Detection of the Mitochondrial Apoptosis‐Induced Channel (MAC) and Its Regulation by Bcl‐2 Family Proteins

Kathleen W. Kinnally1, Sonia Martinez‐Caballero1, Laurent M. Dejean1

1 New York University, College of Dentistry, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.12
DOI:  10.1002/0471140856.tx0212s30
Online Posting Date:  December, 2006
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Abstract

Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis‐induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl‐2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl‐2 family proteins and pharmacological agents. Protocols include the use of time‐lapse video‐microscopy to monitor the onset of apoptosis in living cells and patch‐clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.

Keywords: MAC; mitochondrial apoptosis‐induced channel; apoptosis; patch‐clamp; Bcl‐2; Bax.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Apoptotic Events Using Time‐Lapse Microscopy
  • Support Protocol 1: Cleaning Coverslips for Time‐Lapse Videomicroscopy Experiments
  • Support Protocol 2: Cleaning Rose Chambers
  • Preparation of Isolated Mitochondria and Proteoliposomes Containing Mitochondrial Outer Membranes
  • Basic Protocol 2: Isolation of Large Amounts of Mitochondria from Cultured Cells
  • Alternate Protocol 1: Quick Isolation of Small Amounts of Mitochondria from Cultured Cells
  • Basic Protocol 3: Purification of the Inner and Outer Membranes from Isolated Mitochondria
  • Support Protocol 3: Immunoblot Analysis of Mitochondrial Proteins
  • Basic Protocol 4: Preparation of Proteoliposomes Containing Mitochondrial Outer Membranes
  • Support Protocol 4: Preparation of Small Liposomes of Azolectin
  • Detecting MAC in Isolated Mitochondria and Proteoliposomes
  • Basic Protocol 5: Patch Clamping Isolated Mitochondria
  • Alternate Protocol 2: Patch Clamping Proteoliposomes
  • Alternate Protocol 3: Detection of Cytochrome c Release from Proteoliposomes Containing Control and Apoptotic Outer Membranes
  • Basic Protocol 6: Modulation of Bax Content by Immunoprecipitation
  • Basic Protocol 7: Preparation of Proteoliposomes Containing Solubilized Mitochondrial Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Apoptotic Events Using Time‐Lapse Microscopy

  Materials
  • 90% (v/v) ethanol solution
  • Cells for inoculating (e.g., HeLa, MEFs; see appendix 3B)
  • 10 ml appropriate cell culture medium (e.g., Leibowitz's medium; US Biological) containing 0.011 g/liter phenol red ( appendix 3B; optional) and apoptotic inducer (e.g., 0.5 to 1 µM staurosporine; optional)
  • 1 M HEPES (pH 7.4) solution (see recipe), sterile (optional)
  • Forceps, sterile
  • 25‐mm2 square or round glass coverslip, clean ( protocol 2)
  • 5‐ml petri dish
  • Rose chamber (∼50 × 37–mm with a 23‐mm circular cutout in the center): composed of a 2 to 3 mm thick square sylastic spacer, two aluminum holders, and four screws (see Khodjakov and Rieder, )
  • Screwdriver
  • 28‐G 1‐in. needles (two)
  • 5‐ml syringe
  • Heat block (e.g., WPI; or see Rieder and Cole, )
  • Microscope equipped with epifluorescence plus phase contrast (PC) or differential interface contrast (DIC) optics (e.g., Nikon Eclipse TE300 phase contrast/differential interference contrast microscope)
  • Two shutters (e.g., Uniblitz) and controlling software (e.g., ImageJ, freely available at http://rsb.info.nih.gov/ij)
  • Charge‐coupled device (CCD) camera, e.g., Spot RT Monochrome CCD camera (Diagnostics Instruments)
  • Video conversion software (e.g., AVS video converter version 3 from NCTsoft, http://www.nctsoft.com)
  • Additional reagents and equipment for growing mammalian cells ( appendix 3B)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Cleaning Coverslips for Time‐Lapse Videomicroscopy Experiments

  Materials
  • 1% (v/v) regular detergent solution: 10 ml of regular detergent (e.g., Liqui Nox; Alconox) and 990 ml distilled water; store up to 1 year at room temperature
  • 90% (v/v) ethanol
  • 3.8% (v/v) HCl solution: add 100 ml of concentrated HCl (38% v/v) to 900 ml of distilled water (in that order); store up to 1 year at room temperature
  • 0.1 M EDTA, pH 8.0: mix 200 ml of 0.5 M EDTA, pH 8.0, ( appendix 2A) with 800 ml distilled water; store up to 6 months at 4°C
  • Disposable gloves
  • 600‐ml beakers
  • 100 to 200 square (Corning 25 mm, no. 1.5) or circular (Fisher 25 mm, no.1 circ.) glass coverslips
  • Bath sonicator (e.g., Branson Model 1210)
  • Screw capped jar with a wide mouth
NOTE: For best results always transfer the coverslips into each new solution individually, wearing disposable gloves.

Support Protocol 2: Cleaning Rose Chambers

  Materials
  • 1% (v/v) regular detergent solution: 10 ml of regular detergent (e.g., Liqui Nox; Alconox) and 990 ml distilled water; store up to 1 year at room temperature
  • 0.1 M EDTA, pH 8.0: 200 ml of 0.5 M EDTA, pH 8.0, ( appendix 2A) and 800 ml distilled water; store up to 6 months at 4°C
  • 90% (v/v) ethanol solution
  • Rose chamber (see protocol 1)
  • 600‐ml beakers
  • Bath sonicator (e.g., Branson Model 1210)
  • Microwave
  • Whatman no. 1 filter paper
  • Pyrex jar

Basic Protocol 2: Isolation of Large Amounts of Mitochondria from Cultured Cells

  Materials
  • Tissue culture cells (e.g., FL5.12, MEFs, HeLa; appendix 3B)
  • Appropriate complete tissue culture medium ( appendix 3B)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1× mitochondrial isolation (MI) buffer (see recipe)
  • Protease inhibitor cocktail for mammalian tissue (Sigma)
  • 0.1 M PMSF stock solution: dissolve 1.74 g phenylmethylsulfonylfluoride (PMSF; Sigma) in 10 ml propanol; store up to 3 months at room temperature
  • 50 mg/ml digitonin stock solution: dissolve 50 mg digitonin (e.g., Calbiochem) in 1 ml of dimethylsulfoxide (DMSO; Sigma); store up to 6 months at −20°C
  • Ethanol‐precipitated bovine serum albumin (BSA; e.g., Sigma)
  • 15‐, 50‐, and 500‐ml centrifuge tubes or bottles with caps
  • Refrigerated centrifuge with fixed‐angle rotor
  • Rugged rotator (e.g., Fisher Scientific)
  • 25‐ml glass homogenizer and tight motor‐driven Teflon pestle (e.g., Thomas)
  • −80°C freezer
  • Additional reagents and equipment for determining protein concentration ( appendix 3G)
NOTE: Perform all operations on ice and use ice‐cold buffers and instruments.

Alternate Protocol 1: Quick Isolation of Small Amounts of Mitochondria from Cultured Cells

  • Cells growing in an appropriate complete culture medium (see protocol 4)
  • 2× mitochondrial isolation (MI) buffer (see recipe)
  • Swinging‐bucket rotor
  • Manual glass homogenizer with tight Teflon pestle
NOTE: Perform all operations on ice and use ice‐cold buffers and instruments.

Basic Protocol 3: Purification of the Inner and Outer Membranes from Isolated Mitochondria

  Materials
  • Isolated mitochondria ( protocol 4)
  • 1× and 2× mitochondrial isolation (MI) buffer (see reciperecipes)
  • Lysis buffer (see recipe)
  • 35% (w/v) sucrose solution (see recipe)
  • French press
  • Refrigerated centrifuge and 15‐ml centrifuge tubes
  • 10‐, 20‐, or 50‐ml beaker
  • Small magnetic stirrer
  • Glass homogenizers (e.g., Thomas) with Teflon pestles
  • 10‐cm stainless steel 12‐G cannula on a 10‐ml syringe
  • Refrigerated ultracentrifuge with hanging‐bucket rotor and 5‐ml collapsible ultracentrifuge tubes
  • Additional reagents and equipment for determining protein concentration ( appendix 3G)
NOTE: All operations should be done on ice; using ice‐cold buffers and instruments.

Support Protocol 3: Immunoblot Analysis of Mitochondrial Proteins

  Materials
  • Purified sample of inner and outer membranes or solubilized mitochondrial proteins ( protocol 6 or protocol 13)
  • 2× Laemmli loading buffer (e.g., Bio‐Rad or see SDS sample buffer, 2× in appendix 3F)
  • 13% or 15% (w/v) SDS‐polyacrylamide gel ( appendix 3F)
  • Methanol, HPLC grade (e.g., Fischer)
  • Electrotransfer buffer (see recipe)
  • PBS/Tween: PBS ( appendix 2A) containing 0.2% (w/v) Tween 20
  • PBS/Tween/5% milk: PBS‐Tween supplemented with 5% (w/v) nonfat milk
  • Primary antibody against VDAC (e.g., Calbiochem), COXIV (e.g., Molecular Probes), cytochrome c (e.g., Pharmingen), or Bax (e.g., Santa Cruz)
  • Horseradish peroxidase–conjugated secondary antibody (e.g., Jackson Immunoresearch)
  • ECL Western Blotting Detection Reagents kit (Amersham Biosciences)
  • Semi‐dry transfer cell (e.g., Trans‐Blot SD, Bio‐Rad) with power source
  • PVDF membranes and thick filter papers (e.g., Bio‐Rad)
  • Additional reagents and equipment for performing SDS‐PAGE ( appendix 3F)

Basic Protocol 4: Preparation of Proteoliposomes Containing Mitochondrial Outer Membranes

  Materials
  • Purified mitochondrial outer membrane ( protocol 6)
  • Small azolectin liposomes ( protocol 9)
  • 5 mM HEPES solution (see recipe)
  • Anhydrous calcium sulfate (e.g., Drierite)
  • 0.15 M KCl solution (see recipe)
  • 35 × 60–mm cover glasses (e.g., Gold Seal)
  • 10‐cm petri dishes (e.g., Fisher)

Support Protocol 4: Preparation of Small Liposomes of Azolectin

  Materials
  • Azolectin (L‐α‐phosphatidylcholine from soybean Type IVs; Sigma): store at −20°C, in the dark and under vacuum to avoid oxidation of the lipids
  • Chloroform (e.g., Fischer)
  • 0.15 M KCl solution (see recipe)
  • 30‐ml Corex tubes, washed sequentially with sulfuric acid and distilled water, and dried before use
  • Nitrogen gas tank
  • Microtip sonicator (e.g., Sonicator 60 Sonic Dismembrator with microtip; Fischer)
  • Refrigerated ultracentrifuge and fixed‐angle rotor
  • 10‐ml ultracentrifuge tubes

Basic Protocol 5: Patch Clamping Isolated Mitochondria

  Materials
  • Isolated mitochondria ( protocol 5)
  • Patching media (see recipe)
  • Gradient media (see recipe)
  • 20 mM dibucaine stock solution: dissolve 760 mg dibucaine (e.g., Sigma) in 1 ml distilled water; protect from light and store up to 2 to 3 months at 4°C
  • Horse cytochrome c (e.g., Sigma)
  • Agar bridges (see recipe)
  • 1.0‐mm Borosilicate glass capillaries with filaments (World Precision Instruments)
  • Microelectrode puller (Sutter Instruments Model P‐87) with a 2‐mm box filament and pressure set in position 500; a two‐line program can make suitable micropipets (see Table 2.12.2)
  • Ag/AgCl 2 electrode (Warner Instruments)
  • Micromanipulator
  • 35 × 60–mm glass coverslips and Lucite holder
  • Phase‐contrast microscope with 40× lens and 15× oculars mounted on an antivibration table.
  • Electrophysiology setup including a patch‐clamp amplifier, low pass filter, computer and software, e.g., purchase Axon pClamp (Axon Instruments) or download Strathclyde Electrophysiological package at http://spider.science.strath.ac.uk/physpharm/index.php?pageName=software
    Table 2.2.2   Materials   Two‐Step Program to Pull Microelectrodes Suitable for Patch Clamping Isolated Mitochondria or Proteoliposomes Using a Sutter Micropipet Puller Model P‐87 f   Two‐Step Program to Pull Microelectrodes Suitable for Patch Clamping Isolated Mitochondria or Proteoliposomes Using a Sutter Micropipet Puller Model P‐87

    Heat Pull Velocity Time
    Ramp +20° 20 150
    Ramp +15° 70 40 200

     fSuggested modifications to optimize the program for small tips and low resistance can be obtained by running glass.exe, a program available for free from the Sutter website at http://www.sutter.com/news/software_downloads.html.

Alternate Protocol 2: Patch Clamping Proteoliposomes

  • Proteoliposomes containing mitochondrial outer membranes (see protocol 13)

Alternate Protocol 3: Detection of Cytochrome c Release from Proteoliposomes Containing Control and Apoptotic Outer Membranes

  Materials
  • Purified mitochondrial outer membrane ( protocol 6)
  • Small liposomes of azolectin ( protocol 9)
  • 3 mg/ml horse cytochrome c stock solution : dissolve 3 mg of horse cytochrome c (e.g., Sigma) in 1 ml of PBS ( appendix 2A); store at 4°C for up to 2 to 3 weeks
  • 5 mM HEPES solution (see recipe)
  • Anhydrous calcium sulfate (e.g., Drierite)
  • 0.15 M KCl solution (see recipe)
  • 0.5 M NaCl solution (see recipe)
  • 10 mM Tris solution (see recipe)
  • 35 × 60–mm coverslips (e.g., Gold Seal)
  • 10‐cm petri dishes (e.g., Fischer)
  • 1.5‐ml polyallomer microcentrifuge tubes
  • Light microscope with 20× or 40× magnification
  • Refrigerated ultracentrifuge with fixed‐angle rotor and 1.5‐ml ultracentrifuge tubes

Basic Protocol 6: Modulation of Bax Content by Immunoprecipitation

  Materials
  • Isolated mitochondria ( protocol 4)
  • 1× mitochondrial isolation (MI) buffer (see recipe)
  • 20% (w/v) CHAPS solution: dissolve 200 mg of 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) in 1 ml of 1× MI buffer (see recipe); store up to 6 months at −20°C
  • Immunoprecipitation (IP) buffer (see recipe) supplemented with 2% (w/v) CHAPS
  • Primary antibody against Bax N‐terminus (e.g., Santa Cruz)
  • 1 mg/ml IgG from rabbit or mouse serum (e.g., Sigma) solution: dilute 5 mg of IgG solution in 5 ml of PBS; divide into 100‐µl aliquots and store up to 3 months at −20°C
  • Protein G‐agarose (Calbiochem)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2× Laemmli loading buffer (e.g., Bio‐Rad)
  • Microtip sonicator (e.g., Sonicator 60 Sonic Dismembrator with microtip; Fischer)
  • Refrigerated microcentrifuge
  • Rugged rotator (e.g., Fisher Scientific)
  • Refrigerated ultracentrifuge, fixed‐angle rotor and 1.5‐ml ultracentrifuge tubes
  • Additional equipment and reagents for determining protein concentrations ( appendix 3G)

Basic Protocol 7: Preparation of Proteoliposomes Containing Solubilized Mitochondrial Proteins

  Materials
  • Total mitochondrial lysates or supernatants from immunoprecipitation assays ( protocol 13)
  • IP buffer (see recipe)/2% (w/v) CHAPS
  • Small azolectin liposomes ( protocol 9)
  • IP buffer (see recipe)
  • IP buffer (see recipe)/2% detergent‐removing gel (Pierce)
  • 5 mM HEPES solution (see recipe)
  • 0.15 M KCl solution (see recipe)
  • Anhydrous calcium sulfate (e.g., Drierite)
  • Light microscope with 40× magnification
  • 50 µl‐dialysis chamber and cellulose acetate membranes with a 10‐kDa MWCO (Harvard Apparatus)
  • 500‐ml beaker
  • Magnetic stirrer
  • 35 × 60–mm cover glasses (e.g., Gold Seal)
  • 10‐cm petri dishes (e.g., Fischer)
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Figures

Videos

Literature Cited

Literature Cited
   Antonsson, B. 2004. Mitochondria and the Bcl‐2 family proteins in apoptosis signaling pathways. Mol. Cell Biochem. 256‐257:141‐155.
   Axon Instruments. 1993. The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques. Axon Instruments, Inc., Foster City, CA.
   Danial, N.N. and Korsmeyer, S.J. 2004. Cell death: Critical control points. Cell 116:205‐219.
   De Giorgi, F., Lartigue, L., Bauer, M.K., Schubert, A., Grimm, S., Hanson, G.T., Remington, S.J., Youle, R.J., and Ichas, F. 2002. The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J. 16:607‐609.
   Dejean, L.M., Martinez‐Caballero, S., Guo, L., Hughes, C., Teijido, O., Ducret, T., Ichas, F., Korsmeyer, S.J., Antonsson, B., Jonas, E.A., and Kinnally, K.W. 2005. Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis‐induced channel. Mol. Biol. Cell 16:2424‐2432.
   Dejean, L.M., Martinez‐Caballero, S., and Kinnally, K.W. 2006a. Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis? Cell Death Differ. 13:1387‐1395
   Dejean, L.M., Martinez‐Caballero, S., Manon, S., and Kinnally, K.W. 2006b. Regulation of the mitochondrial apoptosis‐induced channel, MAC, by BCL‐2 family proteins. Biochim. Biophys. Acta 1762:191‐201.
   Desagher, S., Osen‐Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J.C. 1999. Bid‐induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144:891‐901.
   Fadeel, B. and Orrenius, S. 2005. Apoptosis: A basic biological phenomenon with wide‐ranging implications in human disease. J. Intern. Med. 258:479‐517.
   Fisher, R.A. 1935. The logic of inductive inference. J. Roy. Statist. Soc. 98:35‐54.
   Green, D.R. and Kroemer, G. 2004. The pathophysiology of mitochondrial cell death. Science 305:626‐629.
   Grigoriev, S.M., Muro, C., Dejean, L.M., Campo, M.L., Martinez‐Caballero, S., and Kinnally, K.W. 2004. Electrophysiological approaches to the study of protein translocation in mitochondria. Int. Rev. Cytol. 238:227‐274.
   Guihard, G., Bellot, G., Moreau, C., Pradal, G., Ferry, N., Thomy, R., Fichet, P., Meflah, K., and Vallette, F.M. 2004. The mitochondrial apoptosis‐induced channel (MAC) corresponds to a late apoptotic event. J. Biol. Chem. 279:46542‐46550.
   Guo, L., Pietkiewicz, D., Pavlov, E.V., Grigoriev, S.M., Kasianowicz, J.J., Dejean, L.M., Korsmeyer, S.J., Antonsson, B., and Kinnally, K.W. 2004. Effects of cytochrome c on the mitochondrial apoptosis‐induced channel MAC. Am. J. Physiol. Cell Physiol. 286:C1109‐C1117.
   Hille, B. 2001. Ionic Channels of Excitable Membranes. Sinauer Assoc., Sunderland, Mass.
   Juin, P., Cartron, P.F., and Vallette, F.M. 2005. Activation of Bax by BH3 domains during apoptosis: The unfolding of a deadly plot. Cell Cycle 4:637‐642.
   Khodjakov, A. and Rieder, C.L. 2006. Imaging the division process in living tissue culture cells. Methods 38:2‐16.
   Khodjakov, A., Rieder, C., Mannella, C.A., and Kinnally, K.W. 2004. Laser micro‐irradiation of mitochondria: Is there an amplified mitochondrial death signal in neural cells? Mitochondrion 3:217‐227.
   Kinnally, K.W., Antonenko, Y.N., and Zorov, D.B. 1992. Modulation of inner mitochondrial membrane channel activity. J. Bioenerg. Biomembr. 24:99‐110.
   Kuwana, T. and Newmeyer, D.D. 2003. Bcl‐2‐family proteins and the role of mitochondria in apoptosis. Curr. Opin. Cell Biol. 15:691‐699.
   Liu, X., Kim, C.N., Yang, J., Jemmerson, R., and Wang, X. 1996. Induction of apoptotic program in cell‐free extracts: Requirement for dATP and cytochrome c. Cell 86:147‐157.
   Lohret, T.A., Jensen, R.E., and Kinnally, K.W. 1997. Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC. J. Cell Biol. 137:377‐386.
   Lucken‐Ardjomande, S. and Martinou, J.C. 2005. Regulation of Bcl‐2 proteins and of the permeability of the outer mitochondrial membrane. C.R. Biologies 328:616‐631.
   Martinez‐Caballero, S., Dejean, L.M., and Kinnally, K.W. 2004. Some amphiphilic cations block the mitochondrial apoptosis‐induced channel, MAC. FEBS Lett. 568:35‐38.
   Martinez‐Caballero, S., Dejean, L.M., Jonas, E.A., and Kinnally, K.W. 2005. The role of the mitochondrial apoptosis induced channel MAC in cytochrome c release. J. Bioenerg. Biomembr. 37:155‐164.
   Nechushtan, A., Smith, C.L., Hsu, Y.T., and Youle, R.J. 1999. Conformation of the Bax C‐terminus regulates subcellular location and cell death. EMBO J. 18:2330‐2341.
   Pavlov, E.V., Priault, M., Pietkiewicz, D., Cheng, E.H., Antonsson, B., Manon, S., Korsmeyer, S.J., Mannella, C.A., and Kinnally, K.W. 2001. A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J. Cell Biol. 155:725‐731.
   Peixoto, P.M.V., Martinez‐Caballero, S., Grigoriev, S.M., Kinnally, K.W., and Campo, M.L. 2004. The ins and outs of mitochondrial protein import from an electrophysiological point of view. Recent Res. Devel. Biophys. 3:413‐474.
   Rieder, C.L. and Cole, R.W. 1998. Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase. J. Cell Biol. 142:1013‐1022.
   Sharpe, J.C., Arnoult, D., and Youle, R.J. 2004. Control of mitochondrial permeability by Bcl‐2 family members. Biochim. Biophys. Acta 1644:107‐113.
   Sorgato, M.C., Keller, B.U., and Stuhmer, W. 1987. Patch‐clamping of the inner mitochondrial membrane reveals a voltage‐dependent ion channel. Nature 330:498‐500.
   Szabo, I. and Zoratti, M. 1992. The mitochondrial megachannel is the permeability transition pore. J. Bioenerg. Biomembr. 24:111‐117.
   Wei, M.C., Zong, W.X., Cheng, E.H., Lindsten, T., Panoutsakopoulou, V., Ross, A.J., Roth, K.A., MacGregor, G.R., Thompson, C.B., and Korsmeyer, S.J. 2001. Proapoptotic BAX and BAK: A requisite gateway to mitochondrial dysfunction and death. Science 292:727‐730.
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