Uncoupler titrations

high-resolution terminology - matching measurements at high-resolution

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 link	Reference	Year
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. https://doi.org/10.1016/j.biocel.2009.03.013	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. https://doi.org/10.1042/BJ20040095	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. https://doi.org/10.1007/978-1-61779-382-0_3	2012
MiPNet03.02 Chemicals-Media	Selected media and chemicals for respirometry with mitochondrial preparations.	2016-08-30
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. https://doi.org/10.1152/ajpcell.1996.271.6.C2053	1996

Bioblast link	Reference	Year
BEC 2020.1 doi10.26124bec2020-0001.v1	Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1	2020

MitoPedia methods: Respirometry

MitoPedia O2k and high-resolution respirometry: DatLab, Oroboros QM, O2k-Open Support

MitoPedia topics: Uncoupler

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Specific

» Artefacts by single dose uncoupling

» ATP synthase

» CCCP

» Coupling-control protocol

» DNP

» Dyscoupled respiration

» FCCP

» 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

Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002

General (alphabetical order)

» Adenine nucleotide translocase

» Adenylates

» Electron transfer pathway

» Mitochondrial preparations

» mt-membrane potential

» Oxygen flux

» Phosphorylation system

» Proton leak

» Proton slip

» TIP2k

Other keyword lists