Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice

Abstract

A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death.DOI: http://dx.doi.org/10.7554/eLife.00772.001

Aberrant Mitochondrial Fission Is Maladaptive in Desmin Mutation–Induced Cardiac Proteotoxicity

“…Mitochondria in D7 Des Tg and Ntg hearts were isolated as described previously 22, 23. Briefly, hearts were harvested, homogenized in MS‐EGTA buffer (225 mmol/L mannitol, 75 mmol/L sucrose, 5 mmol/L HEPES, and 1 mmol/L EGTA, pH 7.4) and subjected to differential centrifugation.…”

“…Mitochondrial oxygen consumption rate was measured with an XF24 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA) by methods as described previously 22, 23, 24. Heart mitochondria were isolated using MS‐EGTA buffer (225 mmol/L mannitol, 75 mmol/L sucrose, 5 mmol/L HEPES, and 1 mmol/L EGTA, pH 7.4) by differential centrifugation as described above.…”

“…Mitochondrial Ca 2+ retention capacity was measured with Calcium Green 5N (Molecular Probes) as previously described 22, 23. Cardiac mitochondria (100 μg) were suspended in KCl buffer containing 125 mmol/L KCl, 20 mmol/L HEPES, 2 mmol/L MgCl 2 , 2 mmol/L potassium phosphate, and 40 μmol/L EGTA, pH 7.2, 200 nmol/L Calcium Green‐5N (Molecular Probes), 7 mmol/L pyruvate, and 1 mmol/L malate.…”

“…Mitochondrial swelling was induced by challenging the mitochondria with 200 μmol/L CaCl 2 , and the absorbance at 540 nm was monitored 22, 23…”

Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives

“…Induction of the MPT results in a loss of the mitochondrial membrane potential due to dissipation of the proton gradient and accompanying collapse of mitochondrial ATP production, ultimately resulting in cell necrosis [57–60]. It is generally agreed on that the MPT is caused by the opening of a large channel (mitochondrial permeability transition pore), which is composed of various components, including Bax and Bak [61, 62] on the mitochondrial outer membrane and subunits of the ATP synthase on the inner membrane [56]. Additional regulatory components include cyclophilin D [63], modulation of which can influence APAP hepatotoxicity in a dose dependent manner [57, 59, 60].…”