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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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Overexpression of STIM1 in HEK293 cells. (A) Western blot analysis of STIM1 (top band) and GAPDH (bottom band) proteins in HEK-STIM1 (STIM1 overexpressing) cells and control cells (HEK-Zeo cells); lane 1, 10 μg protein; lane 2, 1 μg protein. We estimate STIM1 protein levels to be nearly 100-fold greater than in the HEK-STIM1 cells compared with control cells. (B) Representative traces of TG-induced Ca2+ release and TG-induced Ca2+ entry in HEK-STIM1 cells (dotted line) compared with HEK-Zeo cells (solid line). TG-induced Ca2+ entry was enhanced in HEK-STIM1 cells by an average of 17% in four experiments. (C and E) Time course of outward current at +80 mV and inward current at −110 mV (note different scales) for control HEK-Zeo (E) and HEK-STIM1 cells (C). (D and F) Representative current-voltage relationships immediately after break-in to achieve whole-cell recording and 5 min later in control HEK-Zeo (D) and HEK-STIM1 cells (F). Outwardly rectifying Mg2+-inhibited cation current representing channel activity of TRPM7 disappeared as Mg2+ diffused into the cell from the pipette. (G and H) Inward and outward currents were not significantly altered by overexpression of STIM1; n = 10 cells for each group. Error bars represent SEM.
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fig7: Overexpression of STIM1 in HEK293 cells. (A) Western blot analysis of STIM1 (top band) and GAPDH (bottom band) proteins in HEK-STIM1 (STIM1 overexpressing) cells and control cells (HEK-Zeo cells); lane 1, 10 μg protein; lane 2, 1 μg protein. We estimate STIM1 protein levels to be nearly 100-fold greater than in the HEK-STIM1 cells compared with control cells. (B) Representative traces of TG-induced Ca2+ release and TG-induced Ca2+ entry in HEK-STIM1 cells (dotted line) compared with HEK-Zeo cells (solid line). TG-induced Ca2+ entry was enhanced in HEK-STIM1 cells by an average of 17% in four experiments. (C and E) Time course of outward current at +80 mV and inward current at −110 mV (note different scales) for control HEK-Zeo (E) and HEK-STIM1 cells (C). (D and F) Representative current-voltage relationships immediately after break-in to achieve whole-cell recording and 5 min later in control HEK-Zeo (D) and HEK-STIM1 cells (F). Outwardly rectifying Mg2+-inhibited cation current representing channel activity of TRPM7 disappeared as Mg2+ diffused into the cell from the pipette. (G and H) Inward and outward currents were not significantly altered by overexpression of STIM1; n = 10 cells for each group. Error bars represent SEM.

Mentions: To further examine the role of STIM1 in SOCE in mammalian cells, we generated siRNAs to STIM1 and STIM2 and tested them individually in HEK293 cells on TG-induced Ca2+ influx, previously linked to TRPC1 and TRPC3 proteins (Wu et al., 2000). RT-PCR and Western blot analysis indicated that STIM1 mRNA and protein levels were selectively reduced by the STIM1 siRNA (Fig. 6, A and B). Immunofluorescence localization indicated an expression pattern consistent with plasma membrane and ER localization (Manji et al., 2000) and confirmed the reduction of STIM1 protein by RNAi (Fig. 6 C). Cellular metabolism of the mitochondrial substrate alamarBlue was not altered (unpublished data), indicating the absence of cytotoxicity or mitochondrial stress after STIM1 suppression. In STIM1-suppressed cells, TG-dependent Ca2+ influx was inhibited by 60%, whereas the Ca2+ release transient was unaffected compared with control cells transfected with a nonsilencing scrambled siRNA (Fig. 6 D). In contrast, Ca2+ influx was unaltered in cells treated with the siRNA for STIM2, even though STIM2 mRNA was effectively reduced. In addition to inhibiting TG-evoked Ca2+ influx, knockdown of STIM1 potently inhibited muscarinic receptor-induced Ca2+ influx, but did not reduce IP3-induced Ca2+ release (Fig. 6 E). The store-operated cation entry pathway in HEK293 cells is known to be permeable to Ba2+ (Wu et al., 2000; Trebak et al., 2002). Measurement of Ba2+ influx has the advantage of being a more direct reflection of cation entry via the plasma membrane because it avoids possible effects on cellular buffering of Ca2+ or other Ca2+ regulatory mechanisms. The initial rate of TG-induced Ba2+ entry in cells transfected with STIM1 siRNA was only 17% of control, whereas Ba2+ entry was unaffected in cells transfected with siRNA for STIM2 (Fig. 6 F). In contrast, stable overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry by an average of 17% (Fig. 7 B). Interestingly, the increase in SOC influx was small in comparison to the robust increase in STIM1 protein levels (Fig. 7 A), and definitive SOC current was still not detected, nor was there any change in Mg2+-inhibited cation current (Fig. 7, B–G). Thus, although STIM1 is required for SOC influx in HEK293 cells at the level of Ca2+ (or Ba2+) entry across the plasma membrane, overexpression does not appear to greatly enhance the number of activatable SOC channels.


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)

Overexpression of STIM1 in HEK293 cells. (A) Western blot analysis of STIM1 (top band) and GAPDH (bottom band) proteins in HEK-STIM1 (STIM1 overexpressing) cells and control cells (HEK-Zeo cells); lane 1, 10 μg protein; lane 2, 1 μg protein. We estimate STIM1 protein levels to be nearly 100-fold greater than in the HEK-STIM1 cells compared with control cells. (B) Representative traces of TG-induced Ca2+ release and TG-induced Ca2+ entry in HEK-STIM1 cells (dotted line) compared with HEK-Zeo cells (solid line). TG-induced Ca2+ entry was enhanced in HEK-STIM1 cells by an average of 17% in four experiments. (C and E) Time course of outward current at +80 mV and inward current at −110 mV (note different scales) for control HEK-Zeo (E) and HEK-STIM1 cells (C). (D and F) Representative current-voltage relationships immediately after break-in to achieve whole-cell recording and 5 min later in control HEK-Zeo (D) and HEK-STIM1 cells (F). Outwardly rectifying Mg2+-inhibited cation current representing channel activity of TRPM7 disappeared as Mg2+ diffused into the cell from the pipette. (G and H) Inward and outward currents were not significantly altered by overexpression of STIM1; n = 10 cells for each group. Error bars represent SEM.
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fig7: Overexpression of STIM1 in HEK293 cells. (A) Western blot analysis of STIM1 (top band) and GAPDH (bottom band) proteins in HEK-STIM1 (STIM1 overexpressing) cells and control cells (HEK-Zeo cells); lane 1, 10 μg protein; lane 2, 1 μg protein. We estimate STIM1 protein levels to be nearly 100-fold greater than in the HEK-STIM1 cells compared with control cells. (B) Representative traces of TG-induced Ca2+ release and TG-induced Ca2+ entry in HEK-STIM1 cells (dotted line) compared with HEK-Zeo cells (solid line). TG-induced Ca2+ entry was enhanced in HEK-STIM1 cells by an average of 17% in four experiments. (C and E) Time course of outward current at +80 mV and inward current at −110 mV (note different scales) for control HEK-Zeo (E) and HEK-STIM1 cells (C). (D and F) Representative current-voltage relationships immediately after break-in to achieve whole-cell recording and 5 min later in control HEK-Zeo (D) and HEK-STIM1 cells (F). Outwardly rectifying Mg2+-inhibited cation current representing channel activity of TRPM7 disappeared as Mg2+ diffused into the cell from the pipette. (G and H) Inward and outward currents were not significantly altered by overexpression of STIM1; n = 10 cells for each group. Error bars represent SEM.
Mentions: To further examine the role of STIM1 in SOCE in mammalian cells, we generated siRNAs to STIM1 and STIM2 and tested them individually in HEK293 cells on TG-induced Ca2+ influx, previously linked to TRPC1 and TRPC3 proteins (Wu et al., 2000). RT-PCR and Western blot analysis indicated that STIM1 mRNA and protein levels were selectively reduced by the STIM1 siRNA (Fig. 6, A and B). Immunofluorescence localization indicated an expression pattern consistent with plasma membrane and ER localization (Manji et al., 2000) and confirmed the reduction of STIM1 protein by RNAi (Fig. 6 C). Cellular metabolism of the mitochondrial substrate alamarBlue was not altered (unpublished data), indicating the absence of cytotoxicity or mitochondrial stress after STIM1 suppression. In STIM1-suppressed cells, TG-dependent Ca2+ influx was inhibited by 60%, whereas the Ca2+ release transient was unaffected compared with control cells transfected with a nonsilencing scrambled siRNA (Fig. 6 D). In contrast, Ca2+ influx was unaltered in cells treated with the siRNA for STIM2, even though STIM2 mRNA was effectively reduced. In addition to inhibiting TG-evoked Ca2+ influx, knockdown of STIM1 potently inhibited muscarinic receptor-induced Ca2+ influx, but did not reduce IP3-induced Ca2+ release (Fig. 6 E). The store-operated cation entry pathway in HEK293 cells is known to be permeable to Ba2+ (Wu et al., 2000; Trebak et al., 2002). Measurement of Ba2+ influx has the advantage of being a more direct reflection of cation entry via the plasma membrane because it avoids possible effects on cellular buffering of Ca2+ or other Ca2+ regulatory mechanisms. The initial rate of TG-induced Ba2+ entry in cells transfected with STIM1 siRNA was only 17% of control, whereas Ba2+ entry was unaffected in cells transfected with siRNA for STIM2 (Fig. 6 F). In contrast, stable overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry by an average of 17% (Fig. 7 B). Interestingly, the increase in SOC influx was small in comparison to the robust increase in STIM1 protein levels (Fig. 7 A), and definitive SOC current was still not detected, nor was there any change in Mg2+-inhibited cation current (Fig. 7, B–G). Thus, although STIM1 is required for SOC influx in HEK293 cells at the level of Ca2+ (or Ba2+) entry across the plasma membrane, overexpression does not appear to greatly enhance the number of activatable SOC channels.

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