (YFP-negative; red) and YFP-Stim1 overexpressing Stim1-KO (YFP-positive; black) DT40 cells (17 and 217 YFP-positive and -negative cells, respectively, in A; 17 and 229 YFP-positive and -negative cells, respectively, in B; and 7 and 227 YFP-positive and -negative cells, respectively, in C). Horizontal bars indicate the presence of ThG, anti-IgM, La3+ and Ca2+. Time points for the addition of reagents are indicated by the arrows.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Effect of Orai1 siRNA (siOrai1) and Orai2 siRNA (siOrai2) on CCE and BCR-mediated Ca2+ entry following treatment with ThG. (A) WT DT40 cells were cotransfected with the EYFP expression vector and siOrai1 and/or siOrai2. Cells were treated with ThG in nominally Ca2+-free medium and then Ca2+ was added. Traces shown are the means ± s.e.m. of normalized fluorescence ratios from YFP-positive siOrai1-transfected cells (gray), YFP-positive siOrai1- and siOrai2-transfected cells (red), YFP-transfected cells (blue) or untransfected cells (black). The horizontal bars indicate the presence of ThG and Ca2+. Arrows indicate the times of addition of reagents by exchanging medium. Results were obtained from 4-12 independent experiments, and the total number of cells examined is shown above each column in D. (B) WT DT40 cells were cotransfected with the EYFP expression vector and siOrai1 and/or siOrai2. Cells were treated with ThG in nominally Ca2+-free medium and then 3 µg/ml anti-IgM was added in the presence of La3+ and Ca2+. Traces shown are the means ± s.e.m. of normalized fluorescence ratios from YFP-positive siOrai1- and siOrai2-transfected cells (red), YFP-transfected cells (blue) or untransfected cells (black). The horizontal bars indicate the presence of anti-IgM, ThG, La3+ and Ca2+. Results were obtained from 8-10 independent experiments, and the total number of cells examined is shown above each column in panel D. (C) Responses in YFP-positive siOrai1- and siOrai2-transfected cells (red) or YFP-transfected cells (blue) using an expanded scale for the boxed region in B. (D) Effect of siOrai1 and/or siOrai2 on CCE or B-SOC in WT DT40 cells. Increased fluorescence ratios were induced by the addition of Ca2+ to ThG-pretreated WT DT40 cells (CCE) or by the addition of 3 µg/ml anti-IgM in the presence of La3+ and Ca2+ to ThG-pretreated (B-SOC) WT DT40 cells. Maximal increases in fluorescence ratios following the addition of Ca2+ to 1 µM ThG-treated cells (CCE), or the fluorescence ratios 10 minutes after addition of anti-IgM to ThG-pretreated cells (B-SOC) in the presence of La3+ and Ca2+, are shown. Black, YFP-negative cells; blue, YFP-positive cells in samples transfected only with YFP; gray, YFP-positive cells in siOrai1-transfected samples; red, YFP-positive cells in siOrai1 and siOrai2 transfected samples. The total number of cells examined is shown above each column. **P<0.01.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Dynamic behavior of YFP-Stim1 in DT40 cells. (A-C) Stim1-KO cells were transfected with the YFP-Stim1 expression vector. YFP-Stim1-expressing Stim1-KO cells were incubated in nominally Ca2+-free medium for 5 minutes (A) and stimulated with 3 µg/ml anti-IgM in Ca2+-free medium for 10 minutes (B), followed by addition of ThG and incubation for 5 minutes (C). YFP signals of Stim1 near the plasma membrane were monitored by TIRF microscopy. Panels a-f are TIRF images at the beginning (a) and 5 minutes after (b) incubation in nominally Ca2+-free medium, 5 minutes (c) and 10 minutes (d) after the addition of anti-IgM stimulation and 3 minutes (e) and 5 minutes (f) after the addition of ThG. Small TIRF images (a1, a2, c1, c2 and e1) were obtained at 5 second intervals in the areas indicated in panels a, c and e, respectively. Images in a1 and a2 were obtained approximately 3 minutes after incubation in Ca2+-free medium. Images c1 and c2 were obtained approximately 8 minutes after stimulation with anti-IgM. Image e1 was obtained approximately 1.5 minutes after the addition of ThG. The movement of YFP-Stim1 from the white to blue arrowheads is shown on each image. (D) Time-dependent changes in TIRF fluorescence intensity in YFP-Stim1-expressing cells. The trace shown is the mean ± s.e.m. of percentage change in fluorescent intensity, where values at each time point (1 second intervals) were normalized by the average TIRF fluorescence intensity measured for 5 minutes before the addition of anti-IgM and the intensity at 10 minutes after the addition of ThG as the minimum and maximum, respectively. The results are the mean ± s.e.m. of four cells from independent experiments. The horizontal bars indicate the presence of anti-IgM and ThG. The arrows indicate the times of medium exchange.

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