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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 Drosophila CRAC current by Stim dsRNA treatment. (A) Current development evaluated at −110 mV in selected control cell (untreated). Cells were bathed in S2 external solution with 2 mM Ca2+ and dialyzed with BAPTA-buffered S2 internal solution to induce store depletion passively. Whole-cell recording was initiated at time 0. (B) Leak-subtracted current-voltage relation of maximal Drosophila CRAC current recorded in the same control cell. (C) Typical Stim dsRNA-treated cell; current at −110 mV. (D) Leak-subtracted I-V relation 200 s after establishing the whole cell configuration. (E) CRAC current density in dsRNA-treated and untreated S2 cells. Each point represents CRAC current density (pA/pF) in a single cell, plotted in consecutive order from left to right within six groups of cells: untreated (circles, n = 27 cells in three experiments); cells treated with dsRNA to suppress CG11059 (triangles, n = 21 cells in three experiments); CG1560 (diamonds, n = 45 cells in six experiments); trp-l (inverted triangles, n = 20 cells in two experiments); CG8743 (pentahedrons, n = 16 cells in two experiments); or Stim (squares, n = 77 cells in eight experiments). Groups 1, 2, and 5 include one cell each with current density >6 pA/pF. Horizontal lines indicate the mean value of current density in each group.
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fig3: Suppression of Drosophila CRAC current by Stim dsRNA treatment. (A) Current development evaluated at −110 mV in selected control cell (untreated). Cells were bathed in S2 external solution with 2 mM Ca2+ and dialyzed with BAPTA-buffered S2 internal solution to induce store depletion passively. Whole-cell recording was initiated at time 0. (B) Leak-subtracted current-voltage relation of maximal Drosophila CRAC current recorded in the same control cell. (C) Typical Stim dsRNA-treated cell; current at −110 mV. (D) Leak-subtracted I-V relation 200 s after establishing the whole cell configuration. (E) CRAC current density in dsRNA-treated and untreated S2 cells. Each point represents CRAC current density (pA/pF) in a single cell, plotted in consecutive order from left to right within six groups of cells: untreated (circles, n = 27 cells in three experiments); cells treated with dsRNA to suppress CG11059 (triangles, n = 21 cells in three experiments); CG1560 (diamonds, n = 45 cells in six experiments); trp-l (inverted triangles, n = 20 cells in two experiments); CG8743 (pentahedrons, n = 16 cells in two experiments); or Stim (squares, n = 77 cells in eight experiments). Groups 1, 2, and 5 include one cell each with current density >6 pA/pF. Horizontal lines indicate the mean value of current density in each group.

Mentions: In previous work, we demonstrated that Ca2+ entry evoked in S2 cells by Ca2+ store depletion occurs through store-operated Ca2+-selective channels that are similar in biophysical properties to CRAC channels in T lymphocytes (Yeromin et al., 2004). Fig. 3 (A and B) illustrates the time course of CRAC current development and the characteristic I-V relationship in control cells dialyzed with the Ca2+ chelator BAPTA to deplete Ca2+ stores passively. In cells treated with dsRNA for Stim, CRAC current was effectively suppressed in most cells (Fig. 3, C and D). We performed similar experiments on six groups of cells: control cells that were untreated; two additional control groups in which cells were treated with dsRNA for cell adhesion molecules CG11059 and CG1560; cells treated with dsRNA directed against two trp-related genes that are expressed in S2 cells, trp-l and CG8743; or cells treated with dsRNA for Stim. Suppression of corresponding gene expression was confirmed by RT-PCR (unpublished data). As summarized in Fig. 3 E, CRAC currents were similar in all three control groups and in the trp-l and CG8743-suppressed groups. In the Stim dsRNA-treated cells, CRAC currents were suppressed to undetectable levels in 83% of cells. These experiments demonstrate that Drosophila Stim is required for normal activity of CRAC channels in S2 cells.


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 Drosophila CRAC current by Stim dsRNA treatment. (A) Current development evaluated at −110 mV in selected control cell (untreated). Cells were bathed in S2 external solution with 2 mM Ca2+ and dialyzed with BAPTA-buffered S2 internal solution to induce store depletion passively. Whole-cell recording was initiated at time 0. (B) Leak-subtracted current-voltage relation of maximal Drosophila CRAC current recorded in the same control cell. (C) Typical Stim dsRNA-treated cell; current at −110 mV. (D) Leak-subtracted I-V relation 200 s after establishing the whole cell configuration. (E) CRAC current density in dsRNA-treated and untreated S2 cells. Each point represents CRAC current density (pA/pF) in a single cell, plotted in consecutive order from left to right within six groups of cells: untreated (circles, n = 27 cells in three experiments); cells treated with dsRNA to suppress CG11059 (triangles, n = 21 cells in three experiments); CG1560 (diamonds, n = 45 cells in six experiments); trp-l (inverted triangles, n = 20 cells in two experiments); CG8743 (pentahedrons, n = 16 cells in two experiments); or Stim (squares, n = 77 cells in eight experiments). Groups 1, 2, and 5 include one cell each with current density >6 pA/pF. Horizontal lines indicate the mean value of current density in each group.
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fig3: Suppression of Drosophila CRAC current by Stim dsRNA treatment. (A) Current development evaluated at −110 mV in selected control cell (untreated). Cells were bathed in S2 external solution with 2 mM Ca2+ and dialyzed with BAPTA-buffered S2 internal solution to induce store depletion passively. Whole-cell recording was initiated at time 0. (B) Leak-subtracted current-voltage relation of maximal Drosophila CRAC current recorded in the same control cell. (C) Typical Stim dsRNA-treated cell; current at −110 mV. (D) Leak-subtracted I-V relation 200 s after establishing the whole cell configuration. (E) CRAC current density in dsRNA-treated and untreated S2 cells. Each point represents CRAC current density (pA/pF) in a single cell, plotted in consecutive order from left to right within six groups of cells: untreated (circles, n = 27 cells in three experiments); cells treated with dsRNA to suppress CG11059 (triangles, n = 21 cells in three experiments); CG1560 (diamonds, n = 45 cells in six experiments); trp-l (inverted triangles, n = 20 cells in two experiments); CG8743 (pentahedrons, n = 16 cells in two experiments); or Stim (squares, n = 77 cells in eight experiments). Groups 1, 2, and 5 include one cell each with current density >6 pA/pF. Horizontal lines indicate the mean value of current density in each group.
Mentions: In previous work, we demonstrated that Ca2+ entry evoked in S2 cells by Ca2+ store depletion occurs through store-operated Ca2+-selective channels that are similar in biophysical properties to CRAC channels in T lymphocytes (Yeromin et al., 2004). Fig. 3 (A and B) illustrates the time course of CRAC current development and the characteristic I-V relationship in control cells dialyzed with the Ca2+ chelator BAPTA to deplete Ca2+ stores passively. In cells treated with dsRNA for Stim, CRAC current was effectively suppressed in most cells (Fig. 3, C and D). We performed similar experiments on six groups of cells: control cells that were untreated; two additional control groups in which cells were treated with dsRNA for cell adhesion molecules CG11059 and CG1560; cells treated with dsRNA directed against two trp-related genes that are expressed in S2 cells, trp-l and CG8743; or cells treated with dsRNA for Stim. Suppression of corresponding gene expression was confirmed by RT-PCR (unpublished data). As summarized in Fig. 3 E, CRAC currents were similar in all three control groups and in the trp-l and CG8743-suppressed groups. In the Stim dsRNA-treated cells, CRAC currents were suppressed to undetectable levels in 83% of cells. These experiments demonstrate that Drosophila Stim is required for normal activity of CRAC channels in S2 cells.

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