RT Journal Article
SR Electronic
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
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 510
OP 526
DO 10.1124/mol.111.074658
VO 81
IS 4
A1 Premanand C. Sundivakkam
A1 Marc Freichel
A1 Vandana Singh
A1 Joseph P. Yuan
A1 Stephen M. Vogel
A1 Veit Flockerzi
A1 Asrar B. Malik
A1 Chinnaswamy Tiruppathi
YR 2012
UL http://molpharm.aspetjournals.org/content/81/4/510.abstract
AB 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.