The Ca2+ sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca2+ entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells

Premanand C. Sundivakkam, Marc Freichel, Vandana Singh, Joseph P. Yuan, Stephen M. Vogel, Veit Flockerzi, Asrar B. Malik, Chinnaswamy Tiruppathi

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Abstract

We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+-sensor, and Orai1, a Ca 2+ selective channel, in regulating Ca2+ entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca2+ entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca2+entry secondary to depletion of ER Ca2+ stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1 or Orai3) or expression of the dominant-negative STIM1K684E-K685E mutant in ECs also suppressed Ca2+ entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(-/-)-ECs failed to rescue Ca 2+ entry; however, WT-TRPC4 expression in TRPC4(-/-)-ECs restored Ca2+ entry indicating the requirement for TRPC4 in mediating store-operated Ca2+ entry. Moreover, expression of the dominant-negative Orai1R91W mutant or Orai3E81W mutant in WTECs failed to prevent thrombin-induced Ca2+ entry. In contrast, expression of the dominant-negative TRPC4EE647-648KK mutant in WT-ECs markedly reduced thrombin-induced Ca2+ entry. In ECs expressing YFP-STIM1, ER-store Ca2+ depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(-/-) cells, indicating that mobilization of STIM1 and engagement of its Ca2+ sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca2+ stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca2+-entry channels, which are essential for mediating Ca2+ entry-dependent disruption of the endothelial barrier.

Original languageEnglish
Pages (from-to)510-526
Number of pages17
JournalMolecular Pharmacology
Volume81
Issue number4
DOIs
StatePublished - 1 Apr 2012

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Endothelial Cells
Endoplasmic Reticulum
Thrombin
Transient Receptor Potential Channels
Stromal Interaction Molecule 1
Lung
Membranes

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Sundivakkam, Premanand C. ; Freichel, Marc ; Singh, Vandana ; Yuan, Joseph P. ; Vogel, Stephen M. ; Flockerzi, Veit ; Malik, Asrar B. ; Tiruppathi, Chinnaswamy. / The Ca2+ sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca2+ entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells. In: Molecular Pharmacology. 2012 ; Vol. 81, No. 4. pp. 510-526.
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title = "The Ca2+ sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca2+ entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells",
abstract = "We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+-sensor, and Orai1, a Ca 2+ selective channel, in regulating Ca2+ entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca2+ entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca2+entry secondary to depletion of ER Ca2+ stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1 or Orai3) or expression of the dominant-negative STIM1K684E-K685E mutant in ECs also suppressed Ca2+ entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(-/-)-ECs failed to rescue Ca 2+ entry; however, WT-TRPC4 expression in TRPC4(-/-)-ECs restored Ca2+ entry indicating the requirement for TRPC4 in mediating store-operated Ca2+ entry. Moreover, expression of the dominant-negative Orai1R91W mutant or Orai3E81W mutant in WTECs failed to prevent thrombin-induced Ca2+ entry. In contrast, expression of the dominant-negative TRPC4EE647-648KK mutant in WT-ECs markedly reduced thrombin-induced Ca2+ entry. In ECs expressing YFP-STIM1, ER-store Ca2+ depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(-/-) cells, indicating that mobilization of STIM1 and engagement of its Ca2+ sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca2+ stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca2+-entry channels, which are essential for mediating Ca2+ entry-dependent disruption of the endothelial barrier.",
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The Ca2+ sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca2+ entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells. / Sundivakkam, Premanand C.; Freichel, Marc; Singh, Vandana; Yuan, Joseph P.; Vogel, Stephen M.; Flockerzi, Veit; Malik, Asrar B.; Tiruppathi, Chinnaswamy.

In: Molecular Pharmacology, Vol. 81, No. 4, 01.04.2012, p. 510-526.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The Ca2+ sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca2+ entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells

AU - Sundivakkam, Premanand C.

AU - Freichel, Marc

AU - Singh, Vandana

AU - Yuan, Joseph P.

AU - Vogel, Stephen M.

AU - Flockerzi, Veit

AU - Malik, Asrar B.

AU - Tiruppathi, Chinnaswamy

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AB - We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+-sensor, and Orai1, a Ca 2+ selective channel, in regulating Ca2+ entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca2+ entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca2+entry secondary to depletion of ER Ca2+ stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1 or Orai3) or expression of the dominant-negative STIM1K684E-K685E mutant in ECs also suppressed Ca2+ entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(-/-)-ECs failed to rescue Ca 2+ entry; however, WT-TRPC4 expression in TRPC4(-/-)-ECs restored Ca2+ entry indicating the requirement for TRPC4 in mediating store-operated Ca2+ entry. Moreover, expression of the dominant-negative Orai1R91W mutant or Orai3E81W mutant in WTECs failed to prevent thrombin-induced Ca2+ entry. In contrast, expression of the dominant-negative TRPC4EE647-648KK mutant in WT-ECs markedly reduced thrombin-induced Ca2+ entry. In ECs expressing YFP-STIM1, ER-store Ca2+ depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(-/-) cells, indicating that mobilization of STIM1 and engagement of its Ca2+ sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca2+ stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca2+-entry channels, which are essential for mediating Ca2+ entry-dependent disruption of the endothelial barrier.

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