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
- 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.
- 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).
|Gnaiger 2008 POS||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 2009 Int J Biochem Cell Biol||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|
|Huetter 2004 Biochem J||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 2012 Methods Mol Biol||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|
|Steinlechner-Maran 1996 Am J Physiol Cell Physiol||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|
|BEC 2020.1 doi10.26124bec2020-0001.v1||Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. doi:10.26124/bec:2020-0001.v1.||2020|
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- » Artefacts by single dose uncoupling
- » ATP synthase
- » CCCP
- » Coupling-control protocol
- » DNP
- » Dyscoupled respiration
- » FCCP
- » Gnaiger 2019 MitoFit Preprint Arch
- » Is respiration uncoupled - noncoupled - dyscoupled?
- » Noncoupled respiration: Discussion
- » Uncoupler
- » Uncoupled respiration - see » Noncoupled respiration
- » Uncoupling proteins
- » Uncoupling protein 1
- » Uncoupler titrations - Optimum uncoupler concentration
- Respiratory states and control ratios
- » Biochemical coupling efficiency
- » Coupling-control state
- » Electron-transfer-pathway state
- » Electron-transfer pathway
- ET capacity
- » E-L coupling efficiency
- » Flux control efficiency
- » Flux control ratio
- » LEAK-control ratio
- » LEAK respiration
- » Noncoupled respiration
- » OXPHOS
- » OXPHOS capacity; » State 3
- » OXPHOS-control ratio, P/E ratio
- » Respiratory acceptor control ratio
- » ROUTINE-control ratio
- » ROUTINE respiration
- » ROUTINE state
- » State 3u
- » State 4
- » Uncoupling-control ratio UCR
- Respiratory states and control ratios
- Other keyword lists
MitoPedia concepts: MitoFit Quality Control System
MitoPedia methods: Respirometry
MitoPedia topics: Uncoupler