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STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veliçelebi G, Stauderman KA - J. Cell Biol. (2005)

Bottom Line: RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry.Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells.We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

View Article: PubMed Central - PubMed

Affiliation: Torrey Pines Therapeutics, Inc., La Jolla, CA 92037, USA.

ABSTRACT
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

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Suppression of Ca2+ signal by Stim dsRNA treatment. (A) [Ca2+]i in single cells treated with CG1560 dsRNA (control). Solution exchanges are indicated by solid (S2 Ringer with 2 mM Ca2+), open (Ca2+ free), and gray (Ca2+-free containing 1 μM TG) bars, respectively. Vertical lines indicate the time of solution exchange. (B) Intracellular Ca2+ responses in S2 cells treated with Stim dsRNA. (C) Averaged values ± SEM for control cells (n = 46 cells in two representative experiments, white bars) and Stim dsRNA-treated (n = 197 cells in three representative experiments, gray bars): resting [Ca2+]i; peak [Ca2+]i during the TG-evoked release transient; maximal and sustained (5 min) [Ca2+]i after readdition of 2 mM external Ca2+. The values of maximal [Ca2+]i and sustained [Ca2+]i in control and Stim suppressed cells are significantly different (P < 5 × 10−6).
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fig2: Suppression of Ca2+ signal by Stim dsRNA treatment. (A) [Ca2+]i in single cells treated with CG1560 dsRNA (control). Solution exchanges are indicated by solid (S2 Ringer with 2 mM Ca2+), open (Ca2+ free), and gray (Ca2+-free containing 1 μM TG) bars, respectively. Vertical lines indicate the time of solution exchange. (B) Intracellular Ca2+ responses in S2 cells treated with Stim dsRNA. (C) Averaged values ± SEM for control cells (n = 46 cells in two representative experiments, white bars) and Stim dsRNA-treated (n = 197 cells in three representative experiments, gray bars): resting [Ca2+]i; peak [Ca2+]i during the TG-evoked release transient; maximal and sustained (5 min) [Ca2+]i after readdition of 2 mM external Ca2+. The values of maximal [Ca2+]i and sustained [Ca2+]i in control and Stim suppressed cells are significantly different (P < 5 × 10−6).

Mentions: Population measurements of intracellular Ca2+ would include contributions of cells that had not been affected by gene silencing. Therefore, to clarify effects of gene silencing at the level of single cells we evaluated Ca2+ signaling and CRAC currents in S2 cells that were pretreated with dsRNA. Fig. 2 A illustrates Ca2+ signals in response to TG-evoked store depletion in eight individual control cells treated with dsRNA for the cell adhesion molecule CG1560. Removal of Ca2+ and readdition of Ca2+ produced no significant change in the Ca2+ signal. Addition of TG in zero-Ca2+ solution produced a transient rise in [Ca2+]i due to release of Ca2+ from the ER; the release transient reached a peak that averaged ∼200 nM before declining. Upon readdition of Ca2+, store-operated Ca2+ influx was revealed as a rise in [Ca2+]i that was sustained and often exceeded 1 μM. In cells that were pretreated with Stim dsRNA, resting [Ca2+]i and the release transient were not altered significantly, but the rise in Ca2+ upon readdition of external Ca2+ was prevented in most of the single cells (Fig. 2 B). In contrast, suppression of CG8743 produced a significant elevation of the resting [Ca2+]i to ∼60% higher than in control cells (135 CG1560 dsRNA-treated control cells; 118 CG8743 dsRNA-treated cells; P < 5 × 10−6), which is consistent with results from the initial screen (Fig. 1 F). However, CG8743 dsRNA did not alter the Ca2+-release transient (not depicted) or Ca2+ influx evoked by TG (Fig. 1 E). Fig. 2 C summarizes the results of three control experiments and six experiments with suppression of Stim. Occasionally, cells in the Stim dsRNA group exhibited normal Ca2+ responses similar to control cells, suggesting either that these cells did not effectively take up the dsRNA, that gene suppression in these cells was less efficient, or that some cells with effective gene suppression express normal SOC influx, as found in DT40 cells after knockout of TRPC1 (Mori et al., 2002). These results demonstrate at the single-cell level that suppression of Stim effectively blocks both the early and sustained components of Ca2+ entry evoked by TG.


STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veliçelebi G, Stauderman KA - J. Cell Biol. (2005)

Suppression of Ca2+ signal by Stim dsRNA treatment. (A) [Ca2+]i in single cells treated with CG1560 dsRNA (control). Solution exchanges are indicated by solid (S2 Ringer with 2 mM Ca2+), open (Ca2+ free), and gray (Ca2+-free containing 1 μM TG) bars, respectively. Vertical lines indicate the time of solution exchange. (B) Intracellular Ca2+ responses in S2 cells treated with Stim dsRNA. (C) Averaged values ± SEM for control cells (n = 46 cells in two representative experiments, white bars) and Stim dsRNA-treated (n = 197 cells in three representative experiments, gray bars): resting [Ca2+]i; peak [Ca2+]i during the TG-evoked release transient; maximal and sustained (5 min) [Ca2+]i after readdition of 2 mM external Ca2+. The values of maximal [Ca2+]i and sustained [Ca2+]i in control and Stim suppressed cells are significantly different (P < 5 × 10−6).
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Related In: Results  -  Collection

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fig2: Suppression of Ca2+ signal by Stim dsRNA treatment. (A) [Ca2+]i in single cells treated with CG1560 dsRNA (control). Solution exchanges are indicated by solid (S2 Ringer with 2 mM Ca2+), open (Ca2+ free), and gray (Ca2+-free containing 1 μM TG) bars, respectively. Vertical lines indicate the time of solution exchange. (B) Intracellular Ca2+ responses in S2 cells treated with Stim dsRNA. (C) Averaged values ± SEM for control cells (n = 46 cells in two representative experiments, white bars) and Stim dsRNA-treated (n = 197 cells in three representative experiments, gray bars): resting [Ca2+]i; peak [Ca2+]i during the TG-evoked release transient; maximal and sustained (5 min) [Ca2+]i after readdition of 2 mM external Ca2+. The values of maximal [Ca2+]i and sustained [Ca2+]i in control and Stim suppressed cells are significantly different (P < 5 × 10−6).
Mentions: Population measurements of intracellular Ca2+ would include contributions of cells that had not been affected by gene silencing. Therefore, to clarify effects of gene silencing at the level of single cells we evaluated Ca2+ signaling and CRAC currents in S2 cells that were pretreated with dsRNA. Fig. 2 A illustrates Ca2+ signals in response to TG-evoked store depletion in eight individual control cells treated with dsRNA for the cell adhesion molecule CG1560. Removal of Ca2+ and readdition of Ca2+ produced no significant change in the Ca2+ signal. Addition of TG in zero-Ca2+ solution produced a transient rise in [Ca2+]i due to release of Ca2+ from the ER; the release transient reached a peak that averaged ∼200 nM before declining. Upon readdition of Ca2+, store-operated Ca2+ influx was revealed as a rise in [Ca2+]i that was sustained and often exceeded 1 μM. In cells that were pretreated with Stim dsRNA, resting [Ca2+]i and the release transient were not altered significantly, but the rise in Ca2+ upon readdition of external Ca2+ was prevented in most of the single cells (Fig. 2 B). In contrast, suppression of CG8743 produced a significant elevation of the resting [Ca2+]i to ∼60% higher than in control cells (135 CG1560 dsRNA-treated control cells; 118 CG8743 dsRNA-treated cells; P < 5 × 10−6), which is consistent with results from the initial screen (Fig. 1 F). However, CG8743 dsRNA did not alter the Ca2+-release transient (not depicted) or Ca2+ influx evoked by TG (Fig. 1 E). Fig. 2 C summarizes the results of three control experiments and six experiments with suppression of Stim. Occasionally, cells in the Stim dsRNA group exhibited normal Ca2+ responses similar to control cells, suggesting either that these cells did not effectively take up the dsRNA, that gene suppression in these cells was less efficient, or that some cells with effective gene suppression express normal SOC influx, as found in DT40 cells after knockout of TRPC1 (Mori et al., 2002). These results demonstrate at the single-cell level that suppression of Stim effectively blocks both the early and sustained components of Ca2+ entry evoked by TG.

Bottom Line: RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry.Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells.We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

View Article: PubMed Central - PubMed

Affiliation: Torrey Pines Therapeutics, Inc., La Jolla, CA 92037, USA.

ABSTRACT
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Show MeSH
Related in: MedlinePlus