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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

TFP enhances CSQ1-STIM1 association but inhibits STIM1-Orai1 interaction and SOCE, and deletion of carboxyl-terminal 35 amino acids in CSQ1 attenuates CSQ1 binding to STIM1.(A) Effect of TFP on the association of CSQ1 with STIM1. HEK293 cells were preincubated with (TG+TFP group) or without (TG group) 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with 1 μM TG for another 5 min. Whole cell lysates were immunoprecipitated with anti-CSQ1 mAb and protein-G agarose, and then Western blot was performed with anti-STIM1 mAb (left). CSQ1-STIM1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). (B) Effect of TFP on the association of STIM1 with Orai1, procedure as in A. **p < 0.01 vs. TG group, n = 3 for both A and B. (C) TFP impacts [Ca2+]i response to TG. TG induced Ca2+ mobilization in Ca2+-free HBSS (0.1 mM EGTA) and Ca2+ entry following addition of 1.8 mM Ca2+ in Fura2-loaded HEK293 cells. (D) Averaged delta peak [Ca2+]i (minus the basal [Ca2+]i) ± SD from experiments shown in C. N = 5 independent experiments for each bar, **p<0.01 vs. TG group. (E) Co-immunoprecipitation of exogenous CSQ1 and endogenous STIM1. HEK293 cells expressing empty vectors or exogenous HA-CSQ1, C9 or C35 truncations were suspended in Ca2+-free HBSS and then stimulated with 1 μM TG for 5 min. Immunoprecipitation and Western blot as indicated (left). STIM1-HA-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/HA-Tag (right). (F) Reverse co-immunoprecipitation between exogenous HA-CSQ1 and STIM1. Immunoprecipitation and Western blot as indicated (left). HA-Tag-STIM1 association intensities were quantified as average protein ratios ± SD of HA-Tag/STIM1 (right). (G) Co-immunoprecipitation between STIM1 and Orai1. Immunoprecipitation and Western blot as indicated (left). STIM1-Orai1 association intensities were quantified as average protein ratios ± SD of Orai1/STIM1 (right). *p < 0.05 and **p < 0.01 vs. HA-CSQ1 (E and F) or vector group (G); #p < 0.05 and ##p < 0.01 vs. C9-CSQ1 in all the panels.
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f2: TFP enhances CSQ1-STIM1 association but inhibits STIM1-Orai1 interaction and SOCE, and deletion of carboxyl-terminal 35 amino acids in CSQ1 attenuates CSQ1 binding to STIM1.(A) Effect of TFP on the association of CSQ1 with STIM1. HEK293 cells were preincubated with (TG+TFP group) or without (TG group) 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with 1 μM TG for another 5 min. Whole cell lysates were immunoprecipitated with anti-CSQ1 mAb and protein-G agarose, and then Western blot was performed with anti-STIM1 mAb (left). CSQ1-STIM1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). (B) Effect of TFP on the association of STIM1 with Orai1, procedure as in A. **p < 0.01 vs. TG group, n = 3 for both A and B. (C) TFP impacts [Ca2+]i response to TG. TG induced Ca2+ mobilization in Ca2+-free HBSS (0.1 mM EGTA) and Ca2+ entry following addition of 1.8 mM Ca2+ in Fura2-loaded HEK293 cells. (D) Averaged delta peak [Ca2+]i (minus the basal [Ca2+]i) ± SD from experiments shown in C. N = 5 independent experiments for each bar, **p<0.01 vs. TG group. (E) Co-immunoprecipitation of exogenous CSQ1 and endogenous STIM1. HEK293 cells expressing empty vectors or exogenous HA-CSQ1, C9 or C35 truncations were suspended in Ca2+-free HBSS and then stimulated with 1 μM TG for 5 min. Immunoprecipitation and Western blot as indicated (left). STIM1-HA-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/HA-Tag (right). (F) Reverse co-immunoprecipitation between exogenous HA-CSQ1 and STIM1. Immunoprecipitation and Western blot as indicated (left). HA-Tag-STIM1 association intensities were quantified as average protein ratios ± SD of HA-Tag/STIM1 (right). (G) Co-immunoprecipitation between STIM1 and Orai1. Immunoprecipitation and Western blot as indicated (left). STIM1-Orai1 association intensities were quantified as average protein ratios ± SD of Orai1/STIM1 (right). *p < 0.05 and **p < 0.01 vs. HA-CSQ1 (E and F) or vector group (G); #p < 0.05 and ##p < 0.01 vs. C9-CSQ1 in all the panels.

Mentions: Additionally, pretreatment of HEK293 cells with TFP substantially increased the co-immunoprecipitation of CSQ1 and STIM1 mediated by TG in Ca2+-free medium (Fig. 2A), implying that it is the monomer form of CSQ1 that interacts with STIM1. To investigate whether the CSQ1 interaction with STIM1 influenced STIM1-Orai1 interaction, co-immunoprecipitation between STIM1 and Orai1 was examined and revealed that TFP lowered the STIM1-Orai1 association induced by TG and accordingly reduced TG-induced SOCE (Fig. 2B–D). TFP (20 μM) also caused observable increases in [Ca2+]i in Ca2+-free medium and upon restoring the extracellular Ca2+ presumably because of its inhibition of Ca2+ binding with CSQ. Pretreatment of cells with 20 μM TFP for 10 min significantly suppressed the TG-induced Ca2+ influx but not the Ca2+ release phase. Immunohistochemical localization of endogenous STIM1 and CSQ1 also revealed co-localization of STIM1 and CSQ1 by TG, and further increase in co-localization by TFP (Supplemental Fig. 2).


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)

TFP enhances CSQ1-STIM1 association but inhibits STIM1-Orai1 interaction and SOCE, and deletion of carboxyl-terminal 35 amino acids in CSQ1 attenuates CSQ1 binding to STIM1.(A) Effect of TFP on the association of CSQ1 with STIM1. HEK293 cells were preincubated with (TG+TFP group) or without (TG group) 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with 1 μM TG for another 5 min. Whole cell lysates were immunoprecipitated with anti-CSQ1 mAb and protein-G agarose, and then Western blot was performed with anti-STIM1 mAb (left). CSQ1-STIM1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). (B) Effect of TFP on the association of STIM1 with Orai1, procedure as in A. **p < 0.01 vs. TG group, n = 3 for both A and B. (C) TFP impacts [Ca2+]i response to TG. TG induced Ca2+ mobilization in Ca2+-free HBSS (0.1 mM EGTA) and Ca2+ entry following addition of 1.8 mM Ca2+ in Fura2-loaded HEK293 cells. (D) Averaged delta peak [Ca2+]i (minus the basal [Ca2+]i) ± SD from experiments shown in C. N = 5 independent experiments for each bar, **p<0.01 vs. TG group. (E) Co-immunoprecipitation of exogenous CSQ1 and endogenous STIM1. HEK293 cells expressing empty vectors or exogenous HA-CSQ1, C9 or C35 truncations were suspended in Ca2+-free HBSS and then stimulated with 1 μM TG for 5 min. Immunoprecipitation and Western blot as indicated (left). STIM1-HA-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/HA-Tag (right). (F) Reverse co-immunoprecipitation between exogenous HA-CSQ1 and STIM1. Immunoprecipitation and Western blot as indicated (left). HA-Tag-STIM1 association intensities were quantified as average protein ratios ± SD of HA-Tag/STIM1 (right). (G) Co-immunoprecipitation between STIM1 and Orai1. Immunoprecipitation and Western blot as indicated (left). STIM1-Orai1 association intensities were quantified as average protein ratios ± SD of Orai1/STIM1 (right). *p < 0.05 and **p < 0.01 vs. HA-CSQ1 (E and F) or vector group (G); #p < 0.05 and ##p < 0.01 vs. C9-CSQ1 in all the panels.
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f2: TFP enhances CSQ1-STIM1 association but inhibits STIM1-Orai1 interaction and SOCE, and deletion of carboxyl-terminal 35 amino acids in CSQ1 attenuates CSQ1 binding to STIM1.(A) Effect of TFP on the association of CSQ1 with STIM1. HEK293 cells were preincubated with (TG+TFP group) or without (TG group) 20 μM TFP for 10 min, then were suspended in Ca2+-free HBSS for 5 min and stimulated with 1 μM TG for another 5 min. Whole cell lysates were immunoprecipitated with anti-CSQ1 mAb and protein-G agarose, and then Western blot was performed with anti-STIM1 mAb (left). CSQ1-STIM1 association intensities were quantified as average protein ratios ± SD of STIM1/CSQ1 (right). (B) Effect of TFP on the association of STIM1 with Orai1, procedure as in A. **p < 0.01 vs. TG group, n = 3 for both A and B. (C) TFP impacts [Ca2+]i response to TG. TG induced Ca2+ mobilization in Ca2+-free HBSS (0.1 mM EGTA) and Ca2+ entry following addition of 1.8 mM Ca2+ in Fura2-loaded HEK293 cells. (D) Averaged delta peak [Ca2+]i (minus the basal [Ca2+]i) ± SD from experiments shown in C. N = 5 independent experiments for each bar, **p<0.01 vs. TG group. (E) Co-immunoprecipitation of exogenous CSQ1 and endogenous STIM1. HEK293 cells expressing empty vectors or exogenous HA-CSQ1, C9 or C35 truncations were suspended in Ca2+-free HBSS and then stimulated with 1 μM TG for 5 min. Immunoprecipitation and Western blot as indicated (left). STIM1-HA-CSQ1 association intensities were quantified as average protein ratios ± SD of STIM1/HA-Tag (right). (F) Reverse co-immunoprecipitation between exogenous HA-CSQ1 and STIM1. Immunoprecipitation and Western blot as indicated (left). HA-Tag-STIM1 association intensities were quantified as average protein ratios ± SD of HA-Tag/STIM1 (right). (G) Co-immunoprecipitation between STIM1 and Orai1. Immunoprecipitation and Western blot as indicated (left). STIM1-Orai1 association intensities were quantified as average protein ratios ± SD of Orai1/STIM1 (right). *p < 0.05 and **p < 0.01 vs. HA-CSQ1 (E and F) or vector group (G); #p < 0.05 and ##p < 0.01 vs. C9-CSQ1 in all the panels.
Mentions: Additionally, pretreatment of HEK293 cells with TFP substantially increased the co-immunoprecipitation of CSQ1 and STIM1 mediated by TG in Ca2+-free medium (Fig. 2A), implying that it is the monomer form of CSQ1 that interacts with STIM1. To investigate whether the CSQ1 interaction with STIM1 influenced STIM1-Orai1 interaction, co-immunoprecipitation between STIM1 and Orai1 was examined and revealed that TFP lowered the STIM1-Orai1 association induced by TG and accordingly reduced TG-induced SOCE (Fig. 2B–D). TFP (20 μM) also caused observable increases in [Ca2+]i in Ca2+-free medium and upon restoring the extracellular Ca2+ presumably because of its inhibition of Ca2+ binding with CSQ. Pretreatment of cells with 20 μM TFP for 10 min significantly suppressed the TG-induced Ca2+ influx but not the Ca2+ release phase. Immunohistochemical localization of endogenous STIM1 and CSQ1 also revealed co-localization of STIM1 and CSQ1 by TG, and further increase in co-localization by TFP (Supplemental Fig. 2).

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