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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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The effect of STIM1 suppression on Ca2+ signaling in individual Jurkat T cells. (A) Western blot of 4A5 cell lysates (lane 2), compared with control 2A4 cells (lane 1) showing >50% reduction in STIM1 protein, with no change in the protein levels of GAPDH. (B) The specificity of STIM1 suppression was confirmed by RT-PCR analysis showing a reduction in STIM1, but not STIM2 or GAPDH, mRNA levels in 4A5 cells (lane 2) compared with control 2A4 cells (lane 1). (C) Intracellular Ca2+ responses in 51 Jurkat 2A4 control cells. Cells were bathed in Jurkat Ringer (2 mM Ca2+), low-Ca2+ (0.4 mM) Jurkat Ringer, and Ca2+-free Jurkat Ringer with 1 μM TG, as indicated. The first peak is due to Ca2+ release from internal stores in the presence of TG. The second and third peaks result from Ca2+ entry through CRAC channels upon addition of 0.4 and 2 mM external Ca2+, respectively. Sustained [Ca2+]i was measured 5 min after readdition of 2 mM external Ca2+. (D) Averaged [Ca2+]i in control 2A4 cells from the same experiment. (E) Intracellular Ca2+ responses in 40 STIM1-suppressed 4A5 Jurkat cells. (F) Averaged [Ca2+]i in STIM1-suppressed 4A5 cells from the same experiment as in D. (G) Combined data from three control experiments (164 cells, white bars) and three experiments with STIM1-suppressed cells (141 cells, gray bars). Averaged values of peak and sustained [Ca2+]i are significantly different in STIM1-suppressed cells (P < 8 × 10−6, < 8 × 10−6, and < 2 × 10−5, respectively, by independent two populations t test). (H) Maximal rate of Ca2+ rise upon Ca2+ readdition as an estimate of Ca2+ influx. Representative averaged traces obtained in the same experiments as in A–D are shown (control 2A4 cells, closed squares; STIM1-suppressed 4A5 cells, open squares), along with corresponding differentiated [Ca2+]i traces, d[Ca2+]i/dt (right axis), for control 2A4 cells (black line without symbols) and STIM1-suppressed 4A5 cells (gray line). The peak derivatives correspond to the maximal rate of Ca2+ rise in nM. (I) STIM1 expression and d[Ca2+]i/dt. The maximal rate of [Ca2+]i rise after 0.4 mM or 2 mM Ca2+ readdition in control 2A4 cells (white bars: 164 cells in three experiments); and STIM1-suppressed 4A5 cells (gray bars: 141 cells in three experiments; P < 3 × 10−7 or < 2 × 10−7 for 0.4 and 2 mM Ca2+, respectively).
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fig4: The effect of STIM1 suppression on Ca2+ signaling in individual Jurkat T cells. (A) Western blot of 4A5 cell lysates (lane 2), compared with control 2A4 cells (lane 1) showing >50% reduction in STIM1 protein, with no change in the protein levels of GAPDH. (B) The specificity of STIM1 suppression was confirmed by RT-PCR analysis showing a reduction in STIM1, but not STIM2 or GAPDH, mRNA levels in 4A5 cells (lane 2) compared with control 2A4 cells (lane 1). (C) Intracellular Ca2+ responses in 51 Jurkat 2A4 control cells. Cells were bathed in Jurkat Ringer (2 mM Ca2+), low-Ca2+ (0.4 mM) Jurkat Ringer, and Ca2+-free Jurkat Ringer with 1 μM TG, as indicated. The first peak is due to Ca2+ release from internal stores in the presence of TG. The second and third peaks result from Ca2+ entry through CRAC channels upon addition of 0.4 and 2 mM external Ca2+, respectively. Sustained [Ca2+]i was measured 5 min after readdition of 2 mM external Ca2+. (D) Averaged [Ca2+]i in control 2A4 cells from the same experiment. (E) Intracellular Ca2+ responses in 40 STIM1-suppressed 4A5 Jurkat cells. (F) Averaged [Ca2+]i in STIM1-suppressed 4A5 cells from the same experiment as in D. (G) Combined data from three control experiments (164 cells, white bars) and three experiments with STIM1-suppressed cells (141 cells, gray bars). Averaged values of peak and sustained [Ca2+]i are significantly different in STIM1-suppressed cells (P < 8 × 10−6, < 8 × 10−6, and < 2 × 10−5, respectively, by independent two populations t test). (H) Maximal rate of Ca2+ rise upon Ca2+ readdition as an estimate of Ca2+ influx. Representative averaged traces obtained in the same experiments as in A–D are shown (control 2A4 cells, closed squares; STIM1-suppressed 4A5 cells, open squares), along with corresponding differentiated [Ca2+]i traces, d[Ca2+]i/dt (right axis), for control 2A4 cells (black line without symbols) and STIM1-suppressed 4A5 cells (gray line). The peak derivatives correspond to the maximal rate of Ca2+ rise in nM. (I) STIM1 expression and d[Ca2+]i/dt. The maximal rate of [Ca2+]i rise after 0.4 mM or 2 mM Ca2+ readdition in control 2A4 cells (white bars: 164 cells in three experiments); and STIM1-suppressed 4A5 cells (gray bars: 141 cells in three experiments; P < 3 × 10−7 or < 2 × 10−7 for 0.4 and 2 mM Ca2+, respectively).

Mentions: Mammalian cells express two homologues, STIM1 and STIM2, of Drosophila Stim. Both are single-pass transmembrane proteins that are present in rat, mouse, and human. STIM1 was detected by Western blot in human Jurkat T cells (Fig. 4 A) and in primary human T lymphocytes (not depicted). In these cells, T cell receptor stimulation leads to activation of the CRAC channel and subsequent gene expression and cytokine release (Lewis, 2001). To test the role of STIM1 in T cells, a stable pool of Jurkat cells expressing a short RNA hairpin loop (shRNA) targeting human STIM1 was generated (Jurkat clone 4A5). Stable pools of Jurkat cells expressing a negative control, nonsilencing scrambled shRNA were also generated (Jurkat negative control clone 2A4). In these cells, STIM1 mRNA and protein were reduced by >50%, but there was no change in STIM2 mRNA levels indicating specificity of the STIM1 shRNA (Fig. 4, A and B). Importantly, TG-dependent Ca2+ influx was significantly reduced in the 4A5 Jurkat cells, as seen in individual cells and the averaged responses (Fig. 4, C–G). Removal and readdition of Ca2+ produced no significant change in the Ca2+ signal, indicating that the TG–independent response was negligible (unpublished data). TG-dependent Ca2+ influx rates were evaluated from the maximal rate of rise in the Ca2+ signal, d[Ca2+]i/dt, upon Ca2+ readdition in both Jurkat Ringer and low-Ca2+ Jurkat Ringer (Fig. 4, H and I); STIM1 suppression reduced mean influx rates by 68% and 82%, respectively. These results confirm that Ca2+ influx in Jurkat T cells is effectively inhibited by suppression of STIM1, as seen for Stim 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)

The effect of STIM1 suppression on Ca2+ signaling in individual Jurkat T cells. (A) Western blot of 4A5 cell lysates (lane 2), compared with control 2A4 cells (lane 1) showing >50% reduction in STIM1 protein, with no change in the protein levels of GAPDH. (B) The specificity of STIM1 suppression was confirmed by RT-PCR analysis showing a reduction in STIM1, but not STIM2 or GAPDH, mRNA levels in 4A5 cells (lane 2) compared with control 2A4 cells (lane 1). (C) Intracellular Ca2+ responses in 51 Jurkat 2A4 control cells. Cells were bathed in Jurkat Ringer (2 mM Ca2+), low-Ca2+ (0.4 mM) Jurkat Ringer, and Ca2+-free Jurkat Ringer with 1 μM TG, as indicated. The first peak is due to Ca2+ release from internal stores in the presence of TG. The second and third peaks result from Ca2+ entry through CRAC channels upon addition of 0.4 and 2 mM external Ca2+, respectively. Sustained [Ca2+]i was measured 5 min after readdition of 2 mM external Ca2+. (D) Averaged [Ca2+]i in control 2A4 cells from the same experiment. (E) Intracellular Ca2+ responses in 40 STIM1-suppressed 4A5 Jurkat cells. (F) Averaged [Ca2+]i in STIM1-suppressed 4A5 cells from the same experiment as in D. (G) Combined data from three control experiments (164 cells, white bars) and three experiments with STIM1-suppressed cells (141 cells, gray bars). Averaged values of peak and sustained [Ca2+]i are significantly different in STIM1-suppressed cells (P < 8 × 10−6, < 8 × 10−6, and < 2 × 10−5, respectively, by independent two populations t test). (H) Maximal rate of Ca2+ rise upon Ca2+ readdition as an estimate of Ca2+ influx. Representative averaged traces obtained in the same experiments as in A–D are shown (control 2A4 cells, closed squares; STIM1-suppressed 4A5 cells, open squares), along with corresponding differentiated [Ca2+]i traces, d[Ca2+]i/dt (right axis), for control 2A4 cells (black line without symbols) and STIM1-suppressed 4A5 cells (gray line). The peak derivatives correspond to the maximal rate of Ca2+ rise in nM. (I) STIM1 expression and d[Ca2+]i/dt. The maximal rate of [Ca2+]i rise after 0.4 mM or 2 mM Ca2+ readdition in control 2A4 cells (white bars: 164 cells in three experiments); and STIM1-suppressed 4A5 cells (gray bars: 141 cells in three experiments; P < 3 × 10−7 or < 2 × 10−7 for 0.4 and 2 mM Ca2+, respectively).
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fig4: The effect of STIM1 suppression on Ca2+ signaling in individual Jurkat T cells. (A) Western blot of 4A5 cell lysates (lane 2), compared with control 2A4 cells (lane 1) showing >50% reduction in STIM1 protein, with no change in the protein levels of GAPDH. (B) The specificity of STIM1 suppression was confirmed by RT-PCR analysis showing a reduction in STIM1, but not STIM2 or GAPDH, mRNA levels in 4A5 cells (lane 2) compared with control 2A4 cells (lane 1). (C) Intracellular Ca2+ responses in 51 Jurkat 2A4 control cells. Cells were bathed in Jurkat Ringer (2 mM Ca2+), low-Ca2+ (0.4 mM) Jurkat Ringer, and Ca2+-free Jurkat Ringer with 1 μM TG, as indicated. The first peak is due to Ca2+ release from internal stores in the presence of TG. The second and third peaks result from Ca2+ entry through CRAC channels upon addition of 0.4 and 2 mM external Ca2+, respectively. Sustained [Ca2+]i was measured 5 min after readdition of 2 mM external Ca2+. (D) Averaged [Ca2+]i in control 2A4 cells from the same experiment. (E) Intracellular Ca2+ responses in 40 STIM1-suppressed 4A5 Jurkat cells. (F) Averaged [Ca2+]i in STIM1-suppressed 4A5 cells from the same experiment as in D. (G) Combined data from three control experiments (164 cells, white bars) and three experiments with STIM1-suppressed cells (141 cells, gray bars). Averaged values of peak and sustained [Ca2+]i are significantly different in STIM1-suppressed cells (P < 8 × 10−6, < 8 × 10−6, and < 2 × 10−5, respectively, by independent two populations t test). (H) Maximal rate of Ca2+ rise upon Ca2+ readdition as an estimate of Ca2+ influx. Representative averaged traces obtained in the same experiments as in A–D are shown (control 2A4 cells, closed squares; STIM1-suppressed 4A5 cells, open squares), along with corresponding differentiated [Ca2+]i traces, d[Ca2+]i/dt (right axis), for control 2A4 cells (black line without symbols) and STIM1-suppressed 4A5 cells (gray line). The peak derivatives correspond to the maximal rate of Ca2+ rise in nM. (I) STIM1 expression and d[Ca2+]i/dt. The maximal rate of [Ca2+]i rise after 0.4 mM or 2 mM Ca2+ readdition in control 2A4 cells (white bars: 164 cells in three experiments); and STIM1-suppressed 4A5 cells (gray bars: 141 cells in three experiments; P < 3 × 10−7 or < 2 × 10−7 for 0.4 and 2 mM Ca2+, respectively).
Mentions: Mammalian cells express two homologues, STIM1 and STIM2, of Drosophila Stim. Both are single-pass transmembrane proteins that are present in rat, mouse, and human. STIM1 was detected by Western blot in human Jurkat T cells (Fig. 4 A) and in primary human T lymphocytes (not depicted). In these cells, T cell receptor stimulation leads to activation of the CRAC channel and subsequent gene expression and cytokine release (Lewis, 2001). To test the role of STIM1 in T cells, a stable pool of Jurkat cells expressing a short RNA hairpin loop (shRNA) targeting human STIM1 was generated (Jurkat clone 4A5). Stable pools of Jurkat cells expressing a negative control, nonsilencing scrambled shRNA were also generated (Jurkat negative control clone 2A4). In these cells, STIM1 mRNA and protein were reduced by >50%, but there was no change in STIM2 mRNA levels indicating specificity of the STIM1 shRNA (Fig. 4, A and B). Importantly, TG-dependent Ca2+ influx was significantly reduced in the 4A5 Jurkat cells, as seen in individual cells and the averaged responses (Fig. 4, C–G). Removal and readdition of Ca2+ produced no significant change in the Ca2+ signal, indicating that the TG–independent response was negligible (unpublished data). TG-dependent Ca2+ influx rates were evaluated from the maximal rate of rise in the Ca2+ signal, d[Ca2+]i/dt, upon Ca2+ readdition in both Jurkat Ringer and low-Ca2+ Jurkat Ringer (Fig. 4, H and I); STIM1 suppression reduced mean influx rates by 68% and 82%, respectively. These results confirm that Ca2+ influx in Jurkat T cells is effectively inhibited by suppression of STIM1, as seen for Stim 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