Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

Abstract The ER Ca 2+ sensor STIM1 and the Ca 2+ channel Orai1 are key players in store-operated Ca 2+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca 2+ entry by about 50–60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca 2+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca 2+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PK Ceta is required for Ca 2+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca 2+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca 2+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca 2+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.