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A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

Pavlov EV, Priault M, Pietkiewicz D, Cheng EH, Antonsson B, Manon S, Korsmeyer SJ, Mannella CA, Kinnally KW - J. Cell Biol. (2001)

Bottom Line: Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2.Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax.These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, New York University College of Dentistry, New York, NY 10010, USA.

ABSTRACT
During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

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Detection and characterization of MAC activity and comparison with VDAC and TOM channels. (A) Single-channel current traces reveal MAC activity with 3 nS peak transition size and predominant transitions of 1.5 nS at −40 mV. Lower current traces are time-expanded regions of the top trace (indicated by heavy lines). (B) Current trace of MAC at 80 mV shows 2 nS peak conductance with multiple 1 nS transitions. (C) Current traces of single channels at 20 mV show MAC character is distinct from TOM channel and VDAC. O and C indicate open and closed conductance levels. Patching media was symmetrical 150 mM KCl, 5 mM Hepes-koh/ pH 7.4. Current traces, low pass filtered at 2 kHz (5 kHz sampling), were obtained using patch-clamp procedures as described in Materials and methods. (D) Histograms show the frequency of detecting MAC in independent patches (n) from proteoliposomes prepared with outer membranes purified from mitochondria isolated before (closed, +IL-3 control) or 12 h after IL-3 withdrawal (open, −IL-3 apoptotic) from FL5.12 cells that were parental or expressing incompetent (Bcl-2 mutant) or functional Bcl-2 (Bcl-2). Statistical difference (P < 0.05) between pairs was determined by Fischer's exact statistical test (Fisher, 1935) and indicated by asterisks.
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fig2: Detection and characterization of MAC activity and comparison with VDAC and TOM channels. (A) Single-channel current traces reveal MAC activity with 3 nS peak transition size and predominant transitions of 1.5 nS at −40 mV. Lower current traces are time-expanded regions of the top trace (indicated by heavy lines). (B) Current trace of MAC at 80 mV shows 2 nS peak conductance with multiple 1 nS transitions. (C) Current traces of single channels at 20 mV show MAC character is distinct from TOM channel and VDAC. O and C indicate open and closed conductance levels. Patching media was symmetrical 150 mM KCl, 5 mM Hepes-koh/ pH 7.4. Current traces, low pass filtered at 2 kHz (5 kHz sampling), were obtained using patch-clamp procedures as described in Materials and methods. (D) Histograms show the frequency of detecting MAC in independent patches (n) from proteoliposomes prepared with outer membranes purified from mitochondria isolated before (closed, +IL-3 control) or 12 h after IL-3 withdrawal (open, −IL-3 apoptotic) from FL5.12 cells that were parental or expressing incompetent (Bcl-2 mutant) or functional Bcl-2 (Bcl-2). Statistical difference (P < 0.05) between pairs was determined by Fischer's exact statistical test (Fisher, 1935) and indicated by asterisks.

Mentions: 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 1–1.5 nS (several per second) (Fig. 2, A–C).


A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

Pavlov EV, Priault M, Pietkiewicz D, Cheng EH, Antonsson B, Manon S, Korsmeyer SJ, Mannella CA, Kinnally KW - J. Cell Biol. (2001)

Detection and characterization of MAC activity and comparison with VDAC and TOM channels. (A) Single-channel current traces reveal MAC activity with 3 nS peak transition size and predominant transitions of 1.5 nS at −40 mV. Lower current traces are time-expanded regions of the top trace (indicated by heavy lines). (B) Current trace of MAC at 80 mV shows 2 nS peak conductance with multiple 1 nS transitions. (C) Current traces of single channels at 20 mV show MAC character is distinct from TOM channel and VDAC. O and C indicate open and closed conductance levels. Patching media was symmetrical 150 mM KCl, 5 mM Hepes-koh/ pH 7.4. Current traces, low pass filtered at 2 kHz (5 kHz sampling), were obtained using patch-clamp procedures as described in Materials and methods. (D) Histograms show the frequency of detecting MAC in independent patches (n) from proteoliposomes prepared with outer membranes purified from mitochondria isolated before (closed, +IL-3 control) or 12 h after IL-3 withdrawal (open, −IL-3 apoptotic) from FL5.12 cells that were parental or expressing incompetent (Bcl-2 mutant) or functional Bcl-2 (Bcl-2). Statistical difference (P < 0.05) between pairs was determined by Fischer's exact statistical test (Fisher, 1935) and indicated by asterisks.
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Related In: Results  -  Collection

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fig2: Detection and characterization of MAC activity and comparison with VDAC and TOM channels. (A) Single-channel current traces reveal MAC activity with 3 nS peak transition size and predominant transitions of 1.5 nS at −40 mV. Lower current traces are time-expanded regions of the top trace (indicated by heavy lines). (B) Current trace of MAC at 80 mV shows 2 nS peak conductance with multiple 1 nS transitions. (C) Current traces of single channels at 20 mV show MAC character is distinct from TOM channel and VDAC. O and C indicate open and closed conductance levels. Patching media was symmetrical 150 mM KCl, 5 mM Hepes-koh/ pH 7.4. Current traces, low pass filtered at 2 kHz (5 kHz sampling), were obtained using patch-clamp procedures as described in Materials and methods. (D) Histograms show the frequency of detecting MAC in independent patches (n) from proteoliposomes prepared with outer membranes purified from mitochondria isolated before (closed, +IL-3 control) or 12 h after IL-3 withdrawal (open, −IL-3 apoptotic) from FL5.12 cells that were parental or expressing incompetent (Bcl-2 mutant) or functional Bcl-2 (Bcl-2). Statistical difference (P < 0.05) between pairs was determined by Fischer's exact statistical test (Fisher, 1935) and indicated by asterisks.
Mentions: 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 1–1.5 nS (several per second) (Fig. 2, A–C).

Bottom Line: Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2.Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax.These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, New York University College of Dentistry, New York, NY 10010, USA.

ABSTRACT
During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

Show MeSH
Related in: MedlinePlus