Mitochondrial permeability transition pore: sensitivity to opening and mechanistic dependence on substrate availability

Abstract

Mitochondrial Ca2+ uptake has a key role in cellular Ca2+ homeostasis. Excessive matrix Ca2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH2-linked substrates. The functional consequences of Ca2+-induced mPTP opening were assessed by Ca2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca2+ overload.

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“…The proteins Bax/Bak interact with VDAC and cause the pore to open more readily, which would allow cytochrome C to be exported into the cytosol . However, the protein cyclophilin D (CypD) is generally accepted as a protein found in the MPTP . The importance of CypD was discovered mainly through the effect of the drug cyclosporine A (CsA), which had an inhibiting effect on CypD .…”

“…27 However, the protein cyclophilin D (CypD) is generally accepted as a protein found in the MPTP. 28 The importance of CypD was discovered mainly through the effect of the drug cyclosporine A (CsA), which had an inhibiting effect on CypD. 25 The addition of CsA was found to desensitize the MPTP to Ca 2+ ions, preventing MPTP opening.…”

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“…Thus, in studies of rat heart mitochondria, by applying Ca 2+ pulses at the threshold level for inducing mPTP opening, succinate oxidation sensitized mitochondria to the mPTP opening. By contrast, NADH‐linked substrate oxidation was inhibited upon subsequent partial mPTP opening and membrane depolarization due to the loss of the mitochondrial NADH pool leaking out from the mitochondria . Hence, with succinate (±rotenone), mitochondrial transmembrane electric potential (ΔΨ m ) collapsed, but only after a Ca 2+ ‐activated burst of respiration before mPTP opening.…”

“…Intriguingly, it was recently concluded that the sensitivity of the mPTP opening depended on whichever mitochondrial energy substrates predominate and of these, succinate driven respiration was the major contributor to this process . Thus, in studies of rat heart mitochondria, by applying Ca 2+ pulses at the threshold level for inducing mPTP opening, succinate oxidation sensitized mitochondria to the mPTP opening.…”

“…Transient stimulation of respiration by mCa 2+ uptake was followed by decreased respiration using NADH‐linked substrates while, in contrast, oxidation of the FADH2‐linked substrate, succinate resulted in large Ca 2+ ‐induced increases in mitochondrial oxygen consumption, such that electron flow was sustained during mPTP partial opening and was consistent with both a short uncoupling response and a slower rate of mitochondrial depolarization . Adding the CypD inhibitor, cyclosporin A or using the Ca 2+ uptake inhibitor, ruthenium red prevented the Ca 2+ activation of mitochondria, which then tolerated greater Ca 2+ loads, as well as the associated decline in respiration or collapse of ΔΨ m , confirming the dependence of mPTP opening on mCa 2+ uptake .…”