Store-operated Ca2+ channels formed by TRPC1, TRPC6 and Orai1 and non-store-operated channels formed by TRPC3 are involved in the regulation of NADPH oxidase in HL-60 granulocytes

SummaryCa2+ influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca2+ conditions underlying the oxidative response are clearly delineated. Here, we show that store-operated Ca2+ entry (SOCE) is required at the beginning of NADPH oxidase activation in response to fMLF (N-formyl-l-methionyl-l-leucyl-l-phenylalanine ) in neutrophil-like HL-60 cells. When extracellular Ca2+ is initially removed, early addition of Ca2+ after stimulation causes a complete restoration of Ca2+ entry and H2O2 production. Both Ca2+ entry and H2O2 production are decreased by purported SOCE blockers, 2-aminoethoxydiphenyl borane (2-APB) and SK&F 96365. Endogenously expressed TRPC (transient receptor potential canonical) homologues and Orai1 were investigated for their role in supporting store-operated Ca2+ channels activity. TRPC1, TRPC6 and Orai1 knock-out by siRNA resulted in the inhibition of Ca2+ influx and H2O2 production in response to fMLF and thapsigargin while suppression of TRPC3 had no effect on thapsigargin induced-SOCE. 2-APB and SK&F 96365 were able to amplify the reduction of fMLF-stimulated Ca2+ entry and H2O2 production observed in cells transfected by TRPC3 siRNA. In summary, Ca2+ influx in HL-60 cells relies on different membrane TRPC channels and Orai1 for allowing NADPH oxidase activation. TRPC3 primarily mediates SOCE-independent pathways and TRPC1, TRPC6 and Orai1 exclusively contribute to SOCE.