Caveolin-1 regulates store-operated Ca2+ influx by binding of its scaffolding domain to transient receptor potential channel-1 in endothelial cells.
Caveolin-1 associates with store-operated cation channels (SOC) in endothelial cells. We examined the role of the caveolin-1 scaffolding domain (CSD) in regulating the SOC [i.e., transient receptor potential channel-1 (TRPC1)] in human pulmonary artery endothelial cells (HPAECs). We used the cell-permeant antennapedia (AP)-conjugated CSD peptide, which competes for protein binding partners with caveolin-1, to assess the interactions of caveolin-1 with TRPC1 and its consequences on thrombin-induced Ca2+ influx. We observed that AP-CSD peptide markedly reduced thrombin-induced Ca2+ influx via SOC in HPAECs in contrast to control peptide. AP-CSD also suppressed thapsigargin-induced Ca2+ influx. Streptavidin-bead pull-down assay indicated strong binding of biotin-labeled AP-CSD peptide to TRPC1. Immunoprecipitation studies demonstrated an interaction between endogenous TRPC1 and ectopically expressed hemagglutinin-tagged CSD. Analysis of the deduced TRPC1 amino acid sequence revealed the presence of CSD binding consensus sequence in the TRPC1 C terminus. We also observed that an AP-TRPC1 peptide containing the CSD binding sequence markedly reduced the thrombin-induced Ca2+ influx. We identified the interaction between biotin-labeled AP-TRPC1 C terminus peptide and caveolin-1. Thus, these results demonstrate a crucial role of caveolin-1 scaffolding domain interaction with TRPC1 in regulating Ca2+ influx via SOC.
Kwiatek
Angela M
AM
Department of Pharmacology (M/C868), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.
Minshall
Richard D
RD
Cool
David R
DR
Skidgel
Randal A
RA
Malik
Asrar B
AB
Tiruppathi
Chinnaswamy
C
D000595:Amino Acid Sequence; D015220:Calcium Channels / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D045170:Matrix Attachment Regions; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010539:Permeability; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D050052:TRPC Cation Channels / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology
D015220:Calcium Channels; D051242:Caveolin 1; D010455:Peptides; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D016392:Proto-Oncogene Proteins pp60(c-src); D013917:Thrombin