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Ca2+ acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability
A J Kowaltowski1, R F Castilho
1Departamento de Patologia Clinica, Faculdade de Ciências Medicas, Universidade Estadual de Campinas, Brazil.
Abstract:
Mitochondrial permeability transition (MPT) induced by the thiol cross-linker phenylarsine oxide (PhAsO) in Ca(2+)-depleted mitochondria incubated in the presence of ruthenium red, an inhibitor of the Ca2+ uniporter, is stimulated by the addition of extramitochondrial Ca2+. The presence of extramitochondrial Ca2+ stimulates the reaction of mitochondrial membrane protein thiol groups with PhAsO. Both Ca(2+)-induced increase in mitochondrial membrane permeabilization and protein thiol group reaction with PhAsO are dependent on time (5-10 min to be complete) and the concentration of Ca2+ (1-25 microM). Mitochondrial permeabilization induced by PhAsO (15 microM) and extramitochondrial Ca2+ is inhibited by ADP, cyclosporin A, dibucaine and Mg2+, while mitochondrial permeabilization induced by high concentrations of PhAsO (60 microM) in the absence of Ca2+ is inhibited only by ADP and cyclosporin A. These results suggest that dibucaine and Mg2+ can inhibit mitochondrial permeabilization by antagonizing the effect of Ca2+ on the mitochondrial membrane. Once mitochondrial permeabilization induced by 15 microM PhAsO and extramitochondrial Ca2+ has already occurred, the addition of the Ca2+ chelator EGTA restores mitochondrial membrane potential (MPT pore closure), suggesting that the presence of Ca2+ is essential for the maintenance of the permeability of the mitochondrial membrane to protons (MPT pore opening). In conclusion, the results presented indicate that low Ca2+ concentrations acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability transition induced by PhAsO, due to increased accessibility of protein thiol groups to the reaction with PhAsO and increased probability of MPT pore opening.
Insights
Extramitochondrial calcium (Ca2+) stimulates mitochondrial permeability transition (MPT) induced by phenylarsine oxide (PhAsO) by increasing thiol group reactivity. This calcium effect is essential for maintaining MPT pore opening.
Area of Science:
- Mitochondrial Physiology
- Cellular Signaling
- Biochemistry
Background:
- Mitochondrial permeability transition (MPT) is a critical process affecting cell viability.
- The role of extramitochondrial calcium in modulating MPT, particularly in conjunction with thiol-reactive agents, requires further elucidation.
Purpose of the Study:
- To investigate the effect of extramitochondrial calcium on MPT induction by phenylarsine oxide (PhAsO).
- To determine the mechanisms by which calcium influences PhAsO-induced mitochondrial permeabilization.
Main Methods:
- Utilized Ca(2+)-depleted mitochondria incubated with ruthenium red.
- Assessed MPT induction using PhAsO and monitored changes in mitochondrial membrane potential and protein thiol reactivity.
- Investigated the effects of various inhibitors (ADP, cyclosporin A, dibucaine, Mg2+) and a chelator (EGTA).
Main Results:
- Extramitochondrial Ca(2+) significantly stimulates PhAsO-induced MPT in a time- and concentration-dependent manner.
- Calcium enhances the reaction of PhAsO with mitochondrial membrane protein thiol groups.
- Dibucaine and Mg(2+) inhibit Ca(2+)-dependent MPT by antagonizing calcium's effect, while ADP and cyclosporin A inhibit MPT regardless of Ca(2+) presence.
Conclusions:
- Low extramitochondrial Ca(2+) concentrations promote PhAsO-induced MPT by increasing thiol accessibility and MPT pore opening probability.
- Calcium is essential for maintaining the proton permeability of the MPT pore.
- Specific inhibitors differentially affect calcium-dependent and independent MPT pathways.