Uncoupler titrations

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Uncoupler titrations

Description

In uncoupler titrations various uncouplers, such as CCCP, FCCP or DNP are applied to uncouple mitochondrial electron transfer from phosphorylation (ATP synthase, ANT and phosphate carrier), particularly with the aim to measure ET capacity. ET capacity is maximum oxygen flux measured as noncoupled respiration with optimum uncoupler concentration.

Uncoupler titrations in HRR

DatLab settings

  • Recommendation: Set "Slope smoothing" to 20 for performing and analyzing experiments with biological sample particularly for uncoupler titrations.

Optimum uncoupler concentration

Stepwise titrations of an uncoupler is necessary to achive the optimum concentration for obtaining maximum flux as a measure ET capacity (noncoupled respiration). It is important to avoid inhibition of respiration by too high uncoupler concentrations. The underlying mechanism for the latter is not clear.
The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentration, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estimation of maximum flux or Electron transfer-pathway capacity (for discussion, see Artefacts by single dose uncoupling).
The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample. This is true for various uncouplers, such as CCCP, FCCP and DNP (Steinlechner-Maran 1996 Am J Physiol Cell Physiol). To evaluate the optimum concentration, an uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration (Huetter_2004_Biochem J, Pesta 2012 Methods Mol Biol). Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.
See Steinlechner-Maran et al (1996) for a comparison of uncoupler titrations with FCCP and DNP from the ROUTINE state to the ET state of cell respiration.

Coupling-control protocol

Uncoupler titrations after inhibition of respiration by oligomycin in coupling-control protocols with living cells yield the sequence of ROUTINE respiration, LEAK respiration and ET capacity, followed by inhibition to ROX (Huetter 2004 Biochem J, Gnaiger 2008 POS). The highest accuracy of uncoupler titrations is achieved by titrations with the TIP2k at high concentrations of the stock solution (Gnaiger 2008 POS. Increasing the concentration in small steps, most accurately titrated by the TIP2k, is recommended (0.5 or 0.25 µM steps or even smaller).


References

Bioblast linkReferenceYear
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52.2008
Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45.2009
Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28.2004
Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58.2012
Selected media and chemicals for respirometry with mitochondrial preparations.2016-08-30
Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61.1996
Bioblast linkReferenceYear
Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1:1-44. https://doi.org/10.26124/bec:2020-0001.v12020


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