Trace amounts of Triton X-100 modify the inhibitor sensitivity of the mitochondrial porin.

Transport properties of mitochondrial porin were investigated on the basis of changes in the activity of hexokinase utilizing external ATP. Production of glucose 6-phosphate is inhibited by polyanion both in intact brain mitochondria and in contact point vesicles. Hexokinase activity is restored by solubilization of the enzyme by high ionic strength or 0.5-1% Triton X-100. In very low concentrations (0.001-0.005%) Triton does not mobilize hexokinase from its binding sites but it is able to release polyanion-inhibition completely. This finding provides an explanation for the discrepancy observed in the transport properties of porin when studied 'in situ' or in artificial lipid membranes.

Transport properties and inhibitor sensitivity of isolated and reconstituted porin differ from those of intact mitochondria.

The pore-forming protein porin has been isolated from rat heart mitochondria and reconstituted in phospholipid vesicles of different composition. Rapid release of anions, cations and non-charged molecules has been demonstrated from the proteoliposomes but not from the protein-free liposomes. In spite of its higher molecular mass and charges, the movement of ATP was almost as fast as that of inorganic phosphate. Polyanion (1:2:3 copolymer of methacrylate/maleate/styrene), a potent inhibitor of porin residing in the mitochondrial contact sites decreased the solute movements but did not completely block any of the investigated transport processes (phosphate, chloride, ATP). Alterations of the lipid environment had significant effect: an increase in the proportion of soybean phospholipids to egg yolk phospholipids resulted in a decrease in the amount of transported substance but did not fully inhibit the ion movements. It is concluded that the transport properties of porin reconstituted in artificial phospholipid membranes are different from the characteristics of porin prevailing in the mitochondrial contact sites and additional regulatory factors are suggested to be effective in the intact mitochondria.

Is the mitochondrial Ca2+ uniporter a voltage-modulated transport pathway?

The influence of membrane potential (delta psi) on Ca2+ transport through the Ca2+ uniporter (UP) was investigated in fura-2-loaded rat heart mitochondria at physiologically relevant-submicromolar-external [Ca2+]. In the absence of delta psi the UP could not mediate Ca2+ uptake even when an 8-fold external (approximately 500 nM) to internal (approximately 60 nM) [Ca2+] gradient was present and charge compensation was provided by acetate and the protonophore, CCCP. A small (approximately -120 mV) and transient delta psi (generated by valinomycin) resulted in a rise in matrix [Ca2+] only when external [Ca2+] exceeded 150 nM. At physiologically high (approximately -180 mV) and stable delta psi this threshold value for Ca2+ uptake dropped to 15 nM. The results indicate that (1) at physiological [Ca2+]o, delta psi in addition to being a component of delta mu Ca2+ seems to be necessary for providing a transport-competent conformation for the UP; and (2) below a threshold [Ca2+]o the UP cannot operate even in the presence of a high electric driving force.

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.

Na+/H+ exchange in mitochondria as monitored by BCECF fluorescence.

The recently developed method of loading isolated heart mitochondria with the fluorescent pH indicator, BCECF, was applied to monitor the Na+o/H+i exchange process from the matrix side of the membrane. The Na+-induced changes in the pH of the matrix (pHm) showed that: (i) the Na+o/H+i exchange followed Michaelis-Menten kinetics with respect to external Na+ with a Km of approx. 20 mM; (ii) in contrast to this, the dependence of the exchange rate on the matrix [H+] did not obey the Michaelian model. No Na+-induced alkalinization occurred above a pHm of 7.45 +/- 0.09 (n = 4). Below this value the reciprocal of the transport rate and that of the matrix [H+] deviated upwardly from the straight line. The results suggest that internal H+ might exert allosteric control on the mitochondrial Na+/H+ exchange process.

Phosphate transport protein of rat heart mitochondria: location of its SH-groups and exploration of their environment.

(1) The properties of the SH groups of the phosphate transport protein of rat heart mitochondria were investigated on the basis of inhibition caused by SH reagents under different conditions. (2) The essential thiol groups are located near the external surface, as they are accessible to impermeable reagents from the external space. (3) The environment of the sulfhydryl groups influences their reactivity, as alteration of the external pH affects adversely their reactions with ionizable and non-ionizable SH reagents. (4) Intramitochondrial pH exerts a transmembrane effect: alkalinization augments and acidification diminishes the reaction rate of the sulfhydryl groups on the opposite surface of the membrane. (5) Changes of the concentration of the transported substrate occurring exclusively in the extramitochondrial space do not influence the reactivity of the essential SH groups. (6) It is concluded that in transport studies the phosphate transport protein of heart and liver mitochondria show basic similarity. It is suggested that the amino-acid sequence around the NEM-reactive cysteine (i.e., Lys-41 - Cys-42 - Arg-43) does not participate in substrate binding.

Characterization of the mitochondrial Na+-H+ exchange. The effect of amiloride analogues.

The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria.

The entrapment of the Ca2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ [( Ca2+]m) [(1987) Biochem J. 248, 609-613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6 microM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.

Mitochondrial phosphate carrier. Functional role of its SH groups and interrelations within the carrier unit.

1. The phosphate carrier of isolated rat liver mitochondria was studied. 2. To investigate the effect of the substrate on the external side of the transport protein, a technique was required by which the extramitochondrial phosphate concentration could be changed without any effect on either the phosphate content or the pH value in the intramitochondrial compartment. This was found when non-respiring mitochondria were incubated with the ionophore nigericin. 3. Using this method variation of the phosphate concentration outside the mitochondria did not protect the essential SH groups located at the external surface of the phosphate carrier. 4. Interrelations within the phosphate carrier unit were investigated in mitochondria in which only one of the two essential thiol groups of the carrier unit was free. These mitochondria were however able to carry out phosphate transport. 5. Alterations of the intramitochondrial pH-induced modification of the position of the single free sulfhydryl group at the external surface of the membrane similarly to the changes which occur in intact mitochondria where both SH groups are free [E. Ligeti and A. Fonyó (1984) Eur. J. Biochem. 139, 279-285]. 6. It is suggested that the sulfhydryl groups essential for the transport function do not participate in the formation of the substrate binding site of the carrier protein. They probably have a role in the conformational change necessary for the translocation step. The possibility of two equivalent but independently functioning parts within the carrier unit is raised.

The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action.

The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166).

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria.

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.

Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier.

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.

Proton cycling through the mitochondrial phosphate transporter in energy transduction.

The molecular structure of the phosphate carrier of mitochondria is probably controlled by the H+ ion concentration within the matrix. It is supposed that changes in molecular structure affect the H+- and H2PO4- ion transporting function and may be part of the "gating" mechanism in phosphate transport.

Participation of inorganic phosphate in the chemiosmotic mechanism of mitochondrial energy transduction.

In respiring liver mitochondria in which Pi transport was inhibited and the intramitochondrial Pi value manipulated to vary about 3-fold, K+-ion uptake, H+-ion extrusion and stimulation of respiration were linearly related to the intramitochondrial Pi content. It is suggested that the HPO 2(4)-anion generated during respiration is the nondiffusible anion required for the Donnan distribution and membrane potential in mitochondria. The intramitochondrial H X HPO4-ionic species is the proton donor required for coupled respiration. The electroneural Pi-H+ cotransport closes the proton cycling in respiration.

Phosphate carrier of liver mitochondria: the reaction of its SH groups with mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide and the modulation of reactivity by the energy state of the mitochondria.

The inhibitory effect of three SH reagents, mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide, on Pi transport in rat liver mitochondria was investigated under a variety of conditions. Mersalyl binds at room temperature with both high (Kd less than 10 microM) and low affinity to mitochondria. Inhibition of Pi transport by mersalyl goes in parallel with titration of the high-affinity sites, inhibition being complete when 3.5-4.5 nmol/mg protein is bound to the mitochondria. At concentrations of mersalyl equal to or higher than 10 microM, inhibition of Pi transport occurs in less than 10 sec. At concentrations of mersalyl lower than 10 microM, the rate of reaction with the Pi carrier is considerably decreased. At a concentration of 100 microM, 5,5'-dithio-bis-nitrobenzoate fully inhibits Pi transport in about 1 min at room temperature. Nearly total inhibition is attained when as little as 40-50 pmol/mg is bound to mitochondria. Upon incubation longer than 1 min, additional SH groups, not belonging to the Pi carrier, begin to react. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreases the rate of reaction of mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide with the Pi carrier. Preincubation with Pi has a similar effect. We propose that both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and Pi act by increasing the acidity of the mitochondrial matrix. Protonation of the Pi carrier at the matrix side would change the accessibility of its SH groups at the outer surface of the inner membrane. This might correspond to a membrane-Bohr effect, possibly related to the opening of a gating pore in the Pi carrier.