TY - JOUR T1 - The Ca<sup>2+</sup> Sensor Stromal Interaction Molecule 1 (STIM1) Is Necessary and Sufficient for the Store-Operated Ca<sup>2+</sup> Entry Function of Transient Receptor Potential Canonical (TRPC) 1 and 4 Channels in Endothelial Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 510 LP - 526 DO - 10.1124/mol.111.074658 VL - 81 IS - 4 AU - Premanand C. Sundivakkam AU - Marc Freichel AU - Vandana Singh AU - Joseph P. Yuan AU - Stephen M. Vogel AU - Veit Flockerzi AU - Asrar B. Malik AU - Chinnaswamy Tiruppathi Y1 - 2012/04/01 UR - http://molpharm.aspetjournals.org/content/81/4/510.abstract N2 - We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+-sensor, and Orai1, a Ca2+ 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 Orai1or 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 Ca2+ 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 WT-ECs 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. ER -