Uncoupler

high-resolution terminology - matching measurements at high-resolution

Uncoupler

Description

An uncoupler is a protonophore (FCCP, CCCP, DNP) which cycles across the inner mt-membrane with transport of protons and dissipation of the electrochemical proton gradient. Mild uncoupling may be induced at low uncoupler concentrations, the non-coupled state of ETS capacity is obtained at optimum uncoupler concentration for maximum flux, whereas at higher concentrations an uncoupler-induced inhibition is observed.

See also:

• Non-coupled respiration
• Uncoupler titration
• Optimum uncoupler concentration

Abbreviation: u

MitoPedia topics: Uncoupler 

Optimum uncoupler concentration

Uncoupler titrations

A titration of an uncoupler is necessary to achive the optimum concentration necessary for maximum stimulation of non-coupled respiration (ETS capacity) and to avoid inhibition of respiration by the too high uncoupler concentration. The underlying mechanism for the latter is not clear.

Uncouplers must be titrated carefully up to an optimum concentration for maximum stimulation of flux, since excess concentrations of uncoupler exert a strongly inhibitory effect.

Increasing the concentration in small steps, most accurately titrated by the TIP2k, is recommended (0.5 µM steps or even smaller).

The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentratin, 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 estmation of maximum flux or electron transfer system capacity.

The optimum FCCP (or DNP) concentration for the non-coupled state varies over a large concentration range, depending on the medium ('binding' of FCCP), type and concentration of sample. This is also true for other uncouplers, such as DNP (ref. 1). To evaluate the optimum concentration, a FCCP titration has to be performed initially (refs. 1-2). For subsequent routine applications, we recommend a few titrations starting close to optimum concentration (ref. 3). Optimum FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.

Discussion

• Artefacts by single dose uncoupling

Uncoupler titration

In uncoupler titrations various uncouplers, such as FCCP or DNP [MiPNet03.02] are applied to uncouple mitochondrial electron transfer through Complexes I to IV from phosphorylation (Complex V or ATP synthase, ANT and phosphate transport), particularly with the aim to obtain the non-coupled state E with an optimum uncoupler concentration at maximum oxygen flux

• [MiPNet12.05].

Uncouplers may be used not only in isolated mitochondria or permeabilized tissue preparations, but also in intact cells. Uncouplers are permeable through the cell membrane, and intact cells contain energy substrates for mitochondrial respiration. The non-coupled (uncoupler-activated) state may be compared with ROUTINE respiration of the intact cells, in terms of the R/E or ROUTINE control ratio (compare: uncoupler control ratio, UCR). Or the non-coupled state may be the basis for evaluating LEAK respiration in the mitochondrial resting state induced by the addition of oligomycin (inhibitor of ATP synthase) or atractyloside (inhibitor of ANT), obtaing the L/E or LEAK control ratio (compare respiratory control ratio, RCR).

Uncoupling Control Ratio, UCR

There are strong mathematical arguments to replace the conventional UCR and RCR by the inverse ratios [MiPNet08.09].

1/UCR = ROUTINE respiration / Non-coupled respiration = R/E

1/RCR = LEAK respiration / Non-coupled respiration = L/E

When using uncouplers in mitochondrial preparations (mt-preparations: isolated mitochondria and permeabilized tissue or cells), different applications are distinguished:

1. External energy substrates have to be added to the preparation, since the endogenous substrates of the cytoplasm have been removed. A residual amount of internal mitochondrial substrates may be removed, if necessary, by an initial addition of a very small amount of ADP to the mitochondrial medium (e.g. MiR06) containing inorganic phosphate.
2. In mt-preparations, an uncoupler may be added as a methodological test for plasma membrane permeabilization. If the inital addition of ADP does not exert a stimulatory effect, subsequent addition of uncoupler will increase respiratory flux if permeabilization has not been achieved.
3. The classical respiratory control ratio (RCR=State 3/State 4) may be compared with an uncoupler-induced respiratory control ratio. Uncoupler titrations are initiated in a resting state, to induce an activated, non-coupled state. A typical resting state is State 2, in the absence of adenylates (no ADP, ATP or AMP added), or State 4 in isolated mitochondria, in the presence of ATP after phosphorylation of ADP. Titration of uncoupler then stimulates respiration. If State 3 has been initiated by the addition of a saturating concentration of ADP (which is different in isolated mitochondria versus permeabilized tissue or cell preparations), the experiment may be continued by addition of oligomycin or atractyloside, to return to a LEAK state, followed by uncoupler titration.
4. Respiratory flux in the non-coupled state is compared with State 3 (saturating ADP in the coupled state), to evaluate metabolic flux control by the phosphorylation system over the electron transport capacity. Importantly, flux control by the phosphorylation system depends on the combination of substrates and inhibitors applied to activate various segments of the electron transfer system, and varies in different state of cytochrome ''c'' release.

References

1. 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. 271: C2053-C2061. [link]
2. Garedew A, Hütter E, Haffner B, Gradl P, Gradl L, Jansen-Dürr P, Gnaiger E (2005) High-resolution respirometry for the study of mitochondrial function in health and disease. The OROBOROS Oxygraph-2k. Proc. 11th Congress Eur. Shock Soc., Vienna, Austria (Redl H, ed) Medimond, Bologna: 107-111. [pdf]
3. 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: 919-928. [link]
4. Ashley Naimi et al. (2005) Limitation of aerobic metabolism by the phosphorylation system and mitochondrial respiratory capacity of fibroblasts in vivo. The coupled reference state and reinterpretation of the uncoupling control ratio. MiPNet10.09: 55-57. [link]
5. Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286: H1633–H1641. [link]
6. Kuznetsov AV, Strobl D, Ruttmann E, Königsrainer A, Margreiter R, Gnaiger E (2002) Evaluation of mitochondrial respiratory function in small biopsies of liver. Analyt. Biochem. 305: 186-194. [link].

References

• Steinlechner-Maran_1996_AJP: Uncoupler titrations with FCCP and DNP.
• Huetter_2004_BJ: Uncoupler titrations after inhibition of respiration by oligomycin: ROUTINE respiration, LEAK respiration, ETS capacity
• Gnaiger_2008_POS: Uncoupler titrations with the TIP2k (see also O2k-DemoExperiments)
• Pesta 2012 Methods Mol Biol: Review of ETS capacity, E, in intact cells, and P/E flux control ratios in permeabilized muscle fibres.
• Gnaiger 2009 Int J Biochem Cell Biol: Definition of respiratory coupling states in isolated mitochondria and permeabilized cells: L, P, E (see also MitoPathways and Respiratory States)
• O2k-Publications: Coupling; Membrane Potential
• http://www.bmb.leeds.ac.uk/illingworth/oxphos/poisons.htm

MitoPedia methods: Respirometry 

MitoPedia topics: "Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property. Respiratory state"Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property. 

Labels:

Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Coupling; Membrane Potential"Coupling; Membrane Potential" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 

HRR: Method"Method" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.