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Results

Figure 3. Coupling control and substrate control in brain mitochondria.A, B: FCR and for coupling states LEAK (L), OXPHOS (P), ETS (E) of CI, CI+II and CII.C, D: FCR and for substrates of CI, CI+II and CII within a coupling state (L or P).

Figure 6. A: Oxygen flux in SUIT protocol with three mt-preparations normalizedfor citrate synthase (CS) activity. B: calculated with binding constant for TPP+

K'in = K'out = 11 [4]. C: calculated with K'out = 100 for Smt and K'out = 200 forHmt. and shifts of between states are affected by K'out.

Preparations

Figure 7. Relation between oxygen flux(JO2/CS) and mt-membrane potential.Summary from five SUIT protocols withthree brain preparations. was calcula-ted with K'in = K'out = 11 for all preparations.

Conclusions

A B C

Background

Substrate supply to mitochondria plays a key role in energy metabolism of the brain.Various neurological diseases are associated with specific enzymatic defects in themitochondrial OXPHOS system including the tricarboxylic acid cycle.

Protocols for analysis of substrate-specific OXPHOS defects are traditionally performedin separate assays limited to a small number of titrations.

We developed protocols for multiple substrate-uncoupler-inhibitor titrations (SUIT),monitoring simultaneously mitochondrial membrane potential and respiration in the O2k-MultiSensor system.

Methods

We applied high-resolution respirometry (HRR) combined with an ion selective electrodesystem (OROBOROS Oxygraph-2k MultiSensor system, ISE) using tetraphenylphosphonium(TPP+) as a reporter ion for simultaneous measurement of mitochondrial respiration, JO2, andmitochondrial membrane potential, ΔΨ, at 37 °C in MiRO6, and 1 or 1.5 µM TPP+.

Three mitochondrial preparations were compared from brain (C57Bl/6N mice, 2 months):• Isolated mitochondria (Imt)• Supernatant after 3 min centrifugation of homogenate at 1300 g (Smt)• Crude homogenate (Hmt)

Coupling control and substrate control states [1, 2] were established sequentially in SUITprotocols. In calculations of ΔΨ, corrections were applied for side effects on the signal of theTPP+ electrode induced by titrated chemicals, and for unspecific binding of TPP+ [3, 4].

Figure 1. SUIT protocol with isolated brain mitochondria. A: Recording of oxygenconcentration [µM] (blue line) and volume-specific oxygen flux JO2 [pmol∙s-1∙ml-1] (red line).B: log(TPP+). Decrease of signal corresponds to increase of . C: Coupling/substratecontrol diagram, with respiratory states:

ROX: Residual oxygen consumption without substrate and ADP; oxygen flux is corrected for ROX.L: LEAK (CI) respiration in the presence of Complex I substrates pyruvate, malate and glutamate.State 3 at high, not saturating ADP concentration (0.25 mM).P: OXPHOS (CI) capacity after addition of saturating ADP (2mM).L: LEAK (CI) respiration after ATP synthase inhibition by oligomycin (Omy).E: Electron transport system capacity, ETS (CI), after FCCP titration (uncoupler, u).E: ETS (CI+II) capacity after addition of succinate (10 mM).E: ETS (CII) capacity after inhibition of CI by rotenone (Rot).ROX: After inhibition of CII with malonic acid (Mna) and CIII with antimycin A (Ama).

Figure 5. Side effects of chemicals on the signal of the TPP+ electrode. The trace showstitrations of the chemicals in a SUIT protocol at 1 µM TPP+ without biological sample.Succinate, ADP, FCCP and ethanol-dissolved inhibitors affect the signal of TPP+ beyond thetheoretical dilution effect. Correction for titrations volumes >1 µl is necessary.

Figure 2. Coupling/substrate control diagrams summarizing five SUIT protocols withisolated brain mitochondria. A: Flux control ratios (FCR) normalized relative to ETScapacity with convergent CI+II electron input. B: Corresponding mitochondrialmembrane potential, [mV].

References1. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of

mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837–1845.2. Pesta D, Gnaiger E (2011) High-resolution respirometry. OXPHOS protocols for human cell cultures and

permeabilized fibres from small biopsies of human muscle. In C. Palmeira & A. Moreno (Eds.) Mitochondrial bioenergetics: methods and protocols.

3. http://www.bioblast.at/index.php/MiPNet14.05_TPP-MitoMembranePotential4. Rottenberg H (1984) Membrane potential and surface potential in mitochondria: Uptake and binding of

lipophilic cations. J. Membrane Biol. 81: 127-138.

AcknowledgementsThis work was supported by FEMtech (Austrian Ministry for Transport, Innovation and Technology), and the Center for Translational Molecular Medicine (NL). Contribution to MitoCom Tyrol.

• Our results challenge the simplistic State 3/State 4 paradigm ofmitochondrial respiratory coupling control and inverse regulationof .

• Complementary to coupling, substrate control exerts an influenceon the complex relationship between oxygen flux and .

• JO2 normalized for CS was similar in isolated mitochondria andhomogenates.

• External binding of TPP+ in homogenates affects absolute valuesof and shifts of between states, partially resolved bychanging K'out for calculation in homogenate preparations.

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Substrate Control in Mitochondrial Respiration and Regulation of Mitochondrial Membrane Potential

Zuzana Sumbalová1, Mario Fasching1, Erich Gnaiger1,2

1OROBOROS INSTRUMENTS, Innsbruck, Austria; 2D. Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria

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Fig. 4. SUIT protocols: Inhibition of CI anddecrease of JO2 and (dotted circles).