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. > MiPNet article

Abbreviation: U

MitoPedia topics: Uncoupler 

Gnaiger E (2014) Is respiration uncoupled - noncoupled - dyscoupled? Mitochondr Physiol Network 2014-04-18.

OROBOROS (2014) Mitochondr Physiol Network

Abstract: Coupling of OXPHOS represents a complex concept. Uncoupler titrations provide an invaluable experimental tool.

• O2k-Network Lab: AT Innsbruck Gnaiger E

Labels:

Regulation: Coupling efficiency;uncoupling  Coupling state: LEAK, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Theory 

Is respiration uncoupled - noncoupled - dyscoupled?

or loosely coupled?

Uncoupled respiration

The uncoupled part of respiration in State P pumps protons to compensate for intrinsic uncoupling, which is a property of (a) the inner mt-membrane (proton leak), (b) the proton pumps (proton slip; decoupling), and (c) is regulated by molecular uncouplers (uncoupling protein, UCP1). Uncoupled and dyscoupled respiration are summarized as LEAK respiration. In contrast, noncoupled respiration is induced experimentally for evaluation of ETS capacity.

• Gnaiger 2012 MitoPathways, MiPNet10.04

Uncoupled respiration - intrinsic

Uncoupling is used for intrinsic (physiological) uncoupling, appreciating the fact that we do not (never??) find mitochondria to be fully (mechanistically) coupled. In the ROUTINE (intact cells) and OXPHOS (mt-preparations) state of respiration, mitochondria are both, partially coupled and partially uncoupled. The uncoupled part of respiration in state P is larger than LEAK respiration evaluated in state L after inhibition of ATP synthase or adenine nucleotide translocase. This is due to the increase of mt-membrane potential in state L versus P, causing a corresponding increase of the proton leak driven by the higher proton motive force. As an approximation, however, the difference E-L yields an estimate of the physiological scope of uncoupling, or the pathological scope of dyscoupling.

Uncoupled respiration - experimental

Uncoupling is also used for directed experimental interventions to lower the degree of coupling, typically by application of established uncouplers (experimental use of a pharmacological intervention), less typical by freeze-thawing or mechanical crashing of mitochondrial membranes. Such experimental uncoupling can induce stimulation or inhibition of respiration.

Noncoupled respiration

Noncoupled respiration is distinguished from general (pharmacological or mechanical) uncoupled respiration, to give a label to an effort to reach the fully uncoupled (non-coupled) state without inhibiting respiration. Non-coupled respiration, therefore, yields an estimate of ETS capacity. Experimentally uncoupled respiration may fail to yield an estimate of ETS capacity, due to inhibition of respiration above optimum uncoupler concentrations or insufficient stimulation by sub-optimal uncoupler concentrations. Optimum uncoupler concentrations for evaluation of (non-coupled) ETS capacity require inhibitor titrations (Steinlechner-Maran_1996_AJP; Huetter_2004_BJ; Gnaiger_2008_POS).

Dyscoupled respiration

Dyscoupled respiration is distinguished from intrinsically (physiologically) uncoupled and from extrinsic experimentally uncoupled respiration as an indication of extrinsic uncoupling (pathological, toxicological, pharmacological by agents that are not specifically applied to induce uncoupling, but are tested for their potential dyscoupling effect). Dyscoupling indicates a mitochondrial dysfunction.

Continue the discussion

Experimental

Optimum uncoupler concentration

A titration of an uncoupler is necessary to achive the optimum concentration necessary for maximum stimulation of noncoupled 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 uncoupler (CCCP, FCCP, DNP) concentration for the non-coupled 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 (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.

Uncoupler titration

In uncoupler titrations various uncouplers, such as CCCP, 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.

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 noncoupled (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 acceptor 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 / Noncoupled respiration = R/E

1/RCR = LEAK respiration / Noncoupled 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, noncoupled state. In the absence of adenylates (no ADP, ATP or AMP added), or in State 4 of isolated mitochondria (in the presence of ATP after phosphorylation of ADP), titration of uncoupler stimulates respiration. If OXPHOS 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 noncoupled state is compared with OXPHOS (saturating ADP in the coupled state), to evaluate metabolic flux control by the phosphorylation system over the electron transfer 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 states of cytochrome ''c'' release.

Discussion

• Artefacts by single dose uncoupling

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 Cell Physiol 271: C2053-C2061. - Uncoupler titrations with FCCP and DNP.
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. - Uncoupler titrations after inhibition of respiration by oligomycin: ROUTINE respiration, LEAK respiration, ETS capacity
4. 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.
5. 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.

• 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
• O2k-Publications: Coupling; Membrane Potential
• http://www.bmb.leeds.ac.uk/illingworth/oxphos/poisons.htm
• Gnaiger E. Biochemical coupling efficiency: from 0 to <1. Mitochondr Physiol Network. >> Biochemical coupling efficiency