Abstract

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 STIM1^K684E-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 Orai1^R91W mutant or Orai3^E81W mutant in WT-ECs failed to prevent thrombin-induced Ca²⁺ entry. In contrast, expression of the dominant-negative TRPC4^EE647-648KK 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.