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

Thomas Briston,James M Staddon,Malcolm Roberts,Ben Powney,Gyorgy Szabadkai,Michael R Duchen,Sian Lewis

doi:10.1038/s41598-017-10673-8

Thomas Briston, James M Staddon + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-10673-8

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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.

Highlights

Mitochondria are capable of oxidising numerous substrates based on availability and metabolic demand
MPTP opening was identified as the release of mitochondrially sequestered Ca2+, the failure of mitochondrial Ca2+ uptake and the collapse of the mitochondrial membrane potential
A decrease of ΔΨm occurred significantly earlier in mitochondria energised with glutamate/malate compared to those metabolising succinate (Fig. 1b and d): deviation from the succinate condition occurred following just 1–2 calcium additions and prior to overt mitochondrial permeability transition pore (mPTP) opening

Summary

Introduction

Mitochondria are capable of oxidising numerous substrates based on availability and metabolic demand. The delivery of energetic substrates to mitochondria provides reducing equivalents required for serial reduction of electron transport chain (ETC) redox centres. These redox reactions are coupled to expulsion of protons from the matrix into the intermembrane space (IMS)[1]. To explore the impact of different metabolic conditions on the probability of pore opening, fluorescence-based analysis coupled to high-resolution respirometry was used to simultaneously measure mitochondrial Ca2+ uptake, ΔΨm, reactive oxygen species (ROS) production and respiration. We report variation in mitochondrial sensitivity to Ca2+-induced mPTP opening based on the source of electron flux through the respiratory chain, as driven by different metabolic substrates, identifying mechanisms by which energetic state determines the mitochondrial response to Ca2+ overload

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