Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+ entry in endothelial cells.
The TRPC1 (transient receptor potential canonical-1) channel is a constituent of the nonselective cation channel that mediates Ca2+ entry through store-operated channels (SOCs) in human endothelial cells. We investigated the role of protein kinase Calpha (PKCalpha) phosphorylation of TRPC1 in regulating the opening of SOCs. Thrombin or thapsigargin added to the external medium activated Ca2+ entry after Ca2+ store depletion, which we monitored by changes in cellular Fura 2 fluorescence. Internal application of the metabolism-resistant analog of inositol 1,4,5-trisphosphate (IP3) activated an inward cationic current within 1 min, which we recorded using the whole cell patch clamp technique. La3+ or Gd3+ abolished the current, consistent with the known properties of SOCs. Pharmacological (Gö6976) or genetic (kinase-defective mutant) inhibition of PKCalpha markedly inhibited IP3-induced activation of the current. Thrombin or thapsigargin also activated La3+-sensitive Ca2+ entry in a PKCalpha-dependent manner. We determined the effects of a specific antibody directed against an extracellular epitope of TRPC1 to address the functional importance of TRPC1. External application of the antibody blocked thrombin- or IP3-induced Ca2+ entry. In addition, we showed that addithrombin or thapsigargin induced phosphorylation of TRPC1 within 1 min. Thrombin failed to induce TRPC1 phosphorylation in the absence of PKCalpha activation. Phosphorylation of TRPC1 and the resulting Ca2+ entry were essential for the increase in permeability induced by thrombin in confluent endothelial monolayers. These results demonstrate that PKCalpha phosphorylation of TRPC1 is an important determinant of Ca2+ entry in human endothelial cells.
Ahmmed
Gias U
GU
Department of Pharmacology, College of Medicine, University of Illinois, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.
Mehta
Dolly
D
Vogel
Stephen
S
Holinstat
Michael
M
Paria
Biman C
BC
Tiruppathi
Chinnaswamy
C
Malik
Asrar B
AB
D000906:Antibodies / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D050052:TRPC Cation Channels; D019284:Thapsigargin / Q000494:pharmacology; D014471:Umbilical Veins
D000906:Antibodies; D015220:Calcium Channels; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium