Assembly of the Mitochondrial Apoptosis-induced Channel, MAC
2009; Elsevier BV; Volume: 284; Issue: 18 Linguagem: Inglês
10.1074/jbc.m806610200
ISSN1083-351X
AutoresSonia Martinez‐Caballero, Laurent M. Dejean, Michael Kinnally, Kyoung Joon Oh, Carmen A. Mannella, Kathleen W. Kinnally,
Tópico(s)ATP Synthase and ATPases Research
ResumoAlthough Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane α-helices in Bax and Bak, mature MAC pores would typically contain ∼9 monomers and have diameters of 5.5–6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes. Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane α-helices in Bax and Bak, mature MAC pores would typically contain ∼9 monomers and have diameters of 5.5–6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes. Permeabilization of the mitochondrial outer membrane is the commitment step in intrinsic apoptosis. This process is tightly regulated by Bcl-2 family proteins that control formation of the megachannel mitochondrial apoptosis-induced channel (MAC) 2The abbreviations used are: MAC, mitochondrial apoptosis-induced channel; Bax KO, Bax-/-Bak+/+; Bak KO, Bax+/+Bak-/-; DKO, Bax-/-Bak-/-; KO, knock-out; MEF, mouse embryonic fibroblasts; nS, nanosiemen; STS, staurosporine; t-Bid, truncated BID; VDAC, voltage-dependent anion-selective channel; VDAC1-/-VDAC3-/-, VDAC1VDAC3 KO; ELISA, enzyme-linked immunosorbent assay. 2The abbreviations used are: MAC, mitochondrial apoptosis-induced channel; Bax KO, Bax-/-Bak+/+; Bak KO, Bax+/+Bak-/-; DKO, Bax-/-Bak-/-; KO, knock-out; MEF, mouse embryonic fibroblasts; nS, nanosiemen; STS, staurosporine; t-Bid, truncated BID; VDAC, voltage-dependent anion-selective channel; VDAC1-/-VDAC3-/-, VDAC1VDAC3 KO; ELISA, enzyme-linked immunosorbent assay. in this membrane. MAC formation correlates with release of pro-apoptotic factors, including cytochrome c from the intermembrane space into the cytosol, and initiates apoptosis (1Bernardi P. Krauskopf A. Basso E. Petronilli V. Blachly-Dyson E. Di Lisa F. Forte M.A. FEBS J. 2006; 273: 2077-2099Crossref PubMed Scopus (549) Google Scholar, 2Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4002) Google Scholar, 3Guihard G. Bellot G. Moreau C. Pradal G. Ferry N. Thomy R. Fichet P. Meflah K. Vallette F.M. J. Biol. Chem. 2004; 279: 46542-46550Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 4Kinnally K.W. Antonsson B. Apoptosis. 2007; 12: 857-868Crossref PubMed Scopus (198) Google Scholar, 5Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2761) Google Scholar, 6Dejean L.M. Martinez-Caballero S. Kinnally K.W. Cell Death Differ. 2006; 13: 1387-1395Crossref PubMed Scopus (133) Google Scholar, 7Antonsson B. Conti F. 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Unlike Bax, Bak is normally a resident of the mitochondrial outer membrane and is bound to VDAC2, another outer membrane protein (16Cheng E.H. Sheiko T.V. Fisher J.K. Craigen W.J. Korsmeyer S.J. Science. 2003; 301: 513-517Crossref PubMed Scopus (661) Google Scholar). However, Bak is not available for oligomerization until another pro-apoptotic protein, like t-Bid, disrupts the interaction of Bak with VDAC2. In contrast, most Bax is located in the cytoplasm until an apoptotic signal induces the translocation of Bax to the outer membrane of mitochondria and eventual Bax oligomerization in this same membrane (14Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 17Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1568) Google Scholar). Bax and Bak have multiple putative transmembrane domains; the amphipathic helices 5 and 6 of Bax are predicted to form, at least in part, the pore of the cytochrome c release channel (18Annis M. Soucie E. Dlugosz P. Cruz-Aguado J. Penn L. Leber B. Andrews D. EMBO J. 2005; 24: 2096-2103Crossref PubMed Scopus (321) Google Scholar). Bax lacking helices 5 and 6 does not translocate to mitochondria nor cause cytochrome c release (19Parikh N. Koshy C. Dhayabaran V. Perumalsamy L. Sowdhamini R. Sarin A. BMC Cell Biol. 2007; 8: 16Crossref PubMed Scopus (28) Google Scholar, 20Er E. Lalier L. Cartron P. Oliver L. Vallette F. J. Biol. Chem. 2007; 282: 24938-24947Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Given the structural similarities between Bax and Bak, the same helices may be important in formation of the MAC pore by both proteins (21Moldoveanu T. Liu Q. Tocilj A. Watson M. Shore G. Gehring K. Mol. Cell. 2006; 24: 677-688Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Although Bax and Bak are certainly involved in MAC formation, the exact molecular composition of this channel remains unknown. In this study we report that Bax and Bak are functionally redundant with regard to MAC formation and cytochrome c release in mouse embryonic fibroblasts (MEF). This is true despite the fact that Bak normally resides in the outer membrane, whereas Bax is generally translocated to this membrane to induce MAC formation. Our experimental design bypasses Bax translocation and any underlying autocatalytic mechanism that might be involved (22Lartigue L. Medina C. Schembri L. Chabert P. Zanese M. Tomasello F. Dalibart R. Thoraval D. Crouzet M. Ichas F. De Giorgi F. J. Cell Sci. 2008; 121: 3515-3523Crossref PubMed Scopus (34) Google Scholar). Instead, it focuses on formation of the MAC pore. Early MAC-associated conductance increments are relatively small, suggesting that Bax-dependent formation of the cytochrome c-permeable pore does not occur prior to membrane insertion of Bax. Mathematical modeling of the conductance changes indicates that, if MAC is a circular pore assembled by sequential addition of helices 5 and 6 from Bax and/or Bak monomers, the mature, cytochrome c transport-competent pore is likely a 9–10-mer of these proteins. Cell Lines and Isolation of Mitochondria—Parental (MEF) and derivative (Bax-/-Bak+/+, Bax+/+Bak-/-, Bax-/- Bak-/-, and VDAC1-/-VDAC3-/-) cell lines were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% nonessential amino acids, and 1% l-glutamine (23Baines C.P. Kaiser R.A. Sheiko T. Craigen W.J. Molkentin J.D. Nat. Cell Biol. 2007; 9: 550-555Crossref PubMed Scopus (763) Google Scholar, 24Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3330) Google Scholar). Cells at 80% confluence were harvested with trypsin, and mitochondria were isolated as described previously (25Murphy R.C. Schneider E. Kinnally K.W. FEBS Lett. 2001; 497: 73-76Crossref PubMed Scopus (46) Google Scholar). As indicated, apoptosis was induced with 1 μm staurosporine 16 h before harvesting. FL5.12 cells were grown in Iscove's modified Eagle's medium with 10% fetal bovine serum and 10% WEHI-3B supplement (26Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (966) Google Scholar), and mitochondria were isolated as described previously (13Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-731Crossref PubMed Scopus (237) Google Scholar). Recombinant Proteins—N-terminally His-tagged, full-length mouse BID protein and t-Bid were prepared using the cysteine-less clone, p22BID30S126S, as described previously (27Oh K.J. Barbuto S. Meyer N. Kim R.S. Collier R.J. Korsmeyer S.J. J. Biol. Chem. 2005; 280: 753-767Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 28Oh K.J. Barbuto S. Pitter K. Morash J. Walensky L.D. Korsmeyer S.J. J. Biol. Chem. 2006; 281: 36999-37008Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Monomeric human BAX, truncated for 20 amino acids at the C terminus (rBaxΔC), was expressed in Escherichia coli and purified as described previously (14Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 29Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (559) Google Scholar, 30Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1092) Google Scholar). Immunoblotting—Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and detected by ECL (GE Healthcare). The primary antibodies were against Bax (N-20, Santa Cruz Biotechnology), Bak (anti-N terminus, Millipore Upstate Biotechnology, Inc.), and VDAC1 (31HL, Calbiochem). The secondary antibodies were horse-radish peroxidase-coupled goat anti-rabbit and anti-mouse (Sigma). When stated, pixel densities were quantified by densitometry (Scion Imaging, National Institutes of Health). Cytochrome c Release Assays—Isolated mitochondria (0.2 mg/ml) were incubated with t-Bid (0.02–1 μm), Bid (1.6 μm), or an equivalent amount of vehicle (150 mm KCl, 20 mm HEPES, pH 7, 30% glycerol, 2% octyl glucoside) for 5–30 min at room temperature in 70 mm sucrose, 230 mm mannitol, 1 mm EDTA, pH 7.4, and pelleted at 15,800 × g for 5 min. Alternatively, cytochrome c was measured after permeabilization of cells as described previously (16Cheng E.H. Sheiko T.V. Fisher J.K. Craigen W.J. Korsmeyer S.J. Science. 2003; 301: 513-517Crossref PubMed Scopus (661) Google Scholar). Cytochrome c in the supernatants of cells or isolated mitochondria was quantified by ELISA following the manufacturer's instructions (QuantikineR&D Systems). alamethicin (80 μg/ml) was used as a positive control (31Polster B.M. Kinnally K.W. Fiskum G. J. Biol. Chem. 2001; 276: 37887-37894Abstract Full Text Full Text PDF PubMed Google Scholar). Patch Clamping Techniques—Membrane patches of isolated mitochondria were, if need be, excised after formation of a seal using micropipettes with ∼0.3-μm tips and resistances of 10–30 megohms at room temperature. The micropipettes tips were smaller than those of our earlier studies directly patch clamping mitochondria. Because the cross-sectional area of the membrane patches is smaller, the reported conductances of patch-clamped mitochondria are lower than previously reported before and after induction of apoptosis (13.3 ± 2.5 nS apoptotic versus 6.3 ± 3.6 nS control in 2001 (13Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-731Crossref PubMed Scopus (237) Google Scholar) and 3–5 nS apoptotic verses 0.6 ± 0.4 nS control in this study). Patching media was symmetrical, 150 mm KCl, 5 mm HEPES, 0.23 mm CaCl2, 1 mm EGTA, pH 7.4. Voltages were clamped with an Axopatch 200 amplifier and reported as pipette potentials. Conductance was typically determined from total amplitude histograms of 30 s of current traces at +20 mV. Patches were scored “positive” for MAC when an increase in voltage-independent (±50 mV) conductance of at least 1.5 nS was seen within 30 min of seal development; this typically occurred when t-Bid was included in the micropipette tip. This channel activity is referred to as MAC in mitochondria isolated from Parental cells, MAC-Bak from Bax KO cells, and MAC-Bax from Bak KO cells. The permeability of untreated mitochondria is also reflected in the seal resistances that were not found to be significantly different for the various cell lines (n = 10–23 independent patches for each cell line). Levels of t-Bid used in the patch pipettes for these experiments were 20 nm for Parental and Bax KO cells, 250 nm for Bak KO cells, and 1 μm for DKO cells unless otherwise indicated. pClamp version 8 (Axon Instruments) and WinEDR version 2.3.3 (Strathclyde Electrophysiological Software; courtesy of J. Dempster, University of Strathclyde, UK) were used for current analysis. Sample rate was 5 kHz with 2-kHz filtration. When necessary, simple perfusion of the chamber often resulted in excision of patches. Permeability ratios were calculated from the reversal potential in the presence of a 150:30 mm KCl gradient as described previously (32Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (92) Google Scholar). Dibucaine (Sigma D0638) and cytochrome c (Sigma C-7752) were introduced and removed by perfusion of the bath (0.5 ml volume) with 3–5 ml of patching solution. The channel activity of monomeric human Bax, truncated for 20 amino acids at the C terminus (BaxΔC20), activated by t-Bid (14Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 29Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (559) Google Scholar, 30Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1092) Google Scholar) was characterized in liposomes devoid of other proteins. Micropipette tips were filled with media containing 380 ng/μl monomeric BaxΔC plus 35 ng/μl t-Bid and then backfilled with patching media. Hence, the actual Bax concentration was lower than that loaded in the tips. Seals were formed with these micropipettes on giant liposomes prepared as described previously (32Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (92) Google Scholar, 33Criado M. Keller B.U. FEBS Lett. 1987; 224: 172-176Crossref PubMed Scopus (127) Google Scholar). Mathematical Analysis—The number of helices forming MAC was estimated assuming α-helices had a diameter (D) of 1.2 nm (34Hirokawa T. Uechi J. Sasamoto H. Suwa M. Mitaku S. Protein Eng. 2000; 13: 771-778Crossref PubMed Scopus (14) Google Scholar) and aligned on center perpendicular to the membrane. The resulting polygon had a variable number (n) of vertices, where the center of each helix was located at a vertex of the polygon. The area (A) was approximated by a circle whose area was calculated by Equation 1 as more helical staves were added to the polygon in an approach similar to Sanson et al. (35Sansom M.S. Kerr I.D. Mellor I.R. Eur. Biophys. J. 1991; 20: 229-240Crossref PubMed Scopus (36) Google Scholar), A=πD2(12−2cos(2πn)−1/2)2(Eq. 1) The area was then corrected by adding n times the triangular area (2Ac) between staves in the pore. Ac is defined by subtracting the area of the two arcs Ar (Equation 2) and AR (Equation 3) from the area of the right triangle AΔ (Equation 4) that is defined by the base r and height R + r in Equation 5 (see supplemental Fig. 1S). Ar=((n−2)2nπ)2ππr2=πr2(n−2)/4n(Eq. 2) AR=12nπR2(Eq. 3) AΔ=12base ⋅ height = 12r(r+R)2−r2=12r(r+R)cos(π/n)(Eq. 4) Ac=AΔ−(Ar+AR)=1/2r(r+R)2−r2−(12nπR2+(n−24n)πr2)(Eq. 5) where we define R in Equation 6 as, R=((sin(π/n))−1−1)r(Eq. 6) To determine the relationship between the number (n) of α-helices and the area, these corrected data were best fit (correlation coefficient of 0.9991) in Fig. 7A by the polynomial Equation 7, area=7×10−12n2+9×10−11n−4×10−10(Eq. 7) The cross-sectional pore area was converted into predicted conductance that was corrected for access resistance using the method of Hille (36Hille B. Ionic Channels of Excitable Membranes, 2nd Ed. Sinauer Associates, Inc., Sunderland, MA2001: 291-314Google Scholar) through Equation 8. ρpore=(L+(πa)/2)(ρsol/πa2)(Eq. 8) where ρpore and ρsol are the resistivity of the pore and a solution of 0.15 m KCl; L is pore length (5.5 nm corresponds to the thickness of the outer membrane (37Mannella C.A. Biochim. Biophys. Acta. 1981; 645: 33-40Crossref PubMed Scopus (25) Google Scholar)), and a is the radius of the pore. This equation established the relationship between the conductance and number of α-helices forming the MAC pore (Fig. 7B). MAC formation occurred in incremental steps of conductance (transition size) often of ∼300 pS. These same equations were used to estimate the number of α-helices inserted into the barrel of the MAC pore to generate the observed transitions. For example, an initial transition of ∼300 pS corresponds to insertion of 6–8 helices. However, the relationship between the transition size and the number of α-helices inserted into the barrel is not linear, i.e. transitions of ∼300 pS into moderately larger 1–3-nS channels likely correspond to the insertion of 1 α-helix to the pore (Fig. 7D). The predicted transition sizes generated by insertions of 1, 2, 4, or 6 α-helices into pores of varying conductance (Fig. 7D) were calculated using the method of Hille (36Hille B. Ionic Channels of Excitable Membranes, 2nd Ed. Sinauer Associates, Inc., Sunderland, MA2001: 291-314Google Scholar) and corrected for access resistance (Equation 8), where the radius (a) was calculated from the area in Equation 7. Analyses were done using Excel and Prism 4 from GraphPad Software. The presence of either Bax or Bak is sufficient to allow initiation of the mitochondria-dependent death program, although the absence of both proteins is necessary to inhibit cytochrome c release (38Kandasamy K. Srinivasula S.M. Alnemri E.S. Thompson C.B. Korsmeyer S.J. Bryant J.L. Srivastava R.K. Cancer Res. 2003; 63: 1712-1721PubMed Google Scholar, 39Lindsten T. Ross A.J. King A. Zong W.X. Rathmell J.C. Shiels H.A. Ulrich E. Waymire K.G. Mahar P. Frauwirth K. Chen Y. Wei M. Eng V.M. Adelman D.M. Simon M.C. Ma A. Golden J.A. Evan G. Korsmeyer S.J. MacGregor G.R. Thompson C.B. Mol. Cell. 2000; 6: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (1180) Google Scholar). MEF lines deficient in either or both Bax and Bak were used to define the roles of these Bcl-2 family proteins in MAC formation and cytochrome c release. These cell lines include the Parental (Bax+/+Bak+/+), the single knockouts Bax KO (Bax-/-Bak+/+) and Bak KO (Bax+/+Bak-/-), as well as the double knock-out DKO (Bax-/-Bak-/-). The presence of Bax and Bak in cell extracts of each MEF line was assessed in the Western blots of Fig. 1A. The mitochondrial amounts of Bax and Bak for these cell lines treated without (control) or with (apoptotic) 1 μm staurosporine are shown in the histograms of Fig. 1B relative to VDAC, which is constitutively expressed in the outer membrane. Although the relative levels of Bak remained stable, Bax levels in mitochondria increased almost 2–4-fold after induction of apoptosis. MAC Forms in the Mitochondrial Outer Membrane of Apoptotic MEF Cells Expressing Bax and/or Bak—We previously found MAC could form in Bax KO but not DKO mitochondria (8Dejean L.M. Martinez-Caballero S. Guo L. Hughes C. Teijido O. Ducret T. Ichas F. Korsmeyer S.J. Antonsson B. Jonas E.A. Kinnally K.W. Mol. Biol. Cell. 2005; 16: 2424-2432Crossref PubMed Scopus (194) Google Scholar); these studies were expanded to include Bak KO cells. Activity of the mitochondrial outer membrane channel, MAC, is detected as increased conductance when mitochondria of apoptotic cells are patch-clamped. This increase in membrane permeability ranged from 3 to 5 nS and was statistically significant (p = 0.0001; n = 20–23 patches) for mitochondria from Parental, Bak KO, and Bax KO lines after induction of apoptosis by staurosporine (Fig. 1C). MAC was scored “present” if the increase in conductance of the outer membrane was at least 1.5 nS. These data indicate MAC was formed by 16 h after induction of apoptosis in these cells. In contrast, the conductances of mitochondrial patches from untreated cells and apoptotic DKO cells were essentially identical (p = 0.8; n = 20–23 patches), indicating that MAC formation requires expression of either Bax or Bak (Fig. 1C). MAC is the outer membrane channel associated with cytochrome c release from mitochondria to the cytosol during intrinsic apoptosis. The fraction of cytochrome c released to the cytosol was determined for the four MEF lines following treatment with staurosporine. As shown in Fig. 1D, cytochrome c was released to the cytosol in Parental, Bax KO, and Bak KO cells, but not DKO cells at the time MAC was detected during staurosporine treatment. These results show that expression of either Bax or Bak is necessary for cytochrome c release and MAC formation. Furthermore, these findings indicate that Bax and Bak are functionally redundant with respect to both processes. The MAC activity of MEF mitochondria showed electrophysiological properties similar to those reported previously for mitochondria from other apoptotic cells. As previously found, the peak conductances were large and variable (in the range 1.5–5 nS), showed no obvious dependence on voltage (±50 mV), and were slightly cation-selective (8Dejean L.M. Martinez-Caballero S. Guo L. Hughes C. Teijido O. Ducret T. Ichas F. Korsmeyer S.J. Antonsson B. Jonas E.A. Kinnally K.W. Mol. Biol. Cell. 2005; 16: 2424-2432Crossref PubMed Scopus (194) Google Scholar, 10Guo L. Pietkiewicz D. Pavlov E.V. Grigoriev S.M. Kasianowicz J.J. Dejean L.M. Korsmeyer S.J. Antonsson B. Kinnally K.W. Am. J. Physiol. 2004; 286: C1109-C1117Crossref PubMed Scopus (52) Google Scholar, 13Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-731Crossref PubMed Scopus (237) Google Scholar). To further explore the possibility that MAC activity of mitochondria containing only Bax is different from that of mitochondria containing only Bak, a system triggered by t-Bid was established in which MAC formation could be monitored in real time while cytochrome c is released. Cytochrome c Release Induced by t-Bid Is Dose-dependent and Requires Bax or Bak Expression—The BH3-only protein Bid is cleaved to form activated t-Bid during apoptosis and triggers the release of cytochrome c from mitochondria of cells that express Bax and/or Bak (29Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (559) Google Scholar, 40Jonas E.A. Hickman J.A. Chachar M. Polster B.M. Brandt T.A. Fannjiang Y. Ivanovska I. Basanez G. Kinnally K.W. Zimmerberg J. Hardwick J.M. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13590-13595Crossref PubMed Scopus (87) Google Scholar, 41Kim H. Rafiuddin-Shah M. Tu H.C. Jeffers J.R. Zambetti G.P. Hsieh J.J. Cheng E.H. Nat. Cell Biol. 2006; 8: 1348-1358Crossref PubMed Scopus (698) Google Scholar). Mitochondria from the various MEF cell lines were incubated with different concentrations of t-Bid for up to 30 min, and the amounts of cytochrome c released were measured (Fig. 2). Although uncleaved Bid or vehicle alone had no effect, t-Bid induced a dose-dependent release of cytochrome c with EC50 (effective concentration for release of 50% of cytochrome c) of 7 nm for Parental, 17 nm for Bax KO, and ∼250 nm for Bak KO mitochondria (Table 1 and Fig. 2A). As expected, t-Bid failed to induce cytochrome c release in mitochondria from DKO cells. The kinetics show the release of cytochrome c occurs over minutes (Fig. 2B). The release of cytochrome c from mitochondria lacking either Bax or Bak was delayed compared with that of Parental mitochondria. Increasing the dose of t-Bid from 20 nm to 1 μm increased the extent of release after 30 min most dramatically in mitochondria lacking Bak.TABLE 1Electrophysiological properties of MAC activity induced by t-BidParental (MAC)Bax KO (MAC-Bak)Bak KO (MAC-Bax)VDAC1VDAC3 KO (MAC)FL5.12 (MAC)aData were included for comparison with MAC recorded from mitochondria and proteoliposomes (8, 10, 13)[t-Bid] EC50 (nm) cytochrome c release71724720NAbNA means not applicable[t-Bid] micropipette (nm)20202501000NAMAC assembly frequency (% patches)64 (n = 11)cn = number of independent determinations68 (n = 12)67 (n = 12)75 (n = 12)NAPeak conductance (nS)3.5 ± 1.8 (n = 7)3.1 ± 2.0 (n = 8)3.0 ± 1.9 (n = 8)3.5 ± 1.5 (n = 9)2.5–4.5 (n = 57)Ion selectivity PK/PCl3.1 ± 1.4 (n = 5)3.7 (n = 1)3.4 ± 1.2 (n = 4)3.4 ± 1.5 (n = 7)3.0 ± 0.9Voltage-dependent (±50 mV)NoNoNoNoNoNo. of MAC assembled in under 10 min5 of 76 of 85 of 89 of 9NAPredicted diameter (nm)dSee under “Experimental Procedures”; data are based on peak conductance assuming 1.2-nm helices are inserted normal to the membrane to form circular pores65.65.564–7Predicted no. of helices forming poredSee under “Experimental Procedures”; data are based on peak conductance assuming 1.2-nm helices are inserted normal to the membrane to form circular pores1817171816–21a Data were included for comparison with MAC recorded from mitochondria and proteoliposomes (8Dejean L.M. Martinez-Caballero S. Guo L. Hughes C. Teijido O. Ducret T. Ichas F. Korsmeyer S.J. Antonsson B. Jonas E.A. Kinnally K.W. Mol. Biol. Cell. 2005; 16: 2424-2432Crossref PubMed Scopus (194) Google Scholar, 10Guo L. Pietkiewicz D. Pavlov E.V. Grigoriev S.M. Kasianowicz J.J. Dejean L.M. Korsmeyer S.J. Antonsson B. Kinnally K.W. Am. J. Physiol. 2004; 286: C1109-C1117Crossref PubMed Scopus (52) Google Scholar, 13Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-731Crossref PubMed Scopus (237) Google Scholar)b NA means not applicablec n = number of independent determinationsd See under “Experimental Procedures”; data are based on peak conductance assuming 1.2-nm helices are inserted normal to the membrane to form circular pores Open table in a new tab t-Bid Triggers MAC Formation in Mitochondria Containing Bax and/or Bak—The conditions established for cytochrome c release were applied to patch clamp experiments to monitor MAC formation. t-Bid was included in the micropipette so that this protein could interact with the cytosolic face of the outer membrane patch, and membrane permeability was continuously monitored as current flow. Because of differences in EC50 for cytochrome c release, the t-Bid concentration used to backfill the micropipette tip was 20 nm for mitochondria from Parental and Bax KO cells, 250 nm for Bak KO mitochondria, and 1 μm for DKO mitochondria. MAC formation was detected as increases in patch conductance with time, i.e. increases in current flow, after development of a seal between the membrane patch and the micropipette. Using this approach, MAC typically formed within 10 min of sealing the micropipette on intac
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