Report |
Address correspondence to Kathleen W. Kinnally, Division of Basic Sciences, New York University College of Dentistry, 345 East 24th St., New York, NY 10010. Tel.: (212) 998-9445. Fax: (212) 995-4087. E-mail: kathleen.kinnally{at}nyu.edu
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Abstract |
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Key Words: apoptosis; mitochondria; channels; Bax; patch-clamp
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Introduction |
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The mechanism by which proapoptotic factors are released from mitochondria early in apoptosis is unknown. It has been speculated that a permeability transition of the inner membrane causes swelling of the matrix space. As the inner membrane has a much greater surface area than the outer membrane, the ensuing swelling would rupture the outer membrane and spill cytochrome c into the cytoplasm. However, cytochrome c release has been shown to occur in the absence of a loss of outer membrane integrity in some cell types, suggesting instead a more selective mechanism, e.g., formation of a pore in the outer membrane (Antonsson et al., 1997; Brenner and Kroemer, 2000; Saito et al., 2000; Martinou and Green, 2001; Shimizu et al., 2001). Importantly, the existence of such a channel has not yet been demonstrated by independent criteria, e.g., electrophysiological characteristics.
There is considerable evidence that members of the Bcl-2 protein family exert their pro- and antiapoptotic effects by regulating the release of cytochrome c from mitochondria (Antonsson et al., 1997; Kluck et al., 1997; Yang et al., 1997; Brenner and Kroemer, 2000; Kroemer and Reed, 2000; Saito et al., 2000; Martinou and Green, 2001; Shimizu et al., 2001; Wei et al., 2001). Interestingly, some proteins in the Bcl-2 family have structures similar to pore-forming toxins and can create ion channels in artificial membranes (Antonsson et al., 1997; Schendel et al., 1997; Schlesinger et al., 1997). Furthermore, Bax can induce transport of cytochrome c in liposomes (Saito et al., 2000). However, there is as yet no direct evidence for a Bax-induced pore or channel in the mitochondrial outer membrane that would correspond to the pathway for release of proapoptotic mitochondrial proteins like cytochrome c.
Hematopoietic FL5.12 cells are mammalian cells that enter apoptosis after withdrawal of the growth factor interleukin-3 (IL-3)* (Oltvai et al., 1993; Gross et al., 1998). Bax oligomerizes and translocates into mitochondria within twelve hours after IL-3 withdrawal. These events immediately precede release of proapoptotic factors, e.g., cytochrome c (Gross et al., 1998), which in turn is prevented by the antiapoptotic protein Bcl-2 (Goping et al., 1998; Gross et al., 2000). In this study, patch clamp techniques are used to directly demonstrate that a novel ion channel activity occurs both in the outer membrane of mammalian mitochondria early in apoptosis, and in the mitochondria of yeast expressing human Bax. Electrophysiological characterization of this high-conductance channel and comparison with other known mitochondrial channels, as well as purified Bax channels, are included.
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Results and discussion |
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Ion conductance of the native mitochondrial outer membrane
The ion permeability of outer membranes was determined by directly patch-clamping isolated mitochondria with microelectrodes (Kinnally et al., 1987; Lohret et al., 1997). The average conductance of outer-membrane patches of mitochondria isolated from FL5.12 cells 12 h after IL-3 withdrawal was significantly greater than that of patches from mitochondria of control cells (13.3 ± 2.5 nS versus 6.3 ± 3.6 nS; analysis limited to patches showing obvious channel activity). This increase in ion permeability, coincident with release of proapoptotic proteins from mitochondria, suggests the formation of novel pores in the outer membrane. Interestingly, the outer membrane patches excised from mitochondria were slightly cation-selective under both conditions (PK+/PCl- of 2; eight independent patches). Current recordings obtained by direct patch-clamping of mitochondria from FL5.12 cells could not resolve individual channel openings or closings, presumably due to the high density of ion channels in the native outer membrane, as previously encountered with mouse-liver mitochondria (Kinnally et al., 1987).
Characterization of an apoptosis-induced channel
To characterize single-channel events in outer membranes, they were diluted with additional lipid to form proteoliposomes. These proteoliposomes were prepared by the same methods as those used in the cytochrome c trapping experiments, except that cytochrome c was omitted from the preparation. A novel ion channel activity was readily detected by patch-clamping the proteoliposomes derived from apoptotic mitochondria. Current traces from these patches exhibit numerous deflections corresponding to opening and closure of the channel at constant voltage (Fig. 2). Analysis of many traces indicates that the channel has multiple conductance levels with peak, single-channel openings of 2.5 ± 0.6 nS. This maximum ion conductance corresponds to a pore diameter of 4.0 ± 0.5 nm, assuming a pore length of 7 nm, using the method of Hille (1992). Conductance changes of this magnitude have not previously been described for channels in the mitochondrial outer membrane. Although common, these large current transitions occurred at a lower frequency (less than one per minute) than the smaller transitions of 11.5 nS (several per second) (Fig. 2, AC).
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The electrophysiological experiments were repeated on clones of FL5.12 cells overexpressing Bcl-2, an antiapoptotic protein that prevents release of cytochrome c from mitochondria (Yang et al., 1997). MAC was not detected in cells overexpressing wild-type Bcl-2 after IL-3 withdrawal, but expression of a mutant Bcl-2 (which does not inhibit apoptosis) resulted in levels of MAC detection similar to those of the parental clones. Thus, effects of Bcl-2 on MAC activity parallel its effects on apoptosis. Elevated detection of this channel early in apoptosis and its suppression by functional Bcl-2 supports a role for this channel in release of proapoptotic factors from mitochondria.
It should be noted, as an aside, that no novel channels were detected in the mitochondrial outer membranes of cells overexpressing wild-type Bcl-2. A similar result was obtained previously with mitochondria isolated from a breast epithelial cell line overexpressing Bcl-2 (Murphy et al., 2001), despite the fact that Bcl-2 forms ion channels in artificial bilayers (Schendel et al., 1997; Schlesinger et al., 1997). However, expression of Bcl-2 in breast epithelial cells did affect an inner membrane channel, preventing calcium-activation of the multiple conductance channel, which may represent the mitochondrial permeability transition pore (Murphy et al., 2001).
MAC activity is similar but distinct from pure Bax
Oligomeric recombinant Bax (human) spontaneously inserts and forms high-conductance ion channels in artificial membranes, and causes release of trapped cytochrome c (Antonsson et al., 1997, 2000; Lewis et al., 1998; Saito et al., 2000). Comparison of MAC and Bax ion channel activities suggests several similarities (Table I). Like MAC, Bax channels display multiple conductance levels (Fig. 3) and a slight cation selectivity, but their typical peak current transitions (0.5 nS) are much smaller than that displayed by MAC (
2.5 nS). If MAC is a Bax channel, this important functional difference could relate to specific aspects of in vivo insertion and assembly in the native mitochondrial outer membrane, to posttranslational modification (Zha et al., 2000), or to involvement of additional mitochondrial components (Priault et al., 1999a, b; Shimizu et al., 2000, 2001; Antonsson et al., 2001).
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Conclusions and future prospects
This is the first demonstration using electrophysiological methods of a novel ion channel activity in the outer membrane of mitochondria associated with onset of apoptosis. This channel, named MAC, is the first whose pore size, inferred from single-channel conductance, is sufficient to allow proteins as large as or larger than cytochrome c to diffuse through the outer membrane. This postulated function is consistent with the inability of MAC-containing proteoliposomes to trap cytochrome c.
Bax clearly plays an essential role in MAC activation, forming the channel either by itself or in association with one or more outer-membrane components common to mammalian and yeast mitochondria. Contrary to some reports published previously (Shimizu et al., 2000, 2001), but in support of others (Priault et al., 1999a,b; Antonsson et al., 2001), this component is not VDAC, since VDAC was absent from the yeast strain in which MAC activity was induced by hBax expression. Although antibodies against hBax had no effect on MAC activity, preliminary experiments suggest cytochrome c reduces the ion conductance of this channel, but not of VDAC or TOM (unpublished data). If Bax alone forms the MAC channel, the observed differences in electrophysiological properties between MAC and channels induced by recombinant Bax in synthetic membranes likely arise from fundamental differences in Bax insertion, assembly, and/or regulation. The antiapoptotic role of Bcl-2 may relate to its ability (as demonstrated above) to suppress the activity of MAC, perhaps by associating with Bax in a manner that inhibits Bax oligomerization (Gross et al., 1998). Extension of the electrophysiological approaches described in this report should lead to a fuller understanding of the molecular mechanisms underlying the formation of this newly discovered channel; its regulation by apoptosis-associated proteins (e.g., Bcl-2 family members, caspases, and cytochrome c); and, ultimately, its role in apoptosis.
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Materials and methods |
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Isolation of mitochondria and preparation of proteoliposomes
Mitochondria were isolated from 215 g of FL5.12 cells as described previously (Campo et al., 1992) for outer membrane preparations. Smaller preparations of mitochondria were isolated from 1520 million cells as described previously (Murphy et al., 2001). Yeast mitochondria were prepared by a modification of Daum et al. (1982) as described previously (Lohret et al., 1997). Mitochondrial outer membranes were stripped from inner membranes by French pressing isolated mitochondria using modifications of the method of Decker and Greenawalt (1977). Outer membranes were separated from inner membranes as described by Lohret et al., (1997) and Mannella (1982). Proteoliposomes were formed by a modification of the method of Criado and Keller (1987) (Lohret et al., 1997). Briefly, small liposomes were formed by sonication of lipid (type IV-S soybean L-
-phosphatidylcholine; Sigma-Aldrich) in water. Mitochondrial outer membranes (3035 µg protein) and small liposomes (600 µg lipid) with or without cytochrome c (30 µg) were mixed with 5 mM Hepes, pH 7.4 (50 µl volume), and dotted on a glass slide. Samples were dehydrated (
3 h) and rehydrated overnight with 150 mM KCl, 5 mM Hepes, pH 7.4, at 4°C. For trapping experiments, proteoliposomes were washed in 0.5 M NaCl, 5 mM Hepes, pH 7.4, to remove bound cytochrome c and pelleted at 45,000 rpm for 2 h in a TLA rotor (Beckman Coulter ultracentrifuge). Pellets were resuspended in 10 mM Tris, pH 7.4, and prepared for electrophoresis.
Bax protein
Human Bax- (hBax) lacking 20 amino acids at the COOH end was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified as described previously (Lewis et al., 1998).
Immunoblotting
Proteins were separated by SDS-PAGE and electrotransferred onto PVDF membranes. Indirect immunodetection employed chemiluminescence (Amersham Pharmacia Biotech) using HRP-coupled secondary antibodies. Membrane proteins (0.56 µg/lane) were probed with primary polyclonal antibodies against hBax (Santa Cruz Biotechnology, Inc.; 1:5,000), mammalian VDAC (Stanley et al., 1995), 1:5005000), or yeast VDAC antibodies (Molecular Probes, 1:5000) and a secondary antirabbit antibody (Jackson ImmunoResearch Laboratories; 1:5,000). Cytochrome c (25 ng) was detected with a monoclonal antibody (Research Diagnostics; 1:2,500) and a secondary antimouse antibody (Jackson ImmunoResearch Laboratories; 1:2,500).
Patch-clamping analysis
Patch-clamp procedures and analysis used were described previously (Lohret et al., 1997). Briefly, membrane patches were excised from proteoliposomes after formation of a giga-seal using micropipettes with 0.4-µm diameter tips and resistances of 1020 M
at room temperature. Unless otherwise stated, the solution was symmetrical 150 mM KCl, 5 mM Hepes, pH 7.4. Ion selectivity was determined from the reversal potential after perfusion of the bath with 30 mM KCl, 56 mM sucrose, 184 mM mannitol, 5 mM Hepes, pH 7.4. Voltage clamp was performed with the excised configuration of the patch-clamp technique (Hamill et al., 1981) using a Dagan 3900 patch clamp amplifier in the inside-out mode. Voltages are reported as bath potentials.
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Footnotes |
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* Abbreviations used in this paper: IL-3, interleukin-3; MAC, mitochondrial apoptosisinduced channel; TOM, translocase outer membrane; VDAC, voltage dependent anion-selective channel.
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Acknowledgments |
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This research was supported by National Institutes of Health grant GM57249 and National Science Foundation grants MCB-9513439, MCB-0096206, and INT003797 to K.W. Kinnally and MENRT grant to M. Priault.
Submitted: 12 July 2001
Accepted: 2 October 2001
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