Ca²⁺-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak

The ability of mitochondria to buffer a rapid rise in cytosolic Ca²⁺ is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca²⁺ imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca²⁺ release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca²⁺. When the latter exceeded the organelle buffering capacity, an abrupt mitochondrial inner membrane permeabilization (MIMP) occurred, releasing matrix contents into the cytosol. MIMP was followed by cell death that was independent of Bcl-2 family members and inhibitable by the intracellular Ca²⁺ chelator BAPTA-AM. Cyclosporin A (CsA), capable of blocking the mitochondrial permeability transition (MPT), completely prevented cell death induced by m-3M3FBS. However, CsA acted upstream of mitochondria by preventing Ca²⁺ release from ER stores. Therefore, loss of Ca²⁺ intracellular balance and mitochondrial Ca²⁺ overload followed by MIMP induced a cell death process that is distinct from Bcl-2 family-regulated mitochondrial outer membrane permeabilization (MOMP). Further, the inhibition of cell death by CsA or its analogues can be independent of effects on the MPT.