Ca(2+)-triggered membrane permeability transition in deenergized mitochondria from rat liver
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B V Chernyak1, V N Dedov, Chernyak VYa
1A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation.
Abstract:
The opening of the cyclosporin A-sensitive permeability transition pore (MTP) in deenergized mitochondria was induced only at millimolar Ca2+. Pretreatment of the mitochondria with 'inducers', such as duroquinone and phenylarsine oxide, allowed observing the pore opening at 0.01-0.1 mM Ca2+. Duroquinone caused a rapid (within 20 s) NAD(P)H oxidation which was followed by a slow (20 min) induction of the pore sensitive to low Ca2+. Phenylarsine oxide capable of cross-linking of vicinal SH-groups caused pore formation without the oxidation of NAD(P)H. The pore opening by both 'inducers' was prevented by N-ethylmaleimide. We propose that oxidation or cross-linking of critical dithiol(s) in membrane proteins increase the sensitivity of a putative 'Ca(2+)-sensor' that regulates the permeability transition pore opening.
Insights
Mitochondrial permeability transition pore (MTP) opening, sensitive to cyclosporin A, requires high calcium. Inducers like duroquinone and phenylarsine oxide lower calcium requirements by affecting critical dithiols.
Area of Science:
- Mitochondrial Physiology
- Biochemistry
- Cellular Signaling
Background:
- The mitochondrial permeability transition pore (MTP) is a critical regulator of mitochondrial function and cell death.
- MTP opening is typically observed in deenergized mitochondria at high calcium concentrations (millimolar range).
- Understanding the regulation of MTP opening is crucial for deciphering mitochondrial dysfunction in various pathologies.
Purpose of the Study:
- To investigate the role of specific chemical agents in modulating the calcium sensitivity of MTP opening.
- To elucidate the molecular mechanisms underlying the induction of MTP opening by 'inducers' like duroquinone and phenylarsine oxide.
- To identify key molecular targets involved in the regulation of the permeability transition pore.
Main Methods:
- Mitochondrial isolation and preparation from deenergized sources.
- Induction of MTP opening using varying calcium (Ca2+) concentrations (0.01-0.1 mM and millimolar).
- Treatment with duroquinone and phenylarsine oxide as MTP 'inducers'.
- Measurement of NAD(P)H oxidation kinetics.
- Assessment of MTP pore formation and inhibition using N-ethylmaleimide.
Main Results:
- MTP opening in deenergized mitochondria was observed only at millimolar Ca2+ without pre-treatment.
- Pretreatment with duroquinone or phenylarsine oxide enabled MTP opening at significantly lower Ca2+ concentrations (0.01-0.1 mM).
- Duroquinone induced rapid NAD(P)H oxidation followed by MTP opening, while phenylarsine oxide caused pore formation directly via SH-group cross-linking.
- N-ethylmaleimide effectively prevented MTP pore opening induced by both agents, indicating the involvement of critical dithiol groups.
Conclusions:
- Oxidation or cross-linking of critical dithiol(s) in mitochondrial membrane proteins enhances the sensitivity of the putative Ca2+-sensor regulating MTP opening.
- These findings suggest a novel mechanism for modulating MTP opening, with potential implications for mitochondrial diseases.
- The study highlights the importance of redox-sensitive thiols in the regulation of mitochondrial permeability transition.