Aging Cell (2004) 3, pp3540

Doi: 10.1111/j.1474-9728.2003.00079.x

Blackwell Publishing, Ltd.

REVIEW

Mitochondrial membrane potential and aging

David G. Nicholls
Buck Institute for Age Research, Novato, California, USA

Summary
The mitochondrial membrane potential (or protonmotive force) is the central bioenergetic parameter that controls respiratory rate, ATP synthesis and the generation of reactive oxygen species, and is itself controlled by electron transport and proton leaks. As a consequence of extensive research, there has emerged a consensus as to how these parameters integrate. Despite this consensus, the literature contains contradictory reports on the extent to which these parameters are modified in animal models of aging. This article critically examines the basis for a number of these reports. Key words: membrane potential; mitochondrion; oxidative stress; protonmotive force; superoxide; uncoupling.

Introduction
The mitochondrion sits at the centre of the web of bioenergetic interactions that control much of cell function. Three fundamental 2+ functions, ATP generation, Ca uptake and storage, and the generation and detoxification of reactive oxygen species (ROS), are driven by the mitochondrial membrane potential, m, while many apoptotic signals initiate apoptosis by disturbing mitochondrial function sufficiently to cause cytochrome c release. These central roles, together with the gradual decline with age in muscle function and the indication of increasing oxidative damage to the organelle, underlie the considerable interest in the concept that there is an important mitochondrial component to the aging process.

increases in response. An actual relationship between p and respiration when proton conductance is varied is shown in Fig. 1(A, trace i) for one particular mitochondrial preparation, that from brown adipose tissue (Nicholls, 1974a, 1977). It is notable that no further increase in respiration is seen when p drops below about 180 mV, when respiration becomes essentially uncontrolled and is limited by respiratory chain capacity. Most mitochondria show quantitatively similar relationships as proton conductance is titrated either by increasing ATP turnover or by graded addition of protonophores. What this means is that classic respiratory control, the ability of respiration to be controlled by ATP demand, is not operative below this value. As reviewed elsewhere (Nicholls & Ward, 2000) there are many pitfalls in the determination of mitochondrial membrane potential in intact cells, and if there is an apparent conflict of data between results obtained by monitoring mitochondrial membrane potential with fluorescent probes and monitoring respiratory parameters in an oxygen electrode, the latter is less liable to error and should generally be given priority. However, respiratory control is a hybrid concept, depending on the extent of the native proton leak responsible for state 4 respiration and the maximal capacity of the respiratory chain or ATP synthase. Respiratory control ratios (RCR) should not be used as the sole basis to describe subtle age-related differences in mitochondrial bioenergetics.

Protonmotive force and ATP synthesis

Recent structural information on the membrane-spanning mitochondrial F0 component of the ATP synthase has indicated that the proton-driven motor responsible for rotating the central stalk of the catalytic F1 subunit possesses 10 subunits, suggesting that 10 protons are utilized for each complete rotation of the stalk (Stock et al., 1999). Because three ATP molecules are synthesized per rotation (Stock et al., 2000), this is consistent + with an H /ATP stoichiometry of 3.3. It has long been known that the import of ADP and phosphate, Pi, together with the export of ATP utilizes a further proton owing to the electrogenic nature of the adenine nucleotide translocator (Pfaff et al., 1969); thus 4.3 protons are required for the synthesis and export to the cytoplasm of each molecule of ATP. The importance of this value is that it allows us to calculate the maximum ATP/ADP ratio that can be maintained by mitochondria at any given value of the protonmotive force, p:

Protonmotive force and respiration rate

The only link between the respiratory chain and the ATP synthase is the proton circuit. When respiratory chain activity is in excess, the majority of the control over the rate of respiration is exerted by the conductance of the pathways for proton reentry into the matrix, in particular the ATP synthase, in parallel with any trans-membrane proton leaks. As this conductance increases, the protonmotive force p falls and respiration

Correspondence Dr D. G. Nicholls, Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA. Tel.: +1 415 209 2095; fax: +1 415 209 2232; e-mail: dnicholls@buckinstitute.org Accepted for publication 26 November 2003 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003

Gp,cytoplasm = 4.3Fp
where Gp,cytoplasm is the Gibbs free energy for the reaction ADP + P i = ATP, F is the Faraday constant and p is the protonmotive force. Without delving into the somewhat
35

36 Mitochondrial membrane potentials, D. G. Nicholls

determined, and most studies report the dominant component of p, i.e. the membrane potential, , which generally changes in parallel with p. What this implies is that the phosphorylation state of the ATP pool in the cytoplasm is extremely sensitive to relatively small changes in membrane potential or p. Figure 1(C) shows actual data from rat liver mitochondria showing the change in the maximal extra-mitochondrial ATP/ ADP ratios that can be generated and maintained as a function of p when the latter is varied over a relatively small range. Concerning reports of low potentials in mitochondria from aged rats discussed above (e.g. Hagen et al., 1997), if confirmed these would imply that the maximal ATP/ADP ratio that could be maintained in the cell by these mitochondria would be only 5 about 10 of that in the young animal. It is difficult to see how such cells could survive, and allow survival of the liver of the aging rat. It should be noted in passing that the maximal ATP/ADP ratio that can be maintained by mitochondria decreases sharply as ATP demand increases, owing both to the drop in p associated with the increased respiration (Fig. 1A, trace i) and to the increasing disequilibrium of the ATP synthase and adenine nucleotide translocator as their rates increase to generate and export the increased ATP. Any age-related deterioration in the kinetics of either process would impact upon the rate at which ATP could be supplied at a usable thermodynamic potential.

Protonmotive force and proton leaks

State 4 respiration exists because all mitochondria have some endogenous proton leak. This endogenous leak was originally described for liver and brown adipose tissue mitochondria (Nicholls, 1974a,b) and has subsequently been extensively investigated by Brand and co-workers (Brand et al., 1994, 1999). The current/voltage relationship of mitochondrial proton leaks can be investigated by taking mitochondria in state 4 and progressively limiting the supply of electrons to the respiratory chain, typically by varying substrate concentration or by increasing concentrations of a dehydrogenase inhibitor such as malonate (Fig. 1A, trace ii). A feature that was originally noted (Nicholls, 1974a,b) is that the endogenous proton leak displays non-ohmic characteristics, i.e. the proton conductance is voltage-dependent, such that the proton current decreases disproportionately with p. We have often emphasized the utility of comparing the mitochondrial proton circuit with an equivalent electrical circuit, and in electronics there is a component, the zener diode, that behaves in a closely similar way to the non-ohmic leak, only conducting electrons when the potential exceeds a critical threshold value. Indeed, it is simple to design an elementary electrical circuit that reproduces some of the key features of the mitochondrial proton circuit (Fig. 2). Zener diodes are employed to provide a dissipative short-circuit to control voltage, and a similar role was proposed for the endogenous proton leak (Nicholls, 1974b). Most studies into the non-ohmic proton leak employ complex II substrates such as succinate or, in the case of brown adipose
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Fig. 1 Experimental data relating mitochondrial respiration, protonmotive force, ROS production and ATP synthesis. (A) Experimental relationships between respiration and protonmotive force for brown fat mitochondria that are either exposed to an increasing demand on the proton current (by titration with uncoupler, closed symbols) or whose substrate supply is progressively restricted (by lowering substrate concentration, open symbols). Data from (Nicholls, 1974a,b, 1977). The initial condition (arrowed) is for mitochondria respiring in state 4 in the presence of 20 mM -glycerophosphate; the respiration is due to the operation of the endogenous non-ohmic proton leak (UCP1 is inactive in these experiments). (B) Data from Korshunov et al. (1997) replotted, showing the dependency of mitochondrial hydrogen peroxide generation upon the magnitude of the mitochondrial membrane potential (normalized to the state 4 protonmotive force to allow comparison with A). (C) Experimentally determined ATP/ADP ratios maintained in the extramitochondrial medium by liver mitochondria as a function of protonmotive force.

complex mathematics (Nicholls & Ferguson, 2002), this equation tells us that, at constant Pi, the maximum ATP/ADP ratio that can be maintained by mitochondria decreases by 10-fold for every 14 mV decrease in p. The full protonmotive force is rarely

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Protonmotive force and oxidative stress

It may seem energetically wasteful to dissipate a significant proportion of state 4 respiration to no apparent use. However, as detailed by Brand et al. (1999), the proton leak may play an important role in basal thermogenesis and in the present context may play a critical role in controlling the generation of ROS. Electrons can leak singly from the respiratory chain to molecular oxygen from 1-electron sites within complexes I and III. That in complex III, the outer UQ binding site, is better characterized, but in both complexes the dwell-time of the electron at the site, and hence the probability of leakage to form superoxide, is increased at high membrane potential. This potential-dependent ROS production is shown in Fig. 1(B), where data from Korshunov et al. (1997) are re-plotted. If the endogenous proton leak were absent, then linear extrapolation of the relationship between respiration and p (trace i) to zero respiration would predict a static head p in excess of 250 mV. The shape of the equivalent extrapolation of the rate of ROS production to this potential is unclear, but even a linear extrapolation would result in a dramatic increase in superoxide generation in resting mitochondria. The ability of proton leaks to control p and hence oxidative stress has been developed by Skulachev (1996) and is discussed later in this issue. However, it is appropriate here to discuss the kinetic consequences of an endogenous or imposed proton leak. Taking the conditions for the mitochondria in Fig. 1, roughly 40% of the respiratory capacity is used in state 4 by the proton leak; however, owing to the non-ohmic nature of the leak, as soon as ATP is utilized and p decreases, the leak decreases to a very low rate, allowing 90% of the respiratory capacity to be utilized for ATP production in state 3 when p in this example is about 180 mV. In contrast an ohmic leak, such as is induced by the protonophore FCCP (Nicholls, 1974a), would continue to waste a high proportion of the proton current even though p in state 3 is decreased to levels at which ROS production is very low. In other words, the non-ohmic leak seems to have evolved to limit p and hence ROS production in state 4, when by definition the mitochondria are not required to generate ATP, but shuts down in the transition to state 3 where the proton current is required for the ATP synthase. This auto-regulation would seem to be more than fortuitous. Figure 1 also indicates the fallacy in the live fast and die young concept that rapidly respiring mitochondria generate more ROS. This was apparently founded on the idea that dioxygen can either be reduced safely by four electrons to form water, or by one electron to form superoxide, and that the more rapidly oxygen is reduced the greater would be the chance of superoxide formation. However, the sites of 4- and 1-electron reduction of dioxygen in the respiratory chain are entirely distinct and even in the moderately hypoxic milieu of the cell cytoplasm, oxygen is in comfortable excess; it is thus the electrons that make the choice of reducing dioxygen in quartets or singly. The higher the respiration rate, the lower p and and the less time the electrons are delayed at the critical sites where leakage is possible. Thus fast-respiring mitochondria

Fig. 2 Simple electrical analogue of the mitochondrial proton circuit. The mitochondrial proton circuit can be considered to be closely analogous to an equivalent electrical circuit where the respiratory chain is replaced by an electrical battery and the variable demand on the proton circuit by the ATP synthase is imitated by the variable resistance Rv. This equivalent circuit is designed to illustrate the main features of the proton circuit. The protonmotive force across the mitochondrial membrane falls slightly as the rate of ATP synthesis (and hence the proton current) is increased because the rate at which the protons can be pumped out of the mitochondrion by the complexes is to a first approximation a linear function of the difference between the Gibbs free energy available from the downhill electron transfer through the complex and that needed for the uphill translocation of protons across the membrane. This is modelled by the inclusion of an internal resistance associated with the battery. As the mitochondrion goes from state 4, when Rv is infinite, to state 3, when the proton current is maximal, the voltage (V) in the model falls from 1.3 to 1.0 V. An important component of the model is a zener diode, which passes essentially zero current until a critical voltage threshold is reached, when it becomes highly conducting, effectively limiting the voltage in this example to 1.3 V and preventing the full dangerously high 1.5 V from being developed. A zener diode is a voltage-limiting device and here models the endogenous non-ohmic proton short circuit found in all mitochondria. The price of this voltage-limiting device is a continuous resting current demand, corresponding to mitochondrial state 4 respiration. When the ATP synthase becomes active and the voltage drops below the 1.3 V threshold, the zener diode no longer conducts current and all the output of the battery flows through the ATP synthase.

tissue, -glycerophosphate. The reason for this is that complex I becomes kinetically limited at a lower p than complexes II IV (Nicholls, 1977), so that complex I substrates generate a slightly lower p that is less able to reach the threshold for activation of the non-ohmic leak. This is also of relevance to oxidative stress, as will be discussed below.
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38 Mitochondrial membrane potentials, D. G. Nicholls

operating close to state 3 generate fewer ROS than those respiring slowly close to state 4 (Fig. 1). It must be emphasized that although the data used for Fig. 1(A) (traces i and ii) were obtained using mitochondria from brown adipose tissue possessing large amounts of the prototypic uncoupling protein UCP1, conditions were such that UCP1 remained inactive (purine nucleotides present and fatty acids absent; Nicholls, 1974b). The non-ohmic leak, which is present in all mitochondria, is not associated with any of the established or putative UCPs (Stuart et al., 2001), indeed its molecular nature remains elusive. It must again be emphasized that complex II substrates can readily generate a supra-physiological p (Nicholls, 1977; Hansford et al., 1997) and that rates of superoxide production reported in the presence of these substrates may exaggerate rates occurring in the cell.

Changes in protonmotive force or membrane potential in aging

As detailed by Rottenberg & Wu (1998) (see also Nicholls & Ward, 2000), there are many pitfalls to the determination of m by flow cytometry using fluorescent cationic probes, the most important of which is to ensure that probe loading is insufficient to allow probe aggregation and quenching in the matrix. If this precaution is not taken, the signal becomes insensitive to changes in m while remaining sensitive to changes in plasma membrane potential, p. In an associated study, Rottenberg & Wu (1997) monitored m in lymphocytes from young and old mice and observed more heterogeneity of potential in the old preparation. Interestingly, m in both preparations was enhanced by the mitochondrial permeability pore and calcineurin inhibitor cyclosporin A, with the effect being slightly larger for the old animals. This may reflect a somewhat greater susceptibility to the permeability transition after isolation of the lymphocytes. Loading of hepatocytes with 10 g/mL rhodamine 123 for 30 min (Hagen et al., 1998a, 1999, 2002) would almost certainly cause sufficient probe loading for matrix quenching, such that the reported decrease in signal would represent a decrease in p and not mitochondrial depolarization. Secondly, when precautions are taken to avoid excess loading, the fluorescence signal is an exponential function of the sum of the plasma and mitochondrial membrane potentials (Ward et al., 2000). Thus a 50% decrease in signal, for example that reported by Hagen et al. (2002) for hepatocyte mitochondrial m, would represent only an 18 mV depolarization (and probably of plasma membrane potential, as discussed above) rather than a 50% decline. Determination of state 4 and state 3 respiration is perhaps the least ambiguous means of monitoring mitochondrial function (proton leak, respiratory chain capacity, maintenance of a sufficient for respiratory control). It is therefore difficult to reconcile reports (Hagen et al ., 1997, 1998b, 1999) that major populations of mitochondria in hepatocytes from old rats maintain values of p up to 85 mV below that of cells from young rats while at the same time the isolated mitochondria

displayed high state 3/state 4 RCRs of 45 compared with 9 for the young animals. Cavazzoni et al. (1999) reported extremely low values of m for hepatocytes from both adult and old rats by rhodamine 123 flow cytometry (73 and 86 mV, respectively) although there was insufficient information to judge how they arrived at these values. Interestingly, however, they reported an enormous 25-fold increase in peroxide formation as assayed by dichlorofluorescin oxidation, with no significant change in levels of superoxide (Cavazzoni et al., 1999). High probe loading, and hence quench mode, may be used to monitor changes in m occurring during the period of observation, in which case a mitochondrial depolarization produces an increase in whole-cell signal as probe redistributes from the quenched matrix to the cytoplasm where it fluoresces. Xiong et al. (2002) have used this mode to investigate mitochondrial membrane potential in acutely isolated cerebellar slices from young and old mice. They observed a slightly smaller increase in fluorescence when the old slices were treated with 1 M FCCP (81% compared with 67% for young slices), which would translate into a 5 mV decrease in m if all other factors were constant. However, it is equally possible that cell composition (e.g. the ratio of neurons to glia) and/or mitochondrial density varied in the two preparations, making a quantitative interpretation difficult. With appropriate precautions, rhodamine 123, tetramethylrhodamine methyl ester (TMRM) and related probes may be used to obtain valid information on changes in m in cells. However, other probes such as chloromethyl-tetramethylrosamine (CMTMR, Mitotracker Orange) and chloromethyl-Xrosamine (Mitotracker Red) bind covalently to mitochondria and titrate thiol groups and are invalid as dynamic probes of m (Ferlini et al., 1998; Gilmore & Wilson, 1999; Scorrano et al., 1999). This must be born in mind when assessing reports of aging-related changes in m using these probes (Sugrue et al., 1999). The use of a tetraphenylphosphonium electrode to monitor changes in (Kamo et al., 1979) avoids many of the problems associated with fluorescent probes, although only population studies can be made. Kokoszka et al. (2001) monitored in mouse liver mitochondria preparations and reported a 10 mV decrease in potential in the preparation from old mice relative to young and middle-aged animals. If the old animals possessed an increased proton leak it would be predicted that the partially depolarized preparation would display a faster state 4 respiration, owing to a decreased restraint upon electron transfer (Fig. 1), but state 4 respiration was drastically decreased by almost 50%, whereas there was a lesser effect on state 3 respiration. Citrate synthase activity also declined in the old preparation (Kokoszka et al., 2001). This enzyme is often considered a control for other activities, and the possibility thus arises that the preparation from old mice contained an increased proportion of damaged organelles. Using the distribution of labelled tetraphenylphosphonium, Hagen et al. (1997) reported a dramatic mitochondrial depolarization in subfractions of intact
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Mitochondrial membrane potentials, D. G. Nicholls 39

hepatocytes from old rats. Although mitochondria in 10% of cells maintained the same m (154 mV) as young animals, 65% were depolarized by 60 mV and 25% by more than 80 mV. As discussed above, if these values accurately reflect in vivo values, it is difficult to see how the liver could remain functional in the aging rat.

Conclusions
Much of the subtlety of mitochondrial bioenergetics, both with the isolated organelle and with intact cells, remains to be exploited in studies aimed at revealing age-related changes in function. Highly sophisticated integrated studies of mitochondrial bioenergetics can be performed using metabolic control analysis (Hafner et al., 1990) in which the consequences of slight perturbations in the activity of one bioenergetic parameter for the network of linked parameters can be quantified. As discussed in the article by Merry in this issue (Merry, 2003) it is essential that this state-of-the-art approach is applied to establish true differences in mitochondrial bioenergetics in aging models. It should be possible to link respiratory chain complex activity, ROS generation, glutathione redox status, membrane potential, proton leak and ATP demand in an integrated network and to resolve some of the current confusion that is based in part upon inappropriate methodologies.

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