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.     

Mathematical modeling of mitochondrial adenine nucleotide translocase

We have developed a mathematical model of adenine nucleotide translocase (ANT) function on the basis of the structural and kinetic properties of the transporter. The model takes into account the effect of membrane potential, pH, and magnesium concentration on ATP and ADP exchange velocity. The parameters of the model have been estimated from experimental data. A satisfactory model should take into account the influence of the electric potential difference on both ternary complex formation and translocation processes. To describe the dependence of translocation constants on electric potential we have supposed that ANT molecules carry charged groups. These groups are shifted during the translocation. Using the model we have evaluated the translocator efficiency and predicted the behavior of ANT under physiological conditions.     

The flavonoid quercetin induces changes in mitochondrial permeability by inhibiting adenine nucleotide translocase

This study shows the effects of the flavonoid quercetin on diverse mitochondrial functions, among them membrane permeability. Our findings indicate that the addition of 50 μM quercetin did not produce reactive oxygen derived species; however, it inhibited the oxidative stress induced after the addition of Fe²/H₂O₂ by about 38%. At this concentration, quercetin also promoted a fast calcium release, inhibited oxidative phosphorylation, stimulated oxygen consumption, and decreased membrane potential. In addition 50 μM quercetin inhibited the adenine nucleotide translocase (ANT) by 46%. These effects induced the opening of the permeability transition pore and release of cytochrome c, by its interaction with a component of the non-specific pore complex, fixed to the carrier in the conformation c, as carboxyatractyloside does. Quercetin-induced permeability transition pore opening was inhibited by 0.5 μM cyclosporin A, but, interestingly, the release of cytochrome c was not inhibited by the immunosuppressor, as quercetin was found to disrupt the outer membrane.     

Molecular dynamics simulations of creatine kinase and adenine nucleotide translocase in mitochondrial membrane patch

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies.     

Evidence against direct transfer of the adenine nucleotides by the heart mitochondrial creatine kinase-adenine nucleotide translocase complex

Adenine nucleotide translocase inhibitors, atractyloside and carboxyatractyloside, added to respiration inhibited rat heart mitochondria had no significant effects on the apparent Km's for ATP and ADP and Vmax's of the creatine kinase reaction. The results suggest that there is no sequential, mandatory, direct transfer of the nucleotides between the translocase and the creatine kinase active site. The reaction was run in both the forward and reverse directions in media containing either 0.25 M sucrose or 0.13 M KCl. The apparent Km's, however, were found to be 2–3 times higher in the KCl medium than in sucrose, suggesting the use of the more physiological medium for meaningful kinetic studies.     

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.     

Salicylic acid induces the proton conductance in the inner mitochondrial membrane of lupine cotyledons

The influence of salicylic acid (SA) on generation of membrane potential (Δψ) at the inner membrane of isolated mitochondria from cotyledons of lupine seedlings (Lupinus angustifolius L.) was investigated. The mitochondrial preparations conformed to all criteria of the intactness: the organelles were characterized by the integrity of their membranes and by tight coupling of oxidation and phosphorylation. High functional activity of mitochondria was also evident from their ability to generate Δψ during succinate oxidation and from the long-term maintenance of steady-state transmembrane potential by virtue of electrontransport chain (ETC) operation or ATP hydrolysis after the inhibition of respiratory ETC. The addition of SA to the incubation medium (0.5–1.0 mM) induced a fast and complete dissipation of Δψ after a distinct lag period. The Δψ was not restored by subsequent ATP hydrolysis, indicating that the phytohormone SA induced the proton conductance of the inner membrane. The SA-induced collapse of Δψ was observed under suppression of ETC by anaerobiosis, cyanide, or inhibitory concentrations of the phytohormone. The SAinduced dissipation of Δψ was not reversed by cyclosporine A but was prevented in the presence of dithiothreitol (DTT). Conversely, the incubation of mitochondria in the presence of phenylarsine oxide (PAO) known to oxidize the protein thiol groups also elevated the proton conductance and eliminated Δψ at the inner membrane of lupine mitochondria. The PAO-induced Δψ collapse was not reversed in the presence of ATP, but Δψ was restored after the addition of DTT. These results and the literature data suggest that, under suppressed ETC activity, salicylic acid permeabilizes the inner membrane of mitochondria from cotyledons of lupine seedlings due to opening of a specialized mitochondrial uncoupling channel (MUC) that is permeable to protons and, possibly, to other small cations (K⁺, Ca²⁺). An important role in the induction of MUC belongs apparently to oxidative stress resulting in oxidation of thiol groups in protein molecules that constitute this channel or regulate the channel activity.     

Developmental changes in the adenine nucleotide translocase in the guinea pig

Investigations of developmental changes in energy metabolism in guinea pig liver mitochondria showed that mitochondria from the newborn were well coupled, with respiratory control ratios and membrane energy potentials similar to those obtained with mitochondria from the 1-day-old and the adult. In contrast, there was a 3-fold increase in the rate of mitochondrial respiration and a 2-fold increase in adenine nucleotide content during the first 24 h of extrauterine life. There was no significant change in the ATP/ADP ratio and only a 30% increase in the uncoupled rate of respiration during this same time period. Titrations of the adenine nucleotide translocase with the specific inhibitor, carboxyatractyloside, showed that the newborn had only 50% of the adenine nucleotide translocase activity of the adult. Furthermore, by applying flux control theory to these inhibitor titrations, it was possible to demonstrate that the adenine nucleotide translocase exerted greater control over respiration in the newborn than in the adult, and at maximal rates of coupled respiration the translocase had a control strength of 0.98. The consequences of this finding on cellular energy metabolism are discussed in relation to adaptation of the newborn to extrauterine life.     

Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity

The effect of agaric acid as inducer of mitochondrial permeability transition was studied. It was found that: (i) agaric acid (AA) promoted efflux of accumulated Ca2+, collapse of transmembrane potential, and mitochondrial swelling; (ii) these effects depend on membrane fluidity; (iii) ADP inhibited the effect of AA on Ca2+ efflux, and (iv) AA blocked binding of the sulfhydryl reagent, eosin-5-maleimide, to the adenine nucleotide translocase. It is proposed that AA induces pore opening through binding of the citrate moiety to the ADP/ATP carrier; this interaction must be stabilized by insertion of the alkyl chain in the lipid milieu of the membrane.     

Protein translocase of mitochondrial inner membrane in Trypanosoma brucei

Translocases of mitochondrial inner membrane (TIMs) are multiprotein complexes. The only Tim component so far characterized in kinetoplastid parasites such as Trypanosoma brucei is Tim17 (TbTim17), which is essential for cell survival and mitochondrial protein import. Here, we report that TbTim17 is present in a protein complex of about 1,100 kDa, which is much larger than the TIM complexes found in fungi and mammals. Depletion of TbTim17 in T. brucei impairs the mitochondrial import of cytochrome oxidase subunit IV, an N-terminal signal-containing protein. Pretreatment of isolated mitoplasts with the anti-TbTim17 antibody inhibited import of cytochrome oxidase subunit IV, indicating a direct involvement of the TbTim17 in the import process. Purification of the TbTim17-containing protein complex from the mitochondrial membrane of T. brucei by tandem affinity chromatography revealed that TbTim17 associates with seven unique as well as a few known T. brucei mitochondrial proteins. Depletion of three of these novel proteins, i.e. TbTim47, TbTim54, and TbTim62, significantly decreased mitochondrial protein import in vitro. In vivo targeting of a newly synthesized mitochondrial matrix protein, MRP2, was also inhibited due to depletion of TbTim17, TbTim54, and TbTim62. Co-precipitation analysis confirmed the interaction of TbTim54 and TbTim62 with TbTim17 in vivo. Overall, our data reveal that TbTim17, the single homolog of Tim17/22/23 family proteins, is present in a unique TIM complex consisting of novel proteins in T. brucei and is critical for mitochondrial protein import.     

Dependence of mitochondrial oxidative phosphorylation on activity of the adenine nucleotide translocase

The coupled reactions of electron transport and ATP synthesis for the first two sites of mitochondrial oxidative phosphorylation have been previously reported to be near equilibrium in isolated respiring pigeon heart (Erecińska, M., Veech, R. L., and Wilson, D. F. (1974) Arch. Biochem. Biophys. 160, 412-421) and rat liver mitochondria (Forman, N. G., and Wilson, D. F. (1982) J. Biol. Chem. 257, 12908-12915). Measurements are presented in this paper which demonstrate that the same relationship exists for both forward and reverse electron transport in rat heart mitochondria. This conclusion implies that adenine nucleotide translocation, a partial reaction of the system, is also near equilibrium, contrasting with proposals that the translocase is rate-limiting for oxidative phosphorylation. To resolve this controversy, the respiratory rates of suspensions of isolated rat liver and rat heart mitochondria were controlled by varying either the added [ATP]/[ADP][Pi] ratios ratios or [ADP] (by varying hexokinase in a regenerating system). Titrations with carboxyatractyloside, a high affinity inhibitor of the translocase which is noncompetitive with ADP, were carried out to assess the dependence of the respiratory rate on translocase activity. Plots of respiratory rate versus [carboxyatractyloside] were all strongly sigmoidal. In liver mitochondria, 40%-70% and in heart mitochondria 66% of the sites could be blocked with carboxyatractyloside before a 10% decrease in the respiratory rate was observed. Further analysis showed that liver and heart mitochondria have translocase/cytochrome a ratios of 1.52 and 3.20, respectively, and that at 23 degrees C the maximal turnover numbers for the translocases were 65 s-1 and 23 s-1. In all states of controlled respiration (no added inhibitor), a substantial excess of translocase activity was present, suggesting that the translocase was not normally rate-limiting in oxidative phosphorylation.     

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator

Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death. Permeabilization of the outer mitochondrial membrane (OM) and/or inner membrane (IM) is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, adenine nucleotide translocator (ANT, in the IM) and voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family. Here we discuss that ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca(2+), atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP. In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death. Cell Death and Differentiation (2000) 7, 1146 - 1154     

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.     

Nature of endogenous proton conductance of the inner mitochondrial membrane. Role of Ca2+ transport system in proton transfer

In order to elucidate the nature of endogenous proton conductance of rat liver inner mitochondrial membrane, the dependence of the rate of Ca2+ transport on pH was studied. It was found that the inhibiting effect of H+ is independent of protonation of functional groups of hypothetical Ca2+ carrier, but results from electrogenic transfer of H+ across the membrane, which is highly permeable for the proton. The adsorption of H+ by mitochondria is inhibited by ruthenium red and other specific inhibitors of Ca2+ transport. It is concluded that endogenous proton conductance of the inner mitochondrial membrane depends on the functioning of the same transport system essential for membrane permeability for Ca2+ and other bivalent cations. The correlation observed between the rates of H+ and Ca2+ transport in mitochondria and the ratio of cation mobilities in aqueous solutions is in favour of a "porous" mechanism of cation transport across the mitochondrial membrane.     

Analysis of functional coupling: mitochondrial creatine kinase and adenine nucleotide translocase

The mechanism of functional coupling between mitochondrial creatine kinase (MiCK) and adenine nucleotide translocase (ANT) in isolated heart mitochondria is analyzed. Two alternative mechanisms are studied: 1), dynamic compartmentation of ATP and ADP, which assumes the differences in concentrations of the substrates between intermembrane space and surrounding solution due to some diffusion restriction and 2), direct transfer of the substrates between MiCK and ANT. The mathematical models based on these possible mechanisms were composed and simulation results were compared with the available experimental data. The first model, based on a dynamic compartmentation mechanism, was not sufficient to reproduce the measured values of apparent dissociation constants of MiCK reaction coupled to oxidative phosphorylation. The second model, which assumes the direct transfer of substrates between MiCK and ANT, is shown to be in good agreement with experiments—i.e., the second model reproduced the measured constants and the estimated ADP flux, entering mitochondria after the MiCK reaction. This model is thermodynamically consistent, utilizing the free energy profiles of reactions. The analysis revealed the minimal changes in the free energy profile of the MiCK-ANT interaction required to reproduce the experimental data. A possible free energy profile of the coupled MiCK-ANT system is presented.     

Interaction of mammalian mitochondrial ribosomes with the inner membrane

All of the products of mitochondrial protein biosynthesis in animals are hydrophobic proteins that are localized in the inner membrane. Hence, it is possible that the synthesis of these proteins could occur on ribosomes associated with the inner membrane. To examine this possibility, inner membrane and matrix fractions of bovine mitochondria were examined for the presence of ribosomes using probes for the rRNAs. Between 40 and 50% of the ribosomes were found to fractionate with the inner membrane. About half of the ribosomes associated with the inner membrane could be released by high salt treatment, indicating that they interact with the membrane largely through electrostatic forces. No release of the ribosome was observed upon treatment with puromycin, suggesting that the association observed is not due to insertion of a nascent polypeptide chain into the membrane. A fraction of the ribosomes remained with residual portions of the membranes that cannot be solubilized in the presence of Triton X-100. These ribosomes may be associated with large oligomeric complexes in the membrane.     

Assessment of membrane-bound mammal mitochondrial adenine nucleotide translocase topography by experimental antibodies

To gain insight into the immunogenicity of mitochondrial adenine nucleotide translocase (ANT), we raised antibodies against purified bovine heart ANT by induction of ascitic fluid in male Balb/c mice. We identified the antigenic determinants detected by these antibodies by (1) immunodetection of GST-ANT fusion proteins and selected partial constructs of ANT, (2) immunodetection of chemically synthesized overlapping peptides on solid support, and (3) back-titration ELISA. Results revealed a short epitope spreading of the antibodies, resulting in a small number of antigenic determinants. Thus, each antibody detects one or two major epitopes located in the putative hydrophilic loops M2 and M3. No evidence for the antigenicity of the first 133 amino acids of ANT was obtained. These well-characterized antibodies were used to study the topography of the membrane-bound ANT by back-titration ELISA with mitochondrial membranes. We demonstrated that amino acids 145-150 and 230-237 are fully accessible to the antibodies in native ANT, whereas regions 133-140 and 244-251 are not. Furthermore, we used mitochondria devoid of the outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) to establish the matrix or cytosolic orientation of loops M2 and M3. The results clearly show that these loops have a matrix orientation and thus support the six transmembrane segment model of ANT topography in the inner mitochondrial membrane.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.