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Retrograde regulation of STIM1-Orai1 interaction and store-operated Ca2+ entry by calsequestrin.

Wang L, Zhang L, Li S, Zheng Y, Yan X, Chen M, Wang H, Putney JW, Luo D - Sci Rep (2015)

Bottom Line: In cells with Ca(2+) stores depleted, TFP further increased CSQ1 monomerization and CSQ1-STIM1 interaction, but reduced the association of STIM1 with Orai1 and SOCE.Over-expression of CSQ1 or a C-terminal (amino acid 388-396) deletion mutant significantly promoted the association of CSQ1 with STIM1, but suppressed both STIM1-Orai1 interaction and SOCE, while over-expression of the C-terminal (amino acid 362-396) deletion mutant had no effect.The physical interaction between low polymeric forms of CSQ1 and STIM1 likely acts by interfering with STIM1 oligimerization and inhibits STIM1-Orai1 interaction, providing a brake to SOCE under physiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Capital Medical University, Beijing 100069, P.R. China.

ABSTRACT
Interaction between the endoplasmic reticulum (ER)-located stromal interaction molecue1 (STIM1) and the plasma membrane-located Ca(2+) channel subunit, Orai1, underlies store-operated Ca(2+) entry (SOCE). Calsequestrin1 (CSQ1), a sarcoplasmic reticulum Ca(2+) buffering protein, inhibits SOCE, but the mechanism of action is unknown. We identified an interaction between CSQ1 and STIM1 in HEK293 cells. An increase in monomeric CSQ1 induced by depleted Ca(2+) stores, or trifluoperazine (TFP), a blocker of CSQ folding and aggregation, enhanced the CSQ1-STIM1 interaction. In cells with Ca(2+) stores depleted, TFP further increased CSQ1 monomerization and CSQ1-STIM1 interaction, but reduced the association of STIM1 with Orai1 and SOCE. Over-expression of CSQ1 or a C-terminal (amino acid 388-396) deletion mutant significantly promoted the association of CSQ1 with STIM1, but suppressed both STIM1-Orai1 interaction and SOCE, while over-expression of the C-terminal (amino acid 362-396) deletion mutant had no effect. The physical interaction between low polymeric forms of CSQ1 and STIM1 likely acts by interfering with STIM1 oligimerization and inhibits STIM1-Orai1 interaction, providing a brake to SOCE under physiological conditions. This novel regulatory mechanism for SOCE may also contribute to the pathological Ca(2+) overload in calsequestrin deficient diseases, such as malignant hyperthermia and ventricular tachycardia.

No MeSH data available.


Related in: MedlinePlus

CSQ1 interacts with STIM1 in HEK293 cells.HEK293 cells cultured in FBS-containing media for 24 h were incubated in FBS-free DMEM for 5 h, and then cells were suspended in Ca2+-free Hepes-buffered saline solution (HBSS, 0.1 mM EGTA added) for 5 min and stimulated with or without 1 μM TG for 5 min until harvested. (A) Co-immunoprecipitations between STIM1 and Orai1. Whole cell lysates (WCL) were immunoprecipitated with specific anti-STIM1monoclonal mouse antibody (mAb) and protein-G agarose, then Western blot was performed with specific anti-Orai1mAb (left). Equal amounts of normal mouse IgG was used as an immunoprecipitation antibody substitute for negative control. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control in all the co-immunoprecipitation experiments. STIM1-Orail1 association intensities were quantified as average protein ratios of Orai1/STIM1 (right). (B) Co-immunoprecipitation between CSQ1 and STIM1. Whole cell lysates were immunoprecipitated with specific anti-CSQ1 mAb and protein-G agarose, then Western blot was performed with specific anti-STIM1mAb (left). STIM1-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). **p < 0.01 vs. control, n = 3 for both A and B. (C) Conformation of CSQ1 is influenced by TFP or TG treatment. HEK293 cells were pre-incubated with or without 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with or without 1 μM TG for 5 min. All the cells were cross-linked with 2% formaldehyde for 5 min, lysed and subjected to 10% SDS-PAGE and subsequent Western blotting with anti-CSQ1  mAb. (D) The antibody-reacting bands of CSQ1 were quantified and the average ratios of monomer/polymer CSQ1 are represented as means ± SD. N = 4 for each bar, *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 vs. TG group.
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f1: CSQ1 interacts with STIM1 in HEK293 cells.HEK293 cells cultured in FBS-containing media for 24 h were incubated in FBS-free DMEM for 5 h, and then cells were suspended in Ca2+-free Hepes-buffered saline solution (HBSS, 0.1 mM EGTA added) for 5 min and stimulated with or without 1 μM TG for 5 min until harvested. (A) Co-immunoprecipitations between STIM1 and Orai1. Whole cell lysates (WCL) were immunoprecipitated with specific anti-STIM1monoclonal mouse antibody (mAb) and protein-G agarose, then Western blot was performed with specific anti-Orai1mAb (left). Equal amounts of normal mouse IgG was used as an immunoprecipitation antibody substitute for negative control. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control in all the co-immunoprecipitation experiments. STIM1-Orail1 association intensities were quantified as average protein ratios of Orai1/STIM1 (right). (B) Co-immunoprecipitation between CSQ1 and STIM1. Whole cell lysates were immunoprecipitated with specific anti-CSQ1 mAb and protein-G agarose, then Western blot was performed with specific anti-STIM1mAb (left). STIM1-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). **p < 0.01 vs. control, n = 3 for both A and B. (C) Conformation of CSQ1 is influenced by TFP or TG treatment. HEK293 cells were pre-incubated with or without 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with or without 1 μM TG for 5 min. All the cells were cross-linked with 2% formaldehyde for 5 min, lysed and subjected to 10% SDS-PAGE and subsequent Western blotting with anti-CSQ1  mAb. (D) The antibody-reacting bands of CSQ1 were quantified and the average ratios of monomer/polymer CSQ1 are represented as means ± SD. N = 4 for each bar, *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 vs. TG group.

Mentions: We have investigated the relationship between CSQ1 and the essential components of SOCE in HEK293 cells, a cell line in which functional CSQ1 but not CSQ2 expression has been reported26 (supplemental Fig. 1). As previously shown27, physical interaction between STIM1 and Orai1 was markedly increased upon depletion of Ca2+ stores by 1 μM TG (Fig. 1A), and a robust Ca2+ entry through the gating of SOC channels was observed (data not shown). CSQ1 also co-immunoprecipitated with STIM1, and this association was enhanced by TG stimulation (Fig. 1B). These data indicate that both the CSQ1-STIM1 and STIM1-Orai1 interactions increase with Ca2+ store depletion, suggesting that CSQ1 may be affecting SOCE through its interaction with STIM1.


Retrograde regulation of STIM1-Orai1 interaction and store-operated Ca2+ entry by calsequestrin.

Wang L, Zhang L, Li S, Zheng Y, Yan X, Chen M, Wang H, Putney JW, Luo D - Sci Rep (2015)

CSQ1 interacts with STIM1 in HEK293 cells.HEK293 cells cultured in FBS-containing media for 24 h were incubated in FBS-free DMEM for 5 h, and then cells were suspended in Ca2+-free Hepes-buffered saline solution (HBSS, 0.1 mM EGTA added) for 5 min and stimulated with or without 1 μM TG for 5 min until harvested. (A) Co-immunoprecipitations between STIM1 and Orai1. Whole cell lysates (WCL) were immunoprecipitated with specific anti-STIM1monoclonal mouse antibody (mAb) and protein-G agarose, then Western blot was performed with specific anti-Orai1mAb (left). Equal amounts of normal mouse IgG was used as an immunoprecipitation antibody substitute for negative control. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control in all the co-immunoprecipitation experiments. STIM1-Orail1 association intensities were quantified as average protein ratios of Orai1/STIM1 (right). (B) Co-immunoprecipitation between CSQ1 and STIM1. Whole cell lysates were immunoprecipitated with specific anti-CSQ1 mAb and protein-G agarose, then Western blot was performed with specific anti-STIM1mAb (left). STIM1-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). **p < 0.01 vs. control, n = 3 for both A and B. (C) Conformation of CSQ1 is influenced by TFP or TG treatment. HEK293 cells were pre-incubated with or without 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with or without 1 μM TG for 5 min. All the cells were cross-linked with 2% formaldehyde for 5 min, lysed and subjected to 10% SDS-PAGE and subsequent Western blotting with anti-CSQ1  mAb. (D) The antibody-reacting bands of CSQ1 were quantified and the average ratios of monomer/polymer CSQ1 are represented as means ± SD. N = 4 for each bar, *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 vs. TG group.
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Related In: Results  -  Collection

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f1: CSQ1 interacts with STIM1 in HEK293 cells.HEK293 cells cultured in FBS-containing media for 24 h were incubated in FBS-free DMEM for 5 h, and then cells were suspended in Ca2+-free Hepes-buffered saline solution (HBSS, 0.1 mM EGTA added) for 5 min and stimulated with or without 1 μM TG for 5 min until harvested. (A) Co-immunoprecipitations between STIM1 and Orai1. Whole cell lysates (WCL) were immunoprecipitated with specific anti-STIM1monoclonal mouse antibody (mAb) and protein-G agarose, then Western blot was performed with specific anti-Orai1mAb (left). Equal amounts of normal mouse IgG was used as an immunoprecipitation antibody substitute for negative control. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control in all the co-immunoprecipitation experiments. STIM1-Orail1 association intensities were quantified as average protein ratios of Orai1/STIM1 (right). (B) Co-immunoprecipitation between CSQ1 and STIM1. Whole cell lysates were immunoprecipitated with specific anti-CSQ1 mAb and protein-G agarose, then Western blot was performed with specific anti-STIM1mAb (left). STIM1-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). **p < 0.01 vs. control, n = 3 for both A and B. (C) Conformation of CSQ1 is influenced by TFP or TG treatment. HEK293 cells were pre-incubated with or without 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with or without 1 μM TG for 5 min. All the cells were cross-linked with 2% formaldehyde for 5 min, lysed and subjected to 10% SDS-PAGE and subsequent Western blotting with anti-CSQ1  mAb. (D) The antibody-reacting bands of CSQ1 were quantified and the average ratios of monomer/polymer CSQ1 are represented as means ± SD. N = 4 for each bar, *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 vs. TG group.
Mentions: We have investigated the relationship between CSQ1 and the essential components of SOCE in HEK293 cells, a cell line in which functional CSQ1 but not CSQ2 expression has been reported26 (supplemental Fig. 1). As previously shown27, physical interaction between STIM1 and Orai1 was markedly increased upon depletion of Ca2+ stores by 1 μM TG (Fig. 1A), and a robust Ca2+ entry through the gating of SOC channels was observed (data not shown). CSQ1 also co-immunoprecipitated with STIM1, and this association was enhanced by TG stimulation (Fig. 1B). These data indicate that both the CSQ1-STIM1 and STIM1-Orai1 interactions increase with Ca2+ store depletion, suggesting that CSQ1 may be affecting SOCE through its interaction with STIM1.

Bottom Line: In cells with Ca(2+) stores depleted, TFP further increased CSQ1 monomerization and CSQ1-STIM1 interaction, but reduced the association of STIM1 with Orai1 and SOCE.Over-expression of CSQ1 or a C-terminal (amino acid 388-396) deletion mutant significantly promoted the association of CSQ1 with STIM1, but suppressed both STIM1-Orai1 interaction and SOCE, while over-expression of the C-terminal (amino acid 362-396) deletion mutant had no effect.The physical interaction between low polymeric forms of CSQ1 and STIM1 likely acts by interfering with STIM1 oligimerization and inhibits STIM1-Orai1 interaction, providing a brake to SOCE under physiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Capital Medical University, Beijing 100069, P.R. China.

ABSTRACT
Interaction between the endoplasmic reticulum (ER)-located stromal interaction molecue1 (STIM1) and the plasma membrane-located Ca(2+) channel subunit, Orai1, underlies store-operated Ca(2+) entry (SOCE). Calsequestrin1 (CSQ1), a sarcoplasmic reticulum Ca(2+) buffering protein, inhibits SOCE, but the mechanism of action is unknown. We identified an interaction between CSQ1 and STIM1 in HEK293 cells. An increase in monomeric CSQ1 induced by depleted Ca(2+) stores, or trifluoperazine (TFP), a blocker of CSQ folding and aggregation, enhanced the CSQ1-STIM1 interaction. In cells with Ca(2+) stores depleted, TFP further increased CSQ1 monomerization and CSQ1-STIM1 interaction, but reduced the association of STIM1 with Orai1 and SOCE. Over-expression of CSQ1 or a C-terminal (amino acid 388-396) deletion mutant significantly promoted the association of CSQ1 with STIM1, but suppressed both STIM1-Orai1 interaction and SOCE, while over-expression of the C-terminal (amino acid 362-396) deletion mutant had no effect. The physical interaction between low polymeric forms of CSQ1 and STIM1 likely acts by interfering with STIM1 oligimerization and inhibits STIM1-Orai1 interaction, providing a brake to SOCE under physiological conditions. This novel regulatory mechanism for SOCE may also contribute to the pathological Ca(2+) overload in calsequestrin deficient diseases, such as malignant hyperthermia and ventricular tachycardia.

No MeSH data available.


Related in: MedlinePlus