Sumbalova 2011 Abstract Mitochondrial Medicine
|Sumbalova Z, Fasching M, Gnaiger E (2011) Substrate control in mitochondrial respiration and regulation of mitochondrial membrane potential. Abstract Mitochondrial Medicine Chicago.|
Substrate supply to mitochondria plays an important role in physiological processes in the brain. Various neurological diseases are associated with specific enzymatic defects in the mitochondrial OXPHOS system including the tricarboxylic acid cycle. Diagnostic protocols for the analysis of substrate-specific OXPHOS defects are required, which are traditionally performed in separate assays using large amounts of tissue. Since specific brain regions are differentially susceptible to various insults, there is a need to assay mitochondrial function in small samples, using complex protocols with substrate-uncoupler-inhibitor titrations (SUIT) and monitoring simultaneously different functional parameters in a MultiSensor system. We applied high-resolution respirometry combined with an ion selective electrode system (TPP+; Oroboros Oxygraph-2k MultiSensor system; MiR06 at 37 °C) to measure mitochondrial (mt) respiration, JO2, and mt-membrane potential, ΔΨ, in mouse brain preparations: (i) isolated mitochondria, (ii) homogenate after 3 min centrifugation at 1300 g, and (iii) crude tissue homogenate. Coupling control and substrate control states  were established sequentially in SUIT protocols. For calculation of ΔΨ, corrections were applied for side effects on the signal of the TPP+ electrode induced by titrated chemicals, and for unspecific binding of TPP+ . Our conclusions were corroborated by consistent results obtained with the three preparations on ΔΨ and respiratory fluxes normalized for citrate synthase. Respiratory coupling control ratios and the cytochrome c test indicated that mitochondria were well coupled and the outer mt-membrane was intact in all preparations. ΔΨ dropped by 20-25 mV as flux was increased by coupling control from the resting LEAK state (no ADP) to OXPHOS capacity (high ADP). Flux increased further by 10-40% as ΔΨ was collapsed in the non-coupled state (FCCP). Opposite to this coupling paradigm of an inverse ΔΨ/JO2 relationship, both ΔΨ and JO2 increased when flux was varied by substrate control. Conventional State 3 respiration with Complex I (CI) related substrates (pyruvate, malate, glutamate) was only 70% to 80% of OXPHOS capacity supported by addition of both CI and CII substrates (pyruvate, malate, glutamate, succinate). Under these conditions, ΔΨ increased with an increase of flux. The shift of ΔΨ was 4-6 mV both in the OXPHOS and LEAK state. Our results challenge the simplistic State 3/State 4 paradigm of mitochondrial respiratory coupling control and inverse regulation of ΔΨ. Substrate control is complementary to coupling control of mitochondrial respiration, as emphasized in the ingenious work of Chance and Williams . Supported by FEMtech, a program of the Federal Ministry for Transport, Innovation and Technology (NMVIT, Austria) to promote equal opportunities in research and technology. Contribution to Mitofood COST Action FAO602 and K-Regio project MitoCom Tyrol.
- Gnaiger E (2009) IJBCB 41: 1837-1845.
- TPP-MitoMembranePotential (MiPNet14.05).
- Chance B, Williams GR (1955) JBC 217: 409-427.
• Keywords: mouse brain, mitochondria, membrane potential, TPP+
Organism: Mouse Tissue;cell: Nervous system Preparation: Homogenate, Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k, TPP