Sumbalova 2015 Abstract MiP2015
|High-resolution measurement of mitochondrial membrane potential and respiration – comparison of potentiometric and fluorometric methods.|
The relationship between mitochondrial (mt) membrane potential (Ψmt and respiration remains poorly understood due to methodological limitations and the complexity of interrelations between fluxes (respiration) and forces (mt-membrane potential as the electric component of the protonmotive force). Ψmt reporter cations inhibit and uncouple mitochondrial respiration depending on type, concentration, and the specific electron transfer segment under investigation. Potentiometric signals based on ion-sensitive electrodes (ISE) and optical signals from fluorophors require different calibrations (linear and non-linear), transformations (log-lin), corrections for unspecific binding, and conversion to electric units [mV] of mt-membrane potential. Signals of ISE reflect the log concentration of the free reporter ion (TPP+) outside of mitochondria, providing a quantitative basis for calculation of Ψmt [mV] in the range of high membrane potential. On the other hand, fluorescence may comprise a mixture of signals from a fluorescent dye in its free state and bound to membranes or proteins, and corrections may be difficult for obtaining the concentration of the free dye . A direct comparison of results with these two approaches, application of different Ψmt reporter molecules and combination with respirometry is required for a critical analysis of mitochondrial membrane potential.
In this study of isolated mouse brain mitochondria (37 °C, MiR05), we used the Oxygraph-2k with the O2k-TPP+ ISE-Module with tetraphenylphosphonium (TPP+) as a reporter ion, or with the O2k-Fluo LED2-Module with safranin or tetramethylrhodamine methyl ester perchlorate (TMRM). All signals required quantitatively important corrections for chemical background effects (responses to titration of substances such as ADP in the absence of mitochondria). The sensitivity of the potentiometric or fluorometric signal of these probes to inhibition of mt-respiration was compared in Complex I- (CI-) or CII-linked substrate states in the absence of adenylates (LEAK, L), at saturating ADP (OXPHOS, P), and in uncoupler titrations (ET capacity, E).
CI-linked OXPHOS respiration was inhibited by TPP+ by <5% up to 3 µM, without any inhibitory effect on CII-linked OXPHOS capacity. Both fluorescent dyes, 2 µM Safranin and 1.5 µM TMRM, inhibited substantially (more than 30%) CI-linked OXPHOS respiration, and to a lower extent (around 10%) CII-linked respiration. 1.5 µM TMRM gradually uncoupled CIIL respiration, which is particularly problematic for evaluation of Ψmt. The TPP+ electrode was very sensitive to inhibition of respiration in states CIL and CIP by rotenone, and in state CIIL by malonate. However, the TPP+ signal was practically insensitive to inhibition in the CIIP state when the mt-membrane potential is low already in the uninhibited state. The increase of TPP+ concentration from 1.5 to 3 µM did not change the sensitivity of the response.
Labels: MiParea: Respiration, Instruments;methods, mt-Membrane
Organism: Mouse Tissue;cell: Nervous system Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k, O2k-Fluorometer, TPP, O2k-Protocol Event: D1 MiP2015, SUIT-020, SUIT-020 Fluo mt D033, SUIT-020 Fluo mt D036
1-Pharmacobiochem Lab, Fac Medicine, Comenius Univ Bratislava, Slovakia; 2-Oroboros Instruments, Innsbruck, Austria; 3-Dep Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ, Innsbruck, Austria. – [email protected]
In contrast, the fluorescence signal of 2 µM safranin responded well also to the inhibition of CII-linked OXPHOS respiration . When the concentration of safranin was decreased to 1 µM, the fluorometric sensitivity was lost to detect the response to inhibition of CIIP. The fluorescence signal of 1.5 µM TMRM was less sensitive to inhibition of CIP and CIIL in comparison to safranin or TPP+, and less sensitive to inhibition of CIIP respiration when compared to safranin. The signal of 1 µM TMRM was insensitive to inhibition of CIIP.
Based on the these results, we recommend using the TPP+ electrode for evaluation of Ψmt with CI-linked and CI&II-linked respiration and 2 µM safranin for evaluation of Ψmt with CII-linked respiration, which is more sensitive in the range of low Ψmt compared to TPP+. The fluorometric method should be elaborated for evaluation of Ψmt for each type of mitochondrial preparation and protein concentration  that has to be kept constant for comparison of different samples in any set of experiments. Before reporting results uncritically as Ψmt [mV], inhibitory and uncoupling effects on respiration, sensitivity and linearity, and confounding effects of unspecific binding need to be taken into account for various mt-preparations (isolated mt, homogenate, permeabilized cells and fibres), and for various probe/sample concentration ratios.
References and acknowledgements
- Perevoshchikova IV, Sorochkina AI, Zorov DB, Antonenko YN (2009) Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension. Biochemistry (Mosc) 74:663-71.
- Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81.
- Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469-77.
Supported by Action Austria-Slovakia (ZS) and K-Regio project MitoFit (EG).