Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Energization-dependent endogenous activation of proton conductance in skeletal muscle mitochondria

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.     

Dexamethasone treatment specifically increases the basal proton conductance of rat liver mitochondria

We investigated the role that mitochondrial proton leak may play in the glucocorticoid-induced hypermetabolic state. Sprague-Dawley rats were injected with dexamethasone over a period of 5 days. Liver mitochondria and gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from dexamethasone-treated, pair-fed and control rats. Respiration and membrane potential were measured simultaneously using electrodes sensitive to oxygen and to the potential-dependent probe triphenylmethylphosphonium, respectively. Five days of dexamethasone injection resulted in a marked increase in the basal proton conductance of liver mitochondria, but not in the muscle mitochondrial populations. This effect would have a modest impact on energy expenditure in rats.     

Fatty acids as acute regulators of the proton conductance of hamster brown-fat mitochondria

Possible mechanisms are evaluated for the acute regulation of the hamster brown-fat mitochondrial proton-conductance pathway which is active during non-shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non-thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl-CoA and acylcarnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl-CoA potentiates the conductance increase. An effect of acyl-CoA upon chloride permeability is not specific to brown-fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl-CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown-fat-specific 32000-Mr protein is discussed.     

Influence of bovine serum albumine on the proton conductance of potato mitochondria membranes

Pulsed acid base titrations, according to the procedure of Mitchell and Moyle, have been carried out on potato mitochondria in the presence and absence of Bovine Serum Albumine (BSA). The rate of the pH decay is slower when BSA is present. The buffering capacities of the outer and inner phases, the t1/2 of the pH decay after an acid pulse and the proton conductance of the inner membrane have been measured. The results show that plant mitochondria are relatively impermeable to H⁺ and OH^–, but leakier than animal mitochondria. This may be related to the lower respiratory control ratios generally found with plant mitochondria.Abbreviations EGTA ethylene glycol bis (aminoethyl ether) NN tetraacetic acid - MERCAP sodium mercaptobenzothiazole - TRIS tris (hydroxymethyl) aminomethane - MOPS morpholinopropane sulfonic acid - BSA bovine serum albumine - RC respiratory control ratio     

Intrinsic uncoupling of mitochondrial proton pumps. 1. Non-ohmic conductance cannot account for the nonlinear dependence of static head respiration on delta microH

The passive membrane conductance LH1 of rat liver mitochondria has been measured and compared with the quantity nJesh/delta microHsh (n = H+/e stoichiometry; Jesh = rate of electron transfer in static head) over a delta microH range. The two curves approach each other only in the lower part of the range, while they sharply diverge at large values of delta microH. Thus nJesh/delta microHsh cannot be considered to be a measure of LH1 in the upper delta microH region. Only a fraction of the static head electron flow is accounted for by futile proton cycling via leaks. Contaminating open membrane fragments or completely leaky mitochondria can be responsible for only a small part of the residual rate of oxygen consumption. We conclude that a large part of static head respiration must have yet another cause and propose it to be intrinsic uncoupling of the respiratory chain enzymes.     

Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

The kinetics of proton transport through mammalian UCP1 (uncoupling protein 1) expressed in yeast mitochondria were measured. There was little or no UCP1 activity in the absence of added palmitate, but significant activity in its presence. The activator 4-HNE (4-hydroxy-2-nonenal) had little effect when added alone, but significantly enhanced proton conductance in the presence of added palmitate. Activation of the proton conductance of UCP1 was synergistic: proton conductance in the presence of both palmitate and 4-HNE was significantly greater than the sum of the individual effects. Mitochondria from control yeast transformed with empty vector showed no such synergy, showing that synergy is a property of UCP1. Activation by the 4-HNE analogue trans-cinnamate showed essentially the same characteristics as activation by 4-HNE. Mitochondria from brown adipose tissue also showed synergistic activation of GDP-sensitive proton conductance by palmitate and 4-HNE. These results show that reactive alkenals activate the proton conductance of UCP1 more strongly when fatty acids are also added, with implications for both mechanistic and physiological models of UCP1 activation.     

The nature of controlled respiration and its relationship to protonmotive force and proton conductance in blowfly flight-muscle mitochondria

1. To determine whether controlled (State 4) pyruvate oxidation can support a high energy state, measurements of the redox span NAD-cytochrome c, phosphorylation potential and protonmotive force (the gradient in electrochemical activity of protons across the mitochondrial inner membrane) were made as indices of energy status. For comparison, these three measurements were also made with glycerol 3-phosphate, an alternative substrate. The two substrates gave essentially identical values for the redox span NAD-cytochrome c in State 4, and the phosphorylation potential was of sufficient magnitude to be considered in equilibrium with the redox span over the first two phosphorylation sites. The magnitude of the protonmotive force in State 4 was much less and the implications of this finding are discussed. 2. Measurements made during the controlled (State 4) to active (State 3) transition indicated that with glycerol 3-phosphate as substrate, both the redox span NAD-cytochrome c and the protonmotive force were diminished; the State 4 --> State 3 transition with pyruvate as substrate was accompanied by an increase in the redox span but a decrease in protonmotive force. The contrary behaviour of these two energetic parameters in the presence of pyruvate was ascribed to a transient excess in the flux of protons through the adenosine triphosphatase relative to the protonpumping respiratory chain, in spite of the increased dehydrogenase activity. 3. The lower protonmotive force seen in State 3 relative to State 4 with pyruvate as substrate was due to a diminution of both the electrical (DeltaPsi) and the chemical (DeltapH) components; with glycerol 3-phosphate, the magnitude of the decrease in protonmotive force during the State 4 --> State 3 transition was similar to that seen with pyruvate, but was due to a large decrease in the electrical component (DeltaPsi) and a small rise in the chemical component (DeltapH). The reason for the difference seen in the behaviour of the components of the protonmotive force was investigated but not established. 4. In the presence of oligomycin and ADP, oxidation of pyruvate, but not of glycerol 3-phosphate, supported a greater protonmotive force than in State 4, in keeping with the dehydrogenase activation and increased redox span NAD-cytochrome c found under these conditions. 5. Experiments involving the use of uncoupling agent to stimulate respiration are compared with those in which limiting concentrations of ADP were used. Estimates of the proton conductance of the inner membrane indicate a similar non-linear dependence on uncoupler concentration with the two substrates. 6. A model is proposed as an explanation of the high rates of controlled glycerol 3-phosphate oxidation. The model relies on a high permeability of the inner membrane to protons and other ions being induced by glycerol 3-phosphate oxidation in State 4.     

The regulation of the proton conductance of brown fat mitochondria. Identification of functional and non-functional nucleotide-binding sites

The binding of purine nucleotides to intact brown fat mitochondria is re-examined. In addition to the previously reported high affinity binding site, a low-affinity site is found, which requires several minutes to saturate. Only the high affinity site is functional in regulating the proton and halide permeabilities of the mitochondria. The low affinity site can introduce errors in the use of nucleotide binding to quantitate the Mr 32000 uncoupling protein unique to these mitochondria.     

The effective proton conductance of the inner membrane of mitochondria from brown adipose tissue. Dependency on proton electrochemical potential gradient.

The nucleotide-sensitive H+ (OH-) conducting pathway of mitochondria from the brown-adipose tissue of cold-adapted guinea-pigs passes an effective proton current which is directly proportional to the proton electrochemical gradient. At 23 degrees C and pH 7.0 this conductance is 16 nmol H+ - min-1 - mg-1 - mV-1. Addition of 0.2 mM GDP results in a conductance which is linear and low (0.7 nmol H+ - min-1 - mg-1 - mV-1) until deltamicronH+ exceeds 220 mV. At higher values of deltamicronH+, which can be attained by glycerol 3-phosphate oxidation but not palmitoyl-L-carnitine plus malate oxidation, the membrane conductance greatly increases, effectively limiting the maximal deltamicronH+ to 240 mV. High glycerol 3-phosphate concentrations which have the thermodynamic potential to exceed this value of deltamicronH+ instead create a greatly increased rate of controlled respiration. The generality and significance of this device to limit deltamicronH+, and its relation to the nucleotide-sensitive conductance, are discussed.     

Voltage-dependent proton fluxes in liposomes

Liposomes containing buffered KCl were prepared from bacterial lipids, were diluted into K+-free media and were treated with valinomycin to induce the formation of a diffusion potential (delta psi). Upon formation of such a potential, substantial proton influx was observed, as assayed by the quenching of 9-aminoacridine fluorescence. Complete reversal of fluorescence quenching occurred when the potential was collapsed by addition of KCl or when methylamine was added. Studies of proton influx as a function of the theoretical magnitude of the delta psi indicated that the phenomenon occurred only above a delta psi of about -60 mV. Establishment of a Na+ diffusion potential also resulted in proton influx. Treatment of K+-loaded liposomes with N,N'-dicyclohexylcarbodiimide did not reduce the delta psi-dependent proton influx. Moreover, proton influx could be demonstrated upon imposition of a diffusion potential in liposomes prepared from a synthetic lipid. The proton fluxes associated with generation of a diffusion potential in liposomes may complicate studies of reconstituted systems in which proton translocation should occur, and may affect the magnitude of the electrochemical proton gradient that is operant under some conditions.     

GDP and carboxyatractylate inhibit 4-hydroxynonenal-activated proton conductance to differing degrees in mitochondria from skeletal muscle and heart

The lipid peroxidation product 4-hydroxynonenal (HNE) increases the proton conductance of the inner mitochondrial membrane through effects on uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT); however, the relative contribution of the two carriers to these effects is unclear. To clarify this we isolated mitochondria from skeletal muscle and heart of wild-type and Ucp3 knockout (Ucp3KO) mice. To increase UCP3 expression, some mice were i.p. injected with LPS (12 mg/kg body weight). In spite of the increased UCP3 expression levels, basal proton conductance did not change. HNE increased the proton conductance of skeletal muscle and heart mitochondria. In skeletal muscle, this increase was lower in Ucp3KO mice and higher in LPS-treated wild-type mice, and was partially abolished by GDP (UCPs inhibitor) and completely abolished by carboxyatractylate (ANT inhibitor) or addition of both inhibitors. GDP had no effect on HNE-induced conductance in heart mitochondria, but carboxyatractylate or administration of both inhibitors had a partial effect. GDP-mediated inhibition of HNE-activated proton conductance in skeletal muscle mitochondria was not observed in Ucp3KO mice, indicating that GDP is specific for UCP3, at least in muscle. Carboxyatractylate was able to inhibit UCP3, probably through an indirect mechanism. Our results are consistent with the conclusion that, in skeletal muscle, HNE-induced increase in proton conductance is mediated by UCP3 (30%) and ANT, whereas in the heart the increase is mediated by ANT and other carriers, possibly including UCP3.     

An estimation of the proton conductance of the inner membrane of turnip (Brassica napus L.) mitochondria

The permeability of the inner membrane of turnip mitochondria to H⁺ and OH^- ions has been investigated using an acid pulse technique. The rate of decay of a H⁺ pulse across the inner membrane is exponential having first-order kinetics and gives t 1/2 values of approx 54 s at neutral pH and at 25° C. Valinomycin or 1799 alone have little effect on t 1/2 values, whereas in combination, values of <15 s are observed. Nigericin produces a similar effect. The effective proton conductance of the inner membrane near pH 7 at 25° C is 0.27 nmol H⁺ min^-1 mg protein^-1 mV^-1. The results suggest that at neutral pH, the inner membrane of plant mitochondria is relatively impermeable to H⁺ and OH^- ions.     

Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus)

We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak.     

The current-voltage relationships of liposomes and mitochondria.

Current-voltage relationships were determined for various membrane systems. We show that phospholipid and mitochondrial membranes exhibit linear relations between H+ flux and pH gradients. These membranes, however, exhibited non-linear relationships when the applied voltage was a membrane potential. The current-voltage relationship approximated to an exponential function. This relationship was found to be linearized when the membranes were treated with an electrogenic proton ionophore. The incorporation of cytochrome c oxidase (EC 1.9.3.1) was found to have no effect on the current-voltage characteristics of the phospholipid membranes. When a membrane potential of more than 140 mV was imposed across vesicular and mitochondrial membranes, they exhibited reversible di-electric breakdown. This phenomenon was correlated with the requirement of a permeant ion for the experimental demonstration of proton translocation by so-called 'proton pumps'.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.     

The acute regulation of mitochondrial proton conductance in cells and mitochondria from the brown fat of cold-adapted and warm-adapted guinea pigs

Cells and mitochondria were prepared from the brown adipose tissue of adult guinea-pigs adapted to either 4-7 degrees C or 22-25 degrees C. The cold-adapted cells displayed noradrenaline-stimulated, propranolol-sensitive respiration, but noradrenaline failed to increase the respiration of the warm-adapted cells. Purine-nucleotide-sensitive proton conductance was greater in cold-adapted mitochondria than in warm-adapted controls. At the same time cold-adapted mitochondria were extremely sensitive to the uncoupling effect of endogenous and infused fatty acids, and resembled the mitochondria from the brown adipose tissue of cold-adapted hamsters. Warm-adapted mitochondria were ninefold less sensitive, and resembled liver mitochondria. With cold-adapted, but not warm-adapted mitochondria, respiration increased proportionately to the rate of fatty acid infusion. It is concluded that the presence of the 32000-Mr proton conductance pathway is necessary for the expression of a high sensitivity to fatty acid uncoupling, suggesting that the fatty acids interact directly with this protein to modulate the proton conductance during the acute regulation of thermogenesis.