Inhibition of the mitochondrial inner membrane anion channel by dicyclohexylcarbodiimide. Evidence for a specific transport pathway

Electrophoretic uniport of anions through the inner mitochondrial membrane can be activated by alkaline pH or by depleting the matrix of divalent cations. It has also been suggested that, in the presence of valinomycin and potassium, respiration can also activate anion uniport. We have proposed that a single pathway is responsible for all three of these transport processes (Garlid, K. D., and Beavis, A. D. (1986) Biochim. Biophys. Acta 853, 187-204). We now present evidence that like the "pH-dependent" pore the divalent cation-regulated pore and the "respiration-induced" pore are blocked by N,N'-dicyclohexylcarbodiimide (DCCD). Moreover, the kinetics of inhibition of the latter two pathways are identical and exhibit a second order rate constant of 2.6 X 10(-3) (nmol DCCD/mg)-1.min-1. DCCD inhibits the uniport of Cl-, phosphate, malate, and other lipophobic anions completely, but it has no effect on the classical electroneutral phosphate and dicarboxylate carriers. In Mg2+-depleted mitochondria DCCD partially inhibits the transport of SCN-; however, in Mg2+-containing mitochondria and at low pH, no inhibition is observed. Furthermore, in DCCD-treated mitochondria, even following depletion of Mg2+, the transport of SCN- is independent of pH. These results lead us to conclude that two pathways for anion uniport exist: a specific, regulated pathway which can conduct a wide variety of anions and a nonregulated pathway through the lipid bilayer which only conducts lipid-soluble ions.     

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.     

Regulation of the monovalent cation permeability of brain mitochondria

The Na+ and K+ conductances of rat brain mitochondria were estimated from rates of metabolically dependent swelling and uncoupling of respiration. These were maximal in the presence of EDTA plus Pi. Pi could not be replaced with acetate. Na+ conductance was greater than that of K+ and was therefore examined in greater detail. According to the influences of N-ethylmaleimide, internal Pi (exogenous and perhaps endogenous) promoted Na+ permeability. Treatment with the ionophore A23187 obviated the Pi requirement although EDTA was still necessary. The stimulation by EDTA with Pi or A23187 and inhibition by exogenous Mg2+ suggested endogenous polyvalent cations could also regulate Na+ conductance. The influence of these substances upon endogenous Mg2+ (and Ca2+) levels is consistent with such a role of membrane-bound Mg2+. Low levels of ruthenium red (150 pmol/mg) inhibit Na+ permeation, indicating that the number of 'sites' or 'channels' involved may be small. The Ca2+ uniport is not directly involved in Na+ flow according to its greater sensitivity to inhibition by ruthenium red.     

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons

The inner membrane of mitochondria possesses a pH-regulated anion uniporter which is activated by depletion of matrix divalent cations with A23187 (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). It is now shown that Cl- transport through this pathway is inhibited by Mg2+ and Ca2+. There appear to be two sites for inhibition by Mg2+. One has an IC50 = 38 microM at pH 7.4 and appears to be on the inside since it is only observed in the presence of A23187 (10 nmol/mg). The other has an IC50 = 440 microM at pH 7.4 and appears to be on the outside since it is observed in mitochondria pretreated with very low doses of A23187 (0.25 nmol/mg or less) and in A23187-pretreated mitochondria washed to remove A23187. Ca2+ is found to inhibit anion uniport in the presence or absence of A23187 with an IC50 of about 17 microM. In contrast to these findings Cl- uniport, activated by addition of valinomycin to respiring mitochondria without depleting endogenous Mg2+ is found to be very insensitive to exogenous Mg2+, being inhibited with an IC50 of 3.2 mM. This is explained by examination of the pH dependence of the Mg2+ IC50 in non-respiring mitochondria. The internal IC50 is found to be pH-dependent, rising to about 250 microM at pH 8.4. The external IC50 is also pH-dependent, rising to 2.5 mM or above at pH 8.4. These data are consistent with a model in which Mg2+ can only bind to the protein when it is protonated at a site with a pK of about 6.8 located in the matrix. Thus, both the intrinsic activity of the uniporter and its inhibition by Mg2+ appear to be regulated by matrix protons. This makes the rate of anion uniport much more sensitive to changes in matrix pH which is physiologically advantageous for its proposed role in volume homeostasis.     

Induction of system A amino acid transport through long-term treatment with ouabain: correlation with increased (Na+/K+)-ATPase activity

Mouse embryo fibroblast cells (C3H-10T1/2) and the methylcholanthrene-transformed derivative (MCA-10T1/2) were treated with basal modified Eagle's medium (BME) containing 10% fetal bovine serum and varying concentrations of ouabain ranging from 0.05 mM to 0.7 mM for 16 h in culture. After replacing the ouabain-containing medium with Earl's balanced salts solution, System A amino acid transport activity increased from approximately 40 to 500 pmol AIB accumulated.mg protein-1.min-1 in the C3H-10T1/2 cells and from approximately 300 to 700 pmol AIB accumulated.mg protein-1.min-1 in the MCA-10T1/2 cells. The (Na+/K+)-ATPase pump activity also increased from approximately 12 to 46 nmol Rb+ accumulated.mg protein-1.min-1 in the normal cells and from approximately 20 to 42 nmol Rb+ accumulated.mg protein-1.min-1 in the transformed cells. System A and the (Na+/K+)ATPase activity were maximally increased at approximately 0.4-0.6 mM ouabain in the normal cells in contrast to the transformed cells which were maximally stimulated at a concentration of approximately 0.2 mM ouabain. This treatment with ouabain increased the [Na+]i/[K+]i as measured by atomic absorption spectroscopy, and thereby decreased the Na+ and K+ electrochemical gradients. Our data show that the internal ion gradients inverted at a lower concentration of ouabain in the transformed cells compared to the normal cells. The ouabain-induced increase in pump and System A activity shown here was used as a tool to further investigate the coordinated ion transport regulation in the control of cell growth.     

Kinetics of nucleotide transport in rat heart mitochondria studied by a rapid filtration technique

A rapid filtration technique has been used to measure at room temperature the kinetics of ADP and ATP transport in rat heart mitochondria in the millisecond time range. Transport was stopped by cessation of the nucleotide supply, without the use of a transport inhibitor, thus avoiding any quenching delay. The mitochondria were preincubated for 30 s either in isotonic KCl containing succinate, MgCl2, and Pi (medium P) or in isotonic KCl supplemented only with EDTA and Tris (medium K); they were referred to as energized and resting mitochondria, respectively. The kinetics of [14C]ADP transport in energized mitochondria were apparently monophasic. The plateau value for [14C]ADP uptake reached 4-5 nmol of nucleotide.(mg of protein)-1. Vmax values for [14C]ADP transport of 400-450 nmol exchanged.min-1.(mg of protein)-1 with Km values of the order of 13-15 microM were calculated, consistent with rates of phosphorylation in the presence of succinate of 320-400 nmol of ATP formed.min-1.(mg of protein)-1. The rate of transport of [14C]ATP in energized mitochondria was 5-10 times lower than that of [14C]ADP. Upon uncoupling, the rate of [14C]ATP uptake was enhanced, and that of [14C]ADP uptake was decreased. However, the two rates did not equalize, indicating that transport was not exclusively electrogenic. Transport of [14C]ADP and [14C]ATP by resting mitochondria followed biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

The alpha-adrenergic-mediated activation of the cardiac mitochondrial Ca2+ uniporter and its role in the control of intramitochondrial Ca2+ in vivo.

Administration of methoxamine (10 microM, 2 min) to perfused rat hearts increased the rate at which subsequently isolated mitochondria accumulated Ca2+. Methoxamine did not change significantly the development of delta phi with time or the basal rates of Ca2+ flux on inhibition of the uniporter with Ruthenium Red. With 200 microM-Pi, the rates of Ca2+ uptake at constant delta phi were unaffected by the small variations in endogenous [Pi] between mitochondrial preparations, and were also unaffected by changes in internal Ca2+ over the approximate range 8-43 nmol of Ca2+/mg. At low internal Ca2+ (about 8 nmol/mg of protein) the rates of Ca2+ uptake at constant delta phi were unaffected by addition of 200 microM-Pi. Under these conditions, the uniporter activity and the uniporter conductance were increased by 38-40% by methoxamine pretreatment. The endogenous Ca2+ content of mitochondria from control heart was about 1.8 nmol of Ca2+/mg of protein. Perfusion with agonist increased the Ca2+ content as follows: 10 microM-methoxamine (2 min), 48%; 1 microM-isoprenaline (2 min), 100%; 1 microM-adrenaline (2 min), 140%. The implications of the data for the adrenergic control of oxidative metabolism by intramitochondrial Ca2+ is discussed.     

Ruthenium red inhibits mitochondrial Na+ and K+ uniports induced by magnesium removal.

Removal of bound magnesium from the outer surface of the inner mitochondrial membrane opens up a Na+ and Li+ selective electrophoretic uniport pathway whereas simultaneous depletion of intramitochondrial magnesium induces an electrogenic K+ flux as well. In order to clarify the nature of these cation movements we tested the effect of ruthenium red, a potent and specific inhibitor of the mitochondrial Ca2+ uniporter on different Na+ and K+ uniport-associated phenomena. Ruthenium red efficiently inhibited mitochondrial swelling and depolarization induced by either EDTA in a NaCl-based medium (Na+ uniport) or by EDTA plus A23187 in a KCl-based medium (K+ uniport). For both cation uniports half-maximal inhibition was attained at a ruthenium red concentration as low as 40 nM. Complete inhibition was found above 200 nM. Neither the Na+/H+ nor the K+/H+ exchange was affected by ruthenium red. In light of these observations the possibility is raised that the electrogenic Na+ and K+ fluxes provoked by magnesium reduction or depletion may be mediated through the Ca2+ uniporter. It is suggested that intactness of the mitochondrial magnesium pools is necessary for maintaining the Ca2+ selectivity of the Ca2+ uniporter, and alterations of the membrane-associated magnesium content would make this transport route available also for monovalent cations.     

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.     

Metabolic state of liver mitochondria and lymphocytes after T-activin administration under condition of hypothermia

After 10 days of swimming (10 min per day, water temperature +20 degrees C) the oxygen consumption in rat liver mitochondria increased via the external pathway of NADH oxidation from 3.6 +/- 0.3 to 4.4 +/- 0,2 nmoles O2 x min-1 x mg-1 protein; when the rats were simultaneously injected with an endogenous immunomodulator T-activin (5 micrograms/100 g body weight) daily, this rate increased up to 6.5 +/- 0.6 nmoles O2 x min-1 x mg-1 protein. In the control group, the uncoupled respiration rate is also higher, while the ascorbate+ +TMPD oxidation rate is lower than in the cold- and cold + T-activin-treated groups. The metabolic states of lymphocyte mitochondria did not differ in the three experimental groups. The respiration rates and delta psi m (monitored by diS-C3-(5) fluorescence) of lymphocyte mitochondria in these three groups were also identical.     

Anion uniport in plant mitochondria is mediated by a Mg(2+)-insensitive inner membrane anion channel.

It has long been established that the inner membrane of plant mitochondria is permeable to Cl-. Evidence has also accumulated which suggests that a number of other anions such as Pi and dicarboxylates can also be transported electrophoretically. In this paper, we present evidence that anion uniport in plant mitochondria is mediated via a pH-regulated channel related to the so-called inner membrane anion channel (IMAC) of animal mitochondria. Like IMAC, the channel in potato mitochondria transports a wide variety of anions including NO3-, Cl-, ferrocyanide, 1,2,3-benzene-tricarboxylate, malonate, Pi, alpha-ketoglutarate, malate, adipate, and glucuronate. In the presence of nigericin, anion uniport is sensitive to the medium pH (pIC50 = 7.60, Hill coefficient = 2). In the absence of nigericin, transport rates are much lower and much less sensitive to pH, suggesting that matrix H+ inhibit anion uniport. This conclusion is supported by measurements of H+ flux which reveal that "activation" of anion transport at high pH by nigericin and at low pH by respiration is associated with an efflux of matrix H+. Other inhibitors of IMAC which are found to block anion uniport in potato mitochondria include propranolol (IC50 = 14 microM, Hill coefficient = 1.28), tributyltin (IC50 = 4 nmol/mg, Hill coefficient = 2.0), and the nucleotide analogs Erythrosin B and Cibacron Blue 3GA. The channel in plant mitochondria differs from IMAC in that it is not inhibited by matrix Mg2+, mercurials, or N,N'-dicyclohexylcarbodiimide. The lack of inhibition by Mg2+ suggests that the physiological regulation of the plant channel may differ from IMAC and that the plant IMAC may have functions such as a role in the malate/oxaloacetate shuttle in addition to its proposed role in volume homeostasis.     

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.     

Rapid and extensive release of Ca2+ from energized mitochondria induced by EGTA

A novel mitochondrial Ca2+ release phenomenon is reported. When rat liver mitochondria (oxidizing succinate) are allowed to accumulate Ca2+ in excess of 40 nmol/mg protein and are then treated with excess EGTA, a fraction of the accumulated cation is rapidly (approximately 1 nmol/s/mg protein) released. The size of the released fraction is an apparent function of the extramitochondrial Ca2+ concentration at the time of EGTA addition and can attain a maximal value of approximately 30 nmol/mg protein. Release is inhibited by ruthenium red (I50 approximately 50 pmol/mg protein) and is not dependent on the presence of Na+ or K+ in the medium. During the period of rapid release, O2 consumption is inhibited, membrane potential increases, and apparent H+ accumulation occurs at a ratio of approximately 2H+ per Ca2+ released. It is proposed that this chelator-induced Ca2+ release occurs by reverse uniport with H+ back diffusion to the matrix space providing charge movement compensation.     

Evidence for more than one Ca2+ transport mechanism in mitochondria.

The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.     

EGTA inhibits reverse uniport-dependent Ca2+ release from uncoupled mitochondria. Possible regulation of the Ca2+ uniporter by a Ca2+ binding site on the cytoplasmic side of the inner membrane

When rat liver mitochondria are allowed to accumulate Ca2+, treated with ruthenium red to inhibit reverse activity of the Ca2+ uniporter, and then treated with an uncoupler, they release Ca2+ and endogenous Mg2+ and undergo large amplitude swelling with ultrastructural expansion of the matrix space. These effects are not produced by Ca2+ plus uncoupler alone. Like other "Ca2+-releasing agents" (i.e. N-ethylmaleimide, t-butylhydroperoxide, oxalacetate, etc.), the development of nonspecific permeability produced by ruthenium red plus uncoupler requires accumulated Ca2+ specifically and is antagonized by inhibitors of phospholipase A2. The permeability responses are also antagonized by ionophore A23187, indicating that a rapid pathway for Ca2+ efflux from deenergized mitochondria is necessary to prevent the development of nonspecific permeability. EGTA can be substituted for ruthenium red to produce the nonspecific permeability change in Ca2+-loaded, uncoupler-treated mitochondria. The permeability responses to EGTA plus uncoupler again require accumulated Ca2+ specifically and are antagonized by inhibitors of phospholipase A2 and by ionophore A23187. The equivalent effects of ruthenium red and EGTA on uncoupled, Ca2+-containing mitochondria indicate that reducing the extramitochondrial Ca2+ concentration to the subnanomolar range produces inhibition of reverse uniport activity. It is proposed that inhibition reflect regulation of the uniporter by a Ca2+ binding site which is available from the cytoplasmic side of the inner membrane. EDTA cannot substitute for EGTA to induce nonspecific permeability in Ca2+-loaded, uncoupled mitochondria. Furthermore, EDTA inhibits the response to EGTA with an I50 value of approximately 10 microM. These data suggest that the uniporter regulatory site also binds Mg2+. The data suggest further that Mg2+ binding to the regulatory site is necessary to inhibit reverse uniport activity, even when the site is not occupied by Ca2+.     

Activation of latent K+ uniport in mitochondria treated with the ionophore A23187

Addition of A23187 plus EDTA to energized mitochondria in KCl medium determines a rapid osmotic swelling due to K+ uptake. The swelling is fully reversed by uncoupler, is stimulated by quinine, and is accompanied by membrane depolarization and increased rate of respiration. A23187-treated mitochondria passively swell in K+ thiocyanate at neutral pH, under conditions where the H+-K+ antiporter appears to be silent. These data indicate that A23187 activates electrophoretic K+ flux, supporting the notion that Mg2+ depletion unmasks several ionic conductance pathways whose concerted interplay could provide a sensitive regulation of mitochondrial volume homeostasis.     

Estradiol affect Na-dependent Ca2+ efflux from synaptosomal mitochondria

The effects of gonadal steroid hormone, 17beta-estradiol (E2), in vitro on rat brain mitochondria Ca2+ movement were investigated. Intrasynaptosomal mitochondria Ca2+ uptake via an energy-driven Ca2+ uniporter have Km = 112.73 +/- 7.3 micromol x l(-1) and Vmax = 21.97 +/- 1.7 nmol 45Ca2+ mg(-1). Ca2+ release trough a Na+/Ca2+ antiporter was measured with a Km for Na+ of 43.7 +/- 2.6 mmol x l(-1), and Vmax of 1.5 +/- 0.3 nmol 45Ca2+ mg(-1). Addition of estradiol in preincubation mixture did not affect the uptake of Ca2+ mediated by the ruthenium red-sensitive uniporter, while it produced biphasic effect on Na-dependent Ca2+ efflux. Estradiol at concentrations up to 1 nmol x l(-1) decreased the efflux significantly (63% inhibition with respect to the control), and at concentrations above 10 nmol x l(-1) increased it exponentially. The maximum inhibiting concentration of estradiol (0.5 nmol x l(-1)) increased the affinity of the uniporter (Km reduced by about 30%), without affecting significantly the capacity (Vmax) for Na+. The results presented suggest that estradiol inhibits Na-dependent Ca2+ efflux from mitochondria and acts on mitochondrial retention of Ca2+, which may modulate mitochondrial and consequently synaptosomal content of Ca2+, and in this way exerts its role in the homeostasis of calcium in nerve terminals.     

Kinetic modalities of ATP synthesis. Regulation by the mitochondrial respiratory chain

Two interconvertible kinetic modes are described for ATP synthesis by bovine heart submitochondrial particles. One mode is characterized by low apparent Km values for ADP (6-10 microM) and Pi (less than or equal to 0.25 mM), and a limited capacity for ATP synthesis (apparent Vmax approximately 500 nmol ATP.min-1.mg of protein-1). ATP synthesis occurs predominantly in this mode when the coupled activity of the respiratory chain relative to the number of functional ATP synthase complexes is low. The second kinetic mode is characterized by high apparent Km values for ADP (50-100 microM) and Pi (approximately 2.0 mM) and a high capacity for ATP synthesis (Vmax greater than 1800 nmol ATP.min-1.mg of protein-1). This mode of ATP synthesis predominates when the available free energy relative to the number of functional ATP synthase units is high. These results suggest that energy pressure in mitochondria might regulate ATP synthesis such that at low levels of energy the ATP synthase operates economically (low substrate Km values, low turnover capacity for ATP synthesis), while at high levels of energy these kinetic constraints are relaxed (high substrate Km values, high turnover capacity for ATP synthesis). The implications of these findings are discussed in relation to the cooperative-type kinetics of ATP synthesis and hydrolysis, the differential effects of a number of F0-F1 inhibitors on the rates of ATP synthesis and hydrolysis, and the controversy as to whether protonic energy in mitochondria is localized or delocalized.     

The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient.

1. A method is described for establishing steady-state conditions of calcium transport across the inner membrane of rat liver mitochondria and for determining the current of Ca2+ flowing across the membrane, together with the Ca2+ electrochemical gradient across the native Ca2+ carrier. These parameters were used to quantify the apparent Ca2+ conductance of the native carrier. 2. At 23 degrees C and pH7.0, the apparent Ca2+ conductance of the carrier is close to 1 nmol of Ca2+-min-1-mg of protein-1 mV-1. Proton extrusion by the respiratory chain, rather than the Ca2+ carrier itself, may often be rate-limiting in studies of initial rates of Ca2+ uptake. 3. Under parallel conditions, the endogenous H+ conductance of the membrane is 0.3 nmol of H+-min-1-mg of protein-1-mV-1. 4. Ruthenium Red and La3+ both strongly inhibit the Ca2+ conductance of the carrier, but are without effect on the H+ conductance of the membrane. 5. The apparent Ca2+ conductance of the carrier shows a sigmoidal dependence on the activity of Ca2+ in the medium. At 23 degrees C and pH7.2, half-maximum conductance is obtained at a Ca2+ activity of 4.7 muM. 6. The apparent Ca2+ conductance and the H+ conductance of the inner membrane increase fourfold from 23 degrees to 38 degrees C. The apparent Arrhenius activation energy for Ca2+ transport is 69kJ/mol. The H+ electrochemical gradient maintained in the absence of Ca2+ transport does not vary significantly with temperature. 7. The apparent Ca2+ conductance increases fivefold on increasing the pH of the medium from 6.8 to 8.0. The H+ conductance of the membrane does not vary significantly with pH over this range. 8. Mg2+ has no effect on the apparent Ca2+ conductance when added at concentration up to 1 mM. 9. Results are compared with classical methods of studying Ca2+ transport across the mitochondrial inner membrane.