Matrix magnesium and the permeability of heart mitochondria to potassium ion

Isolated beef heart mitochondria were treated with A23187 in the presence of different concentrations of Mg2+ or EDTA to establish varying levels of total mitochondrial Mg2+. The Mg2+ content was related to the rate of passive swelling of the mitochondria in potassium acetate and other potassium salts in which swelling is presumed to depend on K+ entry via an endogenous K+/H+ antiport. Swelling in these salts does not commence until Mg2+ has been depleted from an initial value of 36 nmol X mg-1 of protein to 8 nmol/mg-1, or less. Below this level, swelling increases linearly with decreasing Mg2+ to a maximum rate at 2 nmol of Mg2+ X mg-1. Rotenone-treated heart mitochondria suspended in 75 mM potassium acetate at pH 7.80 show no delta pH by 5,5-dimethyl-2,4-oxazolidinedione distribution. Distribution of methylamine also shows essentially no delta pH under these conditions when allowance is made for binding of [14C]methylamine by mitochondrial membranes under these conditions. Addition of A23187 results in a small and transient delta pH (delta pH less than 0.14, acid interior) as measured by methylamine distribution. Estimation of the maximum matrix free Mg2+ concentration from the maximum delta pH observed and the external free Mg2+ concentration at equilibrium with A23187 shows that swelling is not initiated until matrix free Mg2+ is decreased to below 150 microM. An independent estimate of free Mg2+ using a null-point procedure gives a lower, but quite similar value (50 microM) for maximum matrix free Mg2+ when swelling commences. The large depletion of total and free Mg2+ that is required to activate swelling in potassium acetate (and presumably K+/H+ antiport activity) does not appear to be compatible with previous indications that free Mg2+ acts as a "carrier brake" to regulate K+ extrusion from the mitochondrion on such an antiport (Garlid, K. D. (1980) J. Biol. Chem. 255, 11273-11279). The removal of a tightly bound component of mitochondrial Mg2+ is closely related to increased K+ permeability and increased passive swelling in potassium salts. This Mg2+ appears to play a role in the maintenance of mitochondrial membrane structure and integrity.     

Bax and heart mitochondria: uncoupling and inhibition of respiration without permeability transition

The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA).It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).     

The uncoupling protein and the potassium channel are activated by hyperosmotic stress in mitochondria from durum wheat seedlings

The plant uncoupling mitochondrial protein (PUMP) and the plant mitochondrial potassium channel (PmitoK_ATP) are two recently discovered energy‐dissipating systems present in plant mitochondria, which may play a role as defence systems under environmental stress. To verify whether hyperosmotic stress affects the two dissipating systems in durum wheat (Triticum durum Desf.), their functioning was studied in early etiolated seedlings maintained under moderate and severe salt (NaCl) and osmotic (mannitol) stress. As measures of mitochondrial stress mitochondrial integrity, membrane potential maintenance and oxygen uptake coupled with ATP synthesis during succinate and proline oxidation were investigated. Both PUMP and PmitoK_ATP were activated under stress conditions. Activation was clearly evident even under moderate stress when proline oxidation was inhibited, although mitochondrial integrity and succinate oxidation were still unaffected. Under severe stress, which significantly affected all the tested indicators of mitochondrial integrity and functionality, PUMP and PmitoK_ATP activation was further enhanced. Interestingly, both systems were activated by reactive oxygen species and were able to control mitochondrial superoxide anion production. These results suggest that PUMP and PmitoK_ATP serve as early antioxidant defence systems in response to hyperosmotic stress and that they are involved in a prolonged response to stress.     

Sodium inhibits permeability transition by decreasing potassium matrix content in rat kidney mitochondria

Inner membrane mitochondria undergo a permeability increase elicited after the opening of a nonspecific pore due to supraphysiological matrix Ca²⁺ load, and the presence of an inducer. Multiple inducers have been used to promote the transition in permeability; among them are carboxyatractyloside (CAT) and reactive oxygen-derived species. In contrast, inhibitors such as ADP and cyclosporin A have been commonly used. In this work, we show that the opening or closure of the nonspecific pore depends on the cationic composition of the incubation medium. It was found that when mitochondria were incubated in either 125 mM KCl or 125 mM LiCl, ADP was essential to maintain selective membrane permeability. Interestingly, the nucleotide was not required when the medium contained 125 mM NaCl. Furthermore, it was established that CAT promotes membrane leakage in K⁺- or Li⁺-incubated mitochondria, while it failed to do so in Na⁺-incubated mitochondria. Evidence is also presented on the ability of Na⁺ to induce resistance in mitochondria against membrane damage by oxidative stress. Mitochondrial Ca²⁺ discharge, swelling, and transmembrane electric gradient were analyzed to establish permeability transition. It is concluded that the protection provided by Na⁺ was accomplished by inducing matrix K⁺ depletion, which, in turn, diminished the free fraction of matrix Ca²⁺.     

Interaction of free fatty acids with mitochondria: coupling, uncoupling and permeability transition

Long chain free fatty acids (FFA) exert, according to their actual concentration, different effects on the energy conserving system of mitochondria. Sub-micromolar concentrations of arachidonic acid (AA) rescue DeltapH-dependent depression of the proton pumping activity of the bc1 complex. This effect appears to be due to a direct interaction of AA with the proton-input mouth of the pump. At micromolar concentrations FFA increase the proton conductance of the inner membrane acting as protonophores. FFA can act as natural uncouplers, causing a mild uncoupling, which prevents reactive oxygen species production in the respiratory resting state. When Ca(2+)-loaded mitochondria are exposed to micromolar concentrations of FFA, the permeability of the inner membrane increases, resulting in matrix swelling, rupture of the outer membrane and release of intermembrane pro-apoptotic proteins. The characteristics of AA-induced swelling appear markedly different in mitochondria isolated from heart or liver. While in the latter it presents the canonical features of the classical permeability transition (PT), in heart mitochondria substantial differences are observed concerning CsA sensitivity, DeltaPsi dependence, reversibility by BSA and specificity for the activating divalent cation. In heart mitochondria, the AA-dependent increase of the inner membrane permeability is affected by ANT ligands such as adenine nucleotides and atractyloside. AA apparently causes a Ca2+-mediated conversion of ANT from a translocator to a channel system. Upon diamide treatment of heart mitochondria, the Ca2+/AA-induced CsA insensitive channel is converted into the classical PT pore. The relevance of these observations in terms of tissue-specific components of the putative PTP and heart ischemic and post-ischemic process is discussed.     

Mitochondrial uncoupling agents. Effects on membrane permeability of molluscan neurons.

Agents which uncouple oxidative phosphorylation in mitochondria were applied to identified neurons in an isolated ganglion of the marine mollusc Navanax inermis. Aromatic monocarboxylic acids, acetanilides, benzamides, benzaldehydes and phenols all caused a rapid, reversible, dose-dependent increase in the membrane potential and conductance of the neurons tested. These events were due primarily to an increase in the membrane's conductance to potassium, relative to chloride. All active compounds also produced a reversible, dose-dependent decrease in the permeability of alkali-cations relative to potassium. The relative activity of congeners in each group of substances was directly correlated with the octanol-water partition coefficients of the various compounds, indicating that hydrophobicity was important in determining drug effect and suggesting that steric requirements were minimal. The results suggest that the observed changes in membrane electrical properties and cation selectivity are due to an increase in the membrane's anionic field strength caused by the hydrophobic interaction of anionic and nonionic agents with the neuronal membrane.     

Energy conservation and uncoupling in mitochondria

Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity-labeling uncoupler 2-azido-4-nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of M_r = 30,000 ± 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin- and uncoupler-sensitive ATP-Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane-impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler-binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane-permeable uncoupler is compared with a membrane-impermeable one.     

Chloride-dependent uncoupling mediated by oligomycin in rat liver mitochondria

In rat liver mitochondria suspended in KCl medium and containing a low concentration of a K(+)-specific cationophore (valinomycin or Triton X-100), oligomycin was shown to induce uncoupling of oxidative phosphorylation, stimulation of adenosine triphosphatase activity, release of the respiratory control, decrease of energy-dependent changes in the fluorescence of the dye 8-anilino-1-naphthalenesulphonic acid and rapid swelling of mitochondria. Oligomycin caused none of the above effects when Br(-) or NO(3) (-) was substituted for Cl(-) as the major anionic species or when Na(+) replaced the K(+). The same concentration of oligomycin that caused uncoupling and swelling slightly improved energy-conserving reactions when the cationophores were omitted. In the presence of KSCN, valinomycin or Triton X-100 by itself caused uncoupling and swelling which was not further enhanced by oligomycin. On the basis of the above results it is suggested that the energy dissipation resulting from the concerted action of the cationophores and oligomycin is connected with the simultaneous transport of K(+) and its counter ion and that oligomycin plays its role in the uncoupling by facilitating the permeation of Cl(-) through the cristae membrane of the mitochondria.     

Proton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration.     

MFN2 retrotranslocation boosts mitophagy by uncoupling mitochondria from the ER

Mitochondrial damage triggers mitochondrial quality control pathways, which act to ensure the health of the mitochondrial network. The turnover of damaged mitochondria by mitophagy is initiated by the Parkinson disease-linked genes PRKN and PINK1, and we recently investigated the role that interorganellar contact sites between the endoplasmic reticulum (ER) and the outer mitochondrial membrane (OMM) play in this pathway. In this punctum, we summarize our findings that show that the ER-OMM tether MFN2 acts as a suppressor of mitophagy through its ability to link the OMM to the ER, potentially limiting the accessibility of other ubiquitination substrates to PINK1 and PRKN. PINK1, PRKN and the AAA-ATPase VCP disrupt contact between mitochondria and the ER via MFN2 ubiquitination, retrotranslocation and turnover from the mitochondrial membrane. Our study provides insight into the role of OMM remodeling in mitophagy.     

Basolateral potassium membrane permeability of A6 cells and cell volume regulation

The K⁺ permeabilities (⁸⁶Rb(K) transport) of the basolateral membranes (J_bK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation.Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca _i (max <1 min) and cell swelling (max at 3–5 min) followed by a regulatory volume decrease (5–30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the ⁸⁶Rb(K) transport and the SCC were partially blocked by Ba²⁺, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3–6 min) stimulation of the ⁸⁶Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca²⁺-free media or quinidine addition did not affect the initial osmotic stimulation of J_bK but prevented its secondary regulation, whereas TEA, glibenclamide and DIDS completely blocked the initial J_bK increase. Under hypo-osmotic conditions, the initial J_bK increase was enhanced by the presence of 1 mm of barium and delayed with higher concentrations (5 mm). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media.We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K⁺ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K⁺ permeability mediated by the observed calcium rise.This work was supported by grants from the Commissariat à l'Energie Atomique and the Centre National de Recherche Scientifique URA 638.     

The action of valinomycin in uncoupling corn mitochondria

Valinomycin in the presence of potassium is a potent uncoupler of corn (Zea mays L.) mitochondria, eliminating respiratory control. Valinomycin produces higher steady state potassium phosphate swelling which can be reversed to give active shrinkage if mersalyl is added to block the Pi⁻/OH⁻ antiporter. Respiration declines concurrently. Uncouplers accelerate the shrinkage and restore the respiration. The same results can be obtained with sodium phosphate if gramicidin D is substituted as ionophore.