Mechanisms of necrotic cell death. Necrotic cell death is initiated by numerous cell insults that include calcium overload, excessive ROS production, hypoxia, ATP depletion, or acidosis. A central feature of necrotic cell death is the rapid loss of mitochondrial membrane potential (Δψ_m), otherwise known as the permeability transition (mPT). mPT is facilitated by the formation of a mitochondrial permeability transition pore (mPTP) traversing the inner and outer mitochondrial membranes (inset 1). Opening of the mPTP allows small ions and metabolites to permeate freely across the inner mitochondrial membrane, essentially shutting down the proton gradient necessary for ATP generation (inset 2). mPT results in the inability of the mitochondria to maintain electrochemical potential, resulting in an increased mitochondrial matrix volume and organelle swelling (and eventually rupture). mPT is catalysed by the inner mitochondrial protein cyclophilin D (CypD; inset 1), with deficiency of this protein leading to resistance to necrosis (but not apoptosis). Another consequence of mitochondrial dysfunction is an increased production of mitochondrial reactive oxygen species (ROS), which when released facilitates mPTP formation as well as damage to DNA and proteins, as well as oxidative modification of lipid components of organelle and plasma membranes. A major instigator of mPTP formation is perturbed calcium homeostasis (inset 3). Many of the channels linked to the control of intracellular Ca²⁺ levels, including the Plasma Membrane ATPase (PMCA) and the Sarcoplasmic Endoreticulum Ca²⁺ ATPase (SERCA), are ATP-dependent, such that after an initial Ca²⁺ insult the subsequent mitochondrial damage and ATP depletion exacerbate elevated intracellular Ca²⁺ levels. Moreover, elevated intracellular calcium activates proteases such as calpain, leading to the degradation of PMCA, further perturbing calcium homeostasis.