Regulation of ATP-induced calcium release in COS-7 cells by calcineurin

Experiments were conducted to examine the role of calcineurin in regulating Ca2+ fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 mu M) of ATP triggered intracellular Ca2+ release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca2+ release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnA Delta CaMAI) inhibited the ATP-induced increase in intracellular Ca2+ concentration ([Ca2+](i)), in both the presence and the absence of extracellular Ca2+. These results suggest that the constitutively active calcineurin prevented Ca2+ release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca2+ release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P-3 receptor [lns(1,4,5)P3R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnA Delta CaMAI and calcineurin B. On the addition of 10 mu M CsA, the inhibited phosphorylation of Ins(1,4,5)P3R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca2+ release through Ins(1,4,5)P3R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.