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

“As previously reported, Bax/Bak1 double-knockout (DKO) MEFs were highly resistant to cell death by the apoptotic inducer staurosporine (Wei et al., 2001), while Wt MEFs showed high levels of killing (Figure 1A).”

“Necroptosis is induced in cells when stimulated with tumor necrosis factor α (TNFα) in the presence of caspase inhibitors, such as zVAD-fmk (Degterev et al., 2005).”

“Recently, mixed lineage kinase domain–like protein (MLKL) and phosphoglycerate mutase family member 5 (PGAM5) were shown to be downstream of the RIP proteins (Sun et al., 2012; Wang et al., 2012).”

“RIP1 is able to interact with RIP3 in controlling necroptosis (Cho et al., 2009; He et al., 2009; Zhang et al., 2009).”

“Indeed, deletion of the gene encoding CypD renders mitochondria resistant to Ca2+ overload– induced swelling and the heart and brain partially resistant to cell death due to ischemic injury (Baines et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005).”

“RIP1 is able to interact with RIP3 in controlling necroptosis (Cho et al., 2009; He et al., 2009; Zhang et al., 2009).”

“Over a decade ago, several reports suggested that Bax and/or Bak directly interacted with presumed components of the MPTP, the adenine nucleotide translocator (ANT) in the inner membrane and the VDAC in the outer membrane (Marzo et al., 1998; Narita et al., 1998; Shimizu et al., 1999).”

“RIP1 is able to interact with RIP3 in controlling necroptosis (Cho et al., 2009; He et al., 2009; Zhang et al., 2009).”

“Indeed, deletion of the gene encoding CypD renders mitochondria resistant to Ca2+ overload– induced swelling and the heart and brain partially resistant to cell death due to ischemic injury (Baines et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005).”

“Bax/Bak regulate cell death independent of ER Ca2+ Bax/Bak1 DKO cells were previously shown to have reduced Ca2+ levels in the endoplasmic reticulum (ER) as a protective mechanism against cell death (Scorrano et al., 2003).”

“To create heart- and liverspecific Bax and Bak1 knockout mice, Baxfl/fl Bak1−/− mice were crossed with α-myosin heavy chain (αMHC)-Cre and albumin-Cre transgenic lines (Postic et al., 1999; Oka et al., 2006).”

“Over a decade ago, several reports suggested that Bax and/or Bak directly interacted with presumed components of the MPTP, the adenine nucleotide translocator (ANT) in the inner membrane and the VDAC in the outer membrane (Marzo et al., 1998; Narita et al., 1998; Shimizu et al., 1999).”

“…that Bax/Bak function merely to regulate the MPTP by binding VDAC or ANT is not consistent with the data from Slc25a4/Slc25a5 (ANT1/2 protein) and Vdac1/2/3 gene–deleted mice, which showed that neither protein is directly required for MPTP formation (Kokoszka et al., 2004; Baines et al., 2007).”

“Indeed, Bcl-2 family members are known to alter membrane characteristics and have channel-like activities of multiple conductance states (Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997).”

“More importantly, purified mitochondria from these DKO MEFs reconstituted with Wt or the nonoligomerizing Bax mutants (Hoppins et al., 2011) each showed partial restoration of MPTP function in the Ca2+ uptake assay (Figure 3E).”

“…that Bax/Bak function merely to regulate the MPTP by binding VDAC or ANT is not consistent with the data from Slc25a4/Slc25a5 (ANT1/2 protein) and Vdac1/2/3 gene–deleted mice, which showed that neither protein is directly required for MPTP formation (Kokoszka et al., 2004; Baines et al., 2007).”

“Recently, mixed lineage kinase domain–like protein (MLKL) and phosphoglycerate mutase family member 5 (PGAM5) were shown to be downstream of the RIP proteins (Sun et al., 2012; Wang et al., 2012).”

“Over a decade ago, several reports suggested that Bax and/or Bak directly interacted with presumed components of the MPTP, the adenine nucleotide translocator (ANT) in the inner membrane and the VDAC in the outer membrane (Marzo et al., 1998; Narita et al., 1998; Shimizu et al., 1999).”

“Indeed, deletion of the gene encoding CypD renders mitochondria resistant to Ca2+ overload– induced swelling and the heart and brain partially resistant to cell death due to ischemic injury (Baines et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005).”

“Indeed, Bcl-2 family members are known to alter membrane characteristics and have channel-like activities of multiple conductance states (Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997).”

“…recent studies have disputed such a direct role, suggesting that Bax/Bak only have a secondary effect, such as by regulating mitochondrial fission/fusion or no effect whatsoever, or just a secondary effect due to organelle rupture (De Marchi et al., 2004; Vaseva et al., 2012; Whelan et al., 2012).”

“DKO MEFs with reconstituted Bax and Bak were also previously described (Kim et al., 2009).”

“Indeed, Bcl-2 family members are known to alter membrane characteristics and have channel-like activities of multiple conductance states (Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997).”

“To create heart- and liverspecific Bax and Bak1 knockout mice, Baxfl/fl Bak1−/− mice were crossed with α-myosin heavy chain (αMHC)-Cre and albumin-Cre transgenic lines (Postic et al., 1999; Oka et al., 2006).”

“Since MPTP formation has been proposed to function as a Ca2+ release mechanism (Elrod et al., 2010; Bernardi and von Stockum, 2012), the observed augmentation in baseline Ca2+ in Bax/Bak-deficient mitochondria, and their greater uptake capacity further suggest that Bax/Bak are required for…”

“…recent studies have disputed such a direct role, suggesting that Bax/Bak only have a secondary effect, such as by regulating mitochondrial fission/fusion or no effect whatsoever, or just a secondary effect due to organelle rupture (De Marchi et al., 2004; Vaseva et al., 2012; Whelan et al., 2012).”

“Ion selectivity was determined through reversal potentials after a 150:30 mM KCl gradient was established across the patch as previously reported (Pavlov et al., 2001).”

“More importantly, purified mitochondria from these DKO MEFs reconstituted with Wt or the nonoligomerizing Bax mutants (Hoppins et al., 2011) each showed partial restoration of MPTP function in the Ca2+ uptake assay (Figure 3E).”

“Histological analysis of the injury induced by myocardial ischemia/reperfusion was described previously (Kaiser et al., 2004).”

“The MPTP has been well characterized by direct patch-clamping of mitochondria and shown to have a specific conductance profile that is distinct from any other channels or permeating activities (Kinnally and Antonsson, 2007).”

“Bax-loxP (fl) Bak1−/− mice were described previously (Takeuchi et al., 2005).”

“Flow cytometry analysis of active Bax has been previously described (Panaretakis et al., 2002).”

“Mitochondrial Ca2+ uptake was measured with Calcium Green-5N (Invitrogen, Grand Island, NY) as previously described (Kwong et al., 2007).”

“Indeed, Nix/BNip3L, which is induced by disease/developmental signaling pathways that enhance cell death, can alter the activity and pro-death characteristics of Bax/ Bak as well as alter MPTP-dependent cell death (Chen et al., 2010).”

“However, restoration with either of the two mutants of Bax that cannot oligomerize, although both still localize to the mitochondria (George et al., 2007; Hoppins et al., 2011), restored ionomycin-induced killing but not apoptotic killing with staurosporine (Figure 3D).”

“Moreover, treatment with gossypol, a BH3-mimetic compound that can convert Bcl-2 and Bcl-xl into pore-forming pro-cell death agents (Lei et al., 2006) restored mitochondrial swelling in the absence of Bax/Bak protein, which was also inhibited with CsA (Figure 4C,D).”

“Seals were formed with these micropipettes on giant liposomes or mitochondria prepared as described previously (Martinez-Caballero et al., 2009).”

“Indeed, we and others have observed that Bax/ Bak1 null MEFs were resistant to this form of cell death (data not shown and Irrinki et al., 2011).”

“…is not a sieve but rather a membrane whose permeability is tightly regulated, despite the presence of other channels such as voltage-dependent anion channel (VDAC, comprising as many as three gene products, Vdac1, Vdac2, and Vdac3) and TOM (Jonas et al., 2004; Martinez-Caballero et al., 2005).”

“As another consideration, the α5/α6 domain of Bax is known to have membrane permeation activity and to permit pore formation on its own, the permeation size of which appears to reach an equilibrium of less than 10 kDa (Fuertes et al., 2010).”

“DOI: 10.7554/eLife.00772 16 of 21 Research article DKO MEFs were infected with a binary adenovirus expressing Bax (Ad-Bax) was previously described and obtained from Dr Bingliang Fang (Kagawa et al., 2000).”

“…recent studies have disputed such a direct role, suggesting that Bax/Bak only have a secondary effect, such as by regulating mitochondrial fission/fusion or no effect whatsoever, or just a secondary effect due to organelle rupture (De Marchi et al., 2004; Vaseva et al., 2012; Whelan et al., 2012).”

“Indeed, increasing concentrations of poloxamer-188 (P-188), which inherently reduces membrane permeability characteristics (Wu et al., 2004), prevented mitochondrial swelling induced by Ca2+ as analyzed in purified mitochondria in solution or by transmission electron microscopy (Figure 4E,F).”

“…is not a sieve but rather a membrane whose permeability is tightly regulated, despite the presence of other channels such as voltage-dependent anion channel (VDAC, comprising as many as three gene products, Vdac1, Vdac2, and Vdac3) and TOM (Jonas et al., 2004; Martinez-Caballero et al., 2005).”

“Cells at 80% confluence were harvested and mitochondria were isolated as previously described (Murphy et al., 2001).”

“However, gossypol, which can convert endogenous Bcl-2 proteins into a pro-death configuration and increase membrane permeability (de Peyster et al., 1986), enhanced or induced ionomycin-dependent cell death in Wt and DKO MEFs, respectively (Figure 7A).”

“DOI: 10.7554/eLife.00772 14 of 21 Research article one of which has a small pore size (0.9 nm) and is homogenous in the outer mitochondrial membrane, which could fulfill the permeability function we observed here to permit nonapoptotic cell death (Lin et al., 2011).”

“Bcl-2 family members appear to generally alter the permeability and rigidity of lipid membranes, and even the Bcl-2 homologue from Caenorhabditis elegans, CED-9 can induce leakiness of reconstituted lipid membranes (Tan et al., 2011).”