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Uncoupling of mitochondria Print E-mail

What is “uncoupling”?

Dr. A.A.Starkov

Weill Medical College Cornell University New York NY USA


The concept of uncoupling was empirically derived from experimental observations with isolated mitochondria. It predates the theoretical assessments of mitochondrial energy transduction which therefore had to explain the mechanism of uncoupling. The basis for the uncoupling lies in the fundamental feature of mitochondrial respiration called the “respiratory control”.

The “respiratory control” phenomenon is illustrated by Figure 1(top) which shows a typical trace of the oxygen consumption by mitochondria recorded with an oxygen-sensitive electrode. In the absence of ADP, the respiration of mitochondria is slow (V1) but its rate is abruptly increased after the addition of ADP (V2). The stimulation is temporary; the respiration rate spontaneously declines and remains slow (V3) until another bolus of ADP is added to the mitochondrial suspension. These transitions in the rate of respiration can be repeated several times until the mitochondrial suspension becomes anaerobic ([O2]=0 on Fig.1). The added ADP was consumed simultaneously with the stimulation of respiration and the ATP was accumulated in the suspension (Fig.1(bottom)), thereby indicating that respiration of mitochondria is “coupled” to the oxidative phosphorylation of ADP.

Figure 1.The Respiratory Control and Uncoupling.

As it appeared that adenine nucleotides “control” the rate of respiration of mitochondria, the phenomenon was termed “adenylate control of the respiration” or more general, “respiratory control”. It was found that the amount of oxygen consumed during this accelerated respiration phase was stoichiometrically related to the amount of added ADP and also depended on the nature of the oxidized respiratory substrate. Further, it was found that Ca2+ and some chemicals can compete with ADP for the control of the mitochondrial respiration, by stimulating it in the absence of ADP and increasing the amount of oxygen consumed during the phosphorylation of a given amount of ADP (Fig.1C,D) or even completely preventing the production of ATP, thereby “un-coupling” the phosphorylation of ADP from the respiration of mitochondria.

    Historically, “uncoupling of oxidative phosphorylation” or simply “uncoupling” meant exactly a loss of coupling between the rate of electron transport in the respiratory chain (respiration) and ATP production (phosphorylation). Compounds capable of stimulating oxygen consumption without a concomitant increase in ATP production were summarily termed “uncouplers.

Energy-dissipating pathways.

The chemiosmotic theory of energy transduction in mitochondria considers the “classical uncoupling” as a manifestation of “energy-dissipating pathways” which comprise all mechanisms increasing non-productive energy expenditure in mitochondria. According to the chemiosmotic theory [1-3], free energy released upon the oxidation of substrates in mitochondria is conserved by transforming it into the electrochemical gradient of protons  (ΔμΗ+) across their inner membrane (IM). The ΔμΗ+ drives metabolically “useful” mitochondrial reactions such as ATP synthesis, maintaining the ion homeostasis, or accumulation of metabolites against the gradient of their concentration. These reactions are catalyzed by the IM-embedded proteins which therefore serve as the “reverse transducers”, converting free energy of the ΔμΗ+ into a form other than heat. On the opposite, an energy-dissipating pathway (EDP) uses the ΔμΗ+ to drive a metabolically “futile” reaction producing heat as the only outcome, thereby reducing the amount of energy stored in the ΔμΗ+.

    In line with this concept, “uncoupling” is understood as a decrease in the ΔμΗ+ caused by energy dissipation due to any futile process. 

Are EDPs “bad” for a cell? Not necessarily. Although EDPs reduce the amount of energy available for other reactions utilizing the ΔμΗ+ and decrease the efficiency of mitochondrial energy transduction, they may be metabolically useful to a cell in many cases, exactly because they reduce the ΔμΗ+ or generate heat. A well-known example of the latter is the protein-catalyzed ΔμΗ+ dissipation in brown fat mitochondria which produces large amount of heat for the purpose of thermoregulation. Limiting the production of reactive oxygen species (ROS) in mitochondria may be another important example of a useful EDP.

The chemiosmotic theory explains the uncoupling as a dissipation of ΔμΗ+ caused by the futile cycling of H+ or other ions across the IM of mitochondria. At the molecular level, three types of uncoupling mechanisms may be defined: protein-independent uncoupling, protein-mediated uncoupling, and innate proton leak of the IM which may represent a mix of both mechanisms.

Figure 2. Mechanisms of Uncoupling. Proton shuttling by weak liposoluble acids.

Protein-independent uncoupling.

Protein-independent uncoupling mechanisms directly increase the permeability of the lipid bilayer component of the IM to H+. One very important class of compounds uncoupling mitochondria by this mechanism is represented by weak lipid-soluble acids. Aromatic compounds like carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) increase the H+-permeability of the lipid bilayer by facilitating H+ transport across the hydrophobic barrier.

Their mechanism of action is well established [4] (Figure 2A). Energized mitochondria build up the ΔΨ and ΔpH across the IM so that the matrix compartment becomes more alkaline and more negatively charged than the external medium. Protonated weak lipophilic acids (A-H+) dissolve in the IM and diffuse through the lipid phase into the matrix compartment where they dissociate into the acid anion (A-) and H+, thereby dissipating ΔpH. However, they do not accumulate in the matrix because their anion is also soluble in the lipid phase due to the charge delocalizing over their aromatic ring.

Figure 2. Mechanisms of Uncoupling. Protein-mediated transport of uncoupler anion.

The acid anion is expelled from mitochondria down the gradient of ΔΨ (more positive outside), thereby dissipating the ΔΨ. This futile proton-shuttling cycle is repeated until there is no more ΔΨ or ΔpH across the IM [4]. There are numerous pharmaceuticals capable of dissipating ΔμΗ+ by this mechanism [5].

Although weak lipophilic acids do not require any proteins for their action, their uncoupling activity can be significantly augmented by the IM-embedded proteins [6] (Figure 2B). Even the uncoupling by “classical” protonophores such as FCCP and 2,4-DNP may involve some IM proteins, although the mechanistic details remain obscure (reviewed in [7]).


Protein-mediated uncoupling pathways.


There are several known uncoupling mechanisms involving the IM proteins. First indications that the IM H+ conductance can be controlled by a protein were obtained by David Nicholls who demonstrated that a mitochondrial protein called “thermogenin” (now known as UCP1) is responsible for the thermogenesis in brown fat tissue, and that it induces the heat production by increasing the H+ conductance of the IM of brown fat mitochondria (reviewed in [8, 9].

UCP1-mediated uncoupling is an archetypical protein-mediated EDP. It demonstrates all features inherent to other known proteins catalyzing the ΔμΗ+ dissipation:

1. Uncoupling is caused by a net increase in the H+ conductance of the IM;

2. Various “co-factors” or “activators” are required for the uncoupling activity, which allows for its modulations at the mitochondrial, cellular, and organism levels;

3. Uncoupling is activated by free long-chain fatty acids (FFA);

4. It is inhibited by purine di- and trinucleotides or by the natural substrates of the uncoupling protein.

5. The molecular mechanism of H+conductance catalyzed by such proteins is uncertain.

 All other proteins known or suspected to uncouple mitochondria conform to these rules, except the ATPase. These proteins include ATP/ADP translocator (ANT), aspartate-glutamate carrier (AGC), UCP2 and UCP3 and possibly other UCPs, and unknown proteins comprising the Ca2+-cycling mechanism(s).

Figure 2. Mechanisms of Uncoupling. Protein-mediated proton conductance.

UCP1, UCP2 and UCP3

The UCP1 is expressed only in brown fat mitochondria, but most tissues express several proteins homologous to the UCP1. These proteins were named “uncoupling proteins”, or UCPs, to signify their perceived capacity to uncouple mitochondria. The naming decision was based only on the amino acid sequence homology to UCP1, not on their functions which are still unknown. To date, 5 such UCPs were identified in mammals; similar proteins were found in other animals and plants [10-15].

All UCPs belong to a family of mitochondrial anion carriers (MACs) which are encoded in humans by the nuclear SLC25 genes [16]. For the exception of UCP1, the function of UCPs and their specific inhibitors are not yet known.

It should be stressed that the MAC family includes other well known transporters homologous to UCP1. Some of them, such as ANT and AGC, are known to uncouple mitochondria, but they were not included in the UCP subfamily (see [10, 16] for a list other mitochondrial MACs homologous to UCP1), perhaps because they already had their names.
   The high degree of structural homology of UCPs to that of UCP1 suggested that they may share functional similarity. The UCP1 uncouples brown fat mitochondria by catalyzing an increase in the IM permeability to protons (Fig.2C); therefore an initial solid expectation was that other UCPs would also uncouple mitochondria by increasing the “proton leak” of the IM. However, experimental data do not support this hypothesis [17].

ANT-mediated uncoupling.

 In the presence of exogenous or endogenous FFA, ANT, AGC, and possibly some other mitochondrial anion carriers can induce a significant increase in the IM H+ conductance and uncouple mitochondria. It is thought that ANT and AGC facilitate the transport of FFA anion from mitochondria (Figure 2B), although the molecular mechanism of this activity is not yet well established. The ANT-mediated FFA-induced uncoupling is inhibited by carboxyatractylate, which is its specific inhibitor, and partially by its substrates ADP and ATP, but not by GDP, whereas AGC-mediated uncoupling is inhibited by its substrates glutamate and aspartate (reviewed in [18]). In general, the uncoupling mediated by these proteins conforms to the rules formulated above for the UCP1.

Toxin-induced substrate cycling by ATPase.

   The arsenate-induced ATP synthase-mediated ADP cycling does not follow these rules. Arsenate is transported by the phosphate carrier into the mitochondrial matrix where it readily substitutes for the phosphate in the ATP synthase-catalyzed phosphorylation of ADP to ATP. However, the arsenate-ADP bond is hydrolyzed by water, so the ADP is regenerated and again enters the ATPase reaction, thereby turning it into a futile cycle [19]. ATP synthase–mediated uncoupling and possible other toxin-induced substrate cycling pathways may be of pathophysiological significance as the mechanisms of toxicity of such compounds.


   The latter operates by catalyzing a futile cycle of Ca
2+ uptake trough a concerted action of a ΔμΗ+-driven Ca2+ uniporter (Figure 2D, “U”) and an electroneutral Ca2+-release pathway (Figure 2D, “E”), resulting in the net increase of H+ backflow and ΔμΗ+ dissipation. In vitro, Ca2+-recycling can be stimulated by FFA, ROS, pro-oxidants and other factors and suppressed by ADP or ATP and some other specific inhibitors. The molecular identity of the proteins involved in Ca2+-recycling is not known.

Figure 2. Mechanisms of Uncoupling. Futile ion-cycling

This mechanism (reviewed in [20]) is thought to play a significant role in excitotoxic cell death and the damage induced by ischemia and reperfusion [20-22]. Eventually, Ca2+-cycling may result in a more permanent uncoupling and damage of mitochondria due to the opening of a pore in the IM, called the “permeability transition pore” (PTP, also known as “mitochondrial permeability transition” (MPT), or “mitochondrial permeability transition pore” (mPTP)) which is also constructed of unknown proteins (reviewed in [20, 23, 24]).

The “proton leak”.

The IM of mitochondria possesses significant innate H+ conductivity which was termed “proton leak”. This phenomenon was first described by Nicholls [25] who demonstrated a unique and intriguing feature of proton leak, the non-ohmic dependence of H+ conductance of the IM on the ΔμΗ+. The H+ conductance of the IM linearly depended on the ΔμΗ+ up to ~180 mV whereas it progressively increased at high, >200 mV values of ΔμΗ+. It appeared that proton leak was limiting the maximum value of ΔμΗ+ at about 230 mV [25].

The molecular mechanism of proton leak remains to be established [17, 26]. Mitochondrial IM is composed of proteins embedded into a lipid membrane; it was estimated that lipids occupy about 50% of the total IM area [27]. The intrinsic IM H+ conductance may be associated with either membrane lipids or proteins, or both.

It is unlikely that the H+ conductance of the membrane lipids represents a significant portion of the total proton leak in the mitochondrial IM. In earlier studies, the H+ conductance of the membrane of protein-free liposomes was compared to the proton leak of the mitochondrial IM. It was found that the H+ conductance was up to 20-times lower than the proton leak in the IM (reviewed in [28]). More recent study estimated the membrane H+ conductance of detergent-free liposomes prepared from the lipids extracted from the liver mitochondrial IM and compared it to the magnitude of proton leak in liver mitochondria [29]. The membrane H+ conductance of liposomes was found to be ~30 fold lower than that of IM, when expressed per mg of phospholipid. In line with earlier studies, authors estimated that H+ conductance through the membrane of the IM can not exceed 5% of the total proton leak [29]. Another study estimated the membrane H+ conductance of liposomes made from the liver IM lipids extracted from 8 different animals. The H+ conductance was the same in all these liposome preparations, in spite of 10-times difference in the IM proton leak between the parent mitochondria from which the lipids were isolated [30]. The most plausible hypothesis would therefore be that some protein component of the IM is responsible for up to 95% of proton leak [17].

Figure 2. Mechanisms of Uncoupling. Proton conductance along a protein/lipid interface.

ANT role in proton leak

What could be that protein component? The answer to this question remains to be found. Recently, ANT has been attracting much interest as potential candidates involved in controlling the IM proton leak [31]. Brand et al. observed that the proton leak was elevated in mitochondria from skeletal muscle tissue rich in the ANT1 isoform of ANT but it was significantly lower in muscle mitochondria isolated from genetically modified ANT1-ablated mice. The H+ conductance also correlated with the amount of expressed ANT in mitochondria from the Drosophila fly. Notably that H+ conductance was independent from ANT activity or FFA-induced ANT-catalyzed uncoupling because it was elevated even in the presence

of ANT inhibitor carboxyatractylate, which blocks both the natural ADP/ATP exchange activity and the FFA-induced uncoupling by ANT. Authors concluded that ANT may be responsible for the 50-75% of all observed proton leak [31]. They further suggested that other MACs can also contribute to the proton leak but their contribution should be much less than that of ANT because of their lower abundance: while the content of ANT is up to the 10% of total mitochondrial protein, that of pyruvate carrier is ~0.3%, UCP2 ~0.01%, and UCP3 is 0.01% (cf. [31]). The pathway of H+ conductance may occur at the protein-lipid interface (Fig.2E) and therefore it requires not the physiological activity of the embedded protein but its presence in the IM [31].

Overall, this is a very plausible hypothesis, although it does not yet explain the mechanism of non-ohmic ΔμH+ dependence of proton leak. In humans, ANT1 is expressed mainly in heart and skeletal muscle, ANT2 is ubiquitous, and ANT3 is abundant in highly proliferative tissues but very weekly expressed in all other tissues (cf [16]). In mice, ANT1 is abundant in muscle and heart and somewhat less in brain and lung mitochondria but absent in liver mitochondria, whereas ANT2 is ubiquitously present in these tissues [32]. The ubiquitous distribution and the abundance of ANT make it a very good candidate on the role of the protein catalyzing the ubiquitous proton leak in the mitochondrial IM. Apparently, any other MAC that is as abundant as ANT may also be expected to contribute to the proton leak.


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