Role of inositol 1,4,5-trisphosphate receptors in apoptosis in DT40 lymphocytes

The role of inositol 1,4,5-trisphosphate receptors (IP3R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP3R constructs. Both full-length type-I IP3R and a truncated construct corresponding to the caspase-3 cleaved "channel-only" fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP3R construct harbored a mutation that inactivates the pore of the Ca2+ channel (D2550A). However, a full-length wild-type IP3R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca2+ in DT40 cells expressing wildtype or pore-dead IP3R mutants. However, in the latter case all the Ca2+ increase originated from Ca2+ entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca2+ chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP(3)Rs or containing the pore-dead mutant. We conclude that nonfunctional IP(3)Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca2+ entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP(3)Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP(3)Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain.