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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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Reduction of Jurkat CRAC current by STIM1 suppression. (A) Current development in selected control 2A4 Jurkat cell. Cells were first bathed in Ca2+-free Jurkat external solution for 5 min and dialyzed with BAPTA-containing Jurkat internal solution. CRAC current was revealed after exchange of Ca2+-free Jurkat solution to 20 mM Ca2+ Jurkat external solution. Maximal current density was evaluated at −110 mV. (B) Leak-subtracted current-voltage relationship of fully developed CRAC current recorded in the same control 2A4 Jurkat cell. (C) Suppression of CRAC current in STIM1-suppressed 4A5 Jurkat cell. (D) Leak-subtracted I-V relationship in the same cell, as in C. (E) CRAC current density in control 2A4 cells (circles, n = 11) and STIM1-suppressed 4A5 cells (squares, n = 11). Horizontal lines indicate the mean value of current density in each group. (P < 3 × 10−6).
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fig5: Reduction of Jurkat CRAC current by STIM1 suppression. (A) Current development in selected control 2A4 Jurkat cell. Cells were first bathed in Ca2+-free Jurkat external solution for 5 min and dialyzed with BAPTA-containing Jurkat internal solution. CRAC current was revealed after exchange of Ca2+-free Jurkat solution to 20 mM Ca2+ Jurkat external solution. Maximal current density was evaluated at −110 mV. (B) Leak-subtracted current-voltage relationship of fully developed CRAC current recorded in the same control 2A4 Jurkat cell. (C) Suppression of CRAC current in STIM1-suppressed 4A5 Jurkat cell. (D) Leak-subtracted I-V relationship in the same cell, as in C. (E) CRAC current density in control 2A4 cells (circles, n = 11) and STIM1-suppressed 4A5 cells (squares, n = 11). Horizontal lines indicate the mean value of current density in each group. (P < 3 × 10−6).

Mentions: Depletion of Ca2+ stores in Jurkat T cells leads to the opening of CRAC channels that can be monitored as CRAC current (Prakriya and Lewis, 2003). STIM1 suppression in 4A5 Jurkat T cells reduced the average CRAC current density evaluated during whole-cell recording. The time course of current and corresponding I-V relations recorded from one control and one STIM1-suppressed cell are shown in Fig. 5 (A–D), and a summary of data is presented in Fig. 5 E. Only ∼9% of STIM1-suppressed cells developed detectable current, compared with 70% of control cells. In cells that exhibited detectable CRAC current, the inactivating Na+ current through CRAC channels upon exposure to divalent-free external solution was also reduced. The ratio of peak Na+ to Ca2+ current was similar in control 2A4 cells (10 ± 1.4, n = 6) and in STIM1-suppressed cells 4A5 cells (12.2 ± 2.8, n = 5). The attenuation of CRAC currents in Jurkat T cells is consistent with Ca2+ imaging results described above.


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)

Reduction of Jurkat CRAC current by STIM1 suppression. (A) Current development in selected control 2A4 Jurkat cell. Cells were first bathed in Ca2+-free Jurkat external solution for 5 min and dialyzed with BAPTA-containing Jurkat internal solution. CRAC current was revealed after exchange of Ca2+-free Jurkat solution to 20 mM Ca2+ Jurkat external solution. Maximal current density was evaluated at −110 mV. (B) Leak-subtracted current-voltage relationship of fully developed CRAC current recorded in the same control 2A4 Jurkat cell. (C) Suppression of CRAC current in STIM1-suppressed 4A5 Jurkat cell. (D) Leak-subtracted I-V relationship in the same cell, as in C. (E) CRAC current density in control 2A4 cells (circles, n = 11) and STIM1-suppressed 4A5 cells (squares, n = 11). Horizontal lines indicate the mean value of current density in each group. (P < 3 × 10−6).
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fig5: Reduction of Jurkat CRAC current by STIM1 suppression. (A) Current development in selected control 2A4 Jurkat cell. Cells were first bathed in Ca2+-free Jurkat external solution for 5 min and dialyzed with BAPTA-containing Jurkat internal solution. CRAC current was revealed after exchange of Ca2+-free Jurkat solution to 20 mM Ca2+ Jurkat external solution. Maximal current density was evaluated at −110 mV. (B) Leak-subtracted current-voltage relationship of fully developed CRAC current recorded in the same control 2A4 Jurkat cell. (C) Suppression of CRAC current in STIM1-suppressed 4A5 Jurkat cell. (D) Leak-subtracted I-V relationship in the same cell, as in C. (E) CRAC current density in control 2A4 cells (circles, n = 11) and STIM1-suppressed 4A5 cells (squares, n = 11). Horizontal lines indicate the mean value of current density in each group. (P < 3 × 10−6).
Mentions: Depletion of Ca2+ stores in Jurkat T cells leads to the opening of CRAC channels that can be monitored as CRAC current (Prakriya and Lewis, 2003). STIM1 suppression in 4A5 Jurkat T cells reduced the average CRAC current density evaluated during whole-cell recording. The time course of current and corresponding I-V relations recorded from one control and one STIM1-suppressed cell are shown in Fig. 5 (A–D), and a summary of data is presented in Fig. 5 E. Only ∼9% of STIM1-suppressed cells developed detectable current, compared with 70% of control cells. In cells that exhibited detectable CRAC current, the inactivating Na+ current through CRAC channels upon exposure to divalent-free external solution was also reduced. The ratio of peak Na+ to Ca2+ current was similar in control 2A4 cells (10 ± 1.4, n = 6) and in STIM1-suppressed cells 4A5 cells (12.2 ± 2.8, n = 5). The attenuation of CRAC currents in Jurkat T cells is consistent with Ca2+ imaging results described above.

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