Intracellular mediators of granulysin-induced cell death

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca²⁺ and K⁺ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca ²⁺ channel blockers, nickel and econazole, and the K⁺ channel blockers, tetra- ethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i), the decrease in intracellular K⁺, and apoptosis. Thapsigargin, which releases Ca²⁺ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca²⁺]_i in Jurkat cells, indicating that the initial increase in [Ca²⁺]_i is from intracel- lular stores. The rise in [Ca²⁺]_i precedes a decrease in intracellular K+, and elevated extracellular K⁺ prevents granulysin mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.