Caveolin‐1 scaffolding domain regulates store‐operated Ca 2+ influx by binding to TRPC1 in endothelial cells

Abstract

Caveolin-1 associates with store-operated cation channels (SOC) in endothelial cells. We examined the importance of the caveolin-1 scaffolding domain (CSD) in regulating the predominant SOC (i.e., transient receptor potential channel-1 [TRPC1]) expressed in human pulmonary artery endothelial cells (HPAEC). We tested a cell permeable antennapedia (AP)-CSD peptide effects on thrombin-induced Ca2+ influx via SOC. AP-CSD peptide markedly reduced thrombin-induced Ca2+ influx via SOC in HPAEC. AP-CSD also suppressed thapsigargin-induced Ca2+ influx. Control-peptide had no effect on thrombin-induced Ca2+ influx. Streptavidin-bead pull-down assays indicated strong binding of biotin labeled AP-CSD peptide to TRPC1. Immunoprecipitation studies further demonstrated the interaction between endogenous TRPC1 and ectopically expressed HA-tagged-CSD. Analysis of the deduced TRPC1 amino acid sequence revealed the presence of CSD binding consensus sequence in the TRPC1-C-terminus. Incubation of HPAEC with AP-TRPC1 peptide containing the CSD binding sequence markedly reduced thrombin-induced Ca2+ influx. We also observed interaction between the biotin-labeled AP-TRPC1 C-terminus peptide and cav-1. These results indicate that the cav-1 scaffolding domain interacts with TRPC1 to regulate SOC Ca2+ influx and thus may regulate nitric oxide production and endothelial permeability.