Triorganotins inhibit the mitochondrial inner membrane anion channel.

The inner membrane of liver and heart mitochondria possesses an anion uniport pathway, known as the inner membrane anion channel (IMAC). IMAC is inhibited by matrix Mg2+, matrix H+, N,N'-dicyclohexycarbodiimide, mercurials and amphiphilic amines such as propranolol. Most of these agents react with a number of different mitochondrial proteins and, therefore, more selective inhibitors have been sought. In this paper, we report the discovery of a new class of inhibitors, triorganotin compounds, which block IMAC completely. One of the most potent, tributyltin (TBT) inhibits malonate uniport via IMAC 95% at 0.9 nmol/mg. The only other mitochondrial protein reported to react with triorganotins, the F1F0ATPase, is inhibited by about 0.75 nmol/mg. The potency of inhibition of IMAC increases with hydrophobicity in the sequence trimethyltin much less than triethyltin much less than tripropyltin less than triphenyltin less than tributyltin; which suggests that the binding site is accessible from the lipid bilayer. It has long been established that triorganotins are anionophores able to catalyze Cl-/OH- exchange; however, TBT is able to inhibit Cl- and NO3- transport via IMAC at doses below those required to catalyze rapid rates of Cl-/OH- exchange. Consistent with previous reports, the data indicate that about 0.8 nmol of TBT per mg of mitochondrial protein is tightly bound and not available to mediate Cl-/OH- exchange. We have also shown that the mercurials, p-chloromercuribenzene sulfonate and mersalyl, which only partially inhibit Cl- and NO3- transport can increase the IC50 for TBT 10-fold. This effect appears to result from a reaction at a previously unidentified mercurial reactive site. The inhibitory dose is also increased by raising the pH and inhibition by TBT can be reversed by S2- and dithiols but not by monothiols.     

Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique.

The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Addition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during anaerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/site ratio for the primary H+ ejection process during electron transport.     

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.     

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.     

Stimulation of mitochondrial calcium ion efflux by thiol-specific reagents and by thyroxine. The relationship to adenosine diphosphate retention and to mitochondrial permeability.

Respiring rat heart mitochondria were loaded with Ca2+ and then treated with Ruthenium Red. The factors affecting the subsequent Ca2+-efflux were studied. Addition of rotenone or antimycin led to a decline of efflux except at pH values above 7.2, provided the load was less than about 80 nmol per mg of protein. Oligomycin reversed the effect of the respiratory inhibitors. Independently of respiration, efflux was stimulated by the uncoupler trifluoromethyltetrachlorbenzimadazole, by mersalyl and by thyroid hormones. The stimulated efflux could be diminished by ADP, with Mg2+ as cofactor if efflux was rapid. With respiration in progress, efflux could be stimulated by N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoate). The effects of mersalyl and of thyroid hormones could be diminished with dithiothreitol. In the absence of stimulating agents, the Ca2+ efflux was proportional to the load up to some critical amount, this critical amount was decreased by the agents. Thyroxine and mersalyl caused not only loss of Ca2+, but also simultaneous, but not necessarily proportional, loss of internal adenine nucleotides. Both efflux rates were kept at a low value by bongkrekic acid added before the stimulating agent. It is concluded that Ca2+ efflux is a measure of a permeability controlled by the binding of ADP (an Mg2+) to the inner membrane, and that this in turn depends on the maintenance of certain thiol gropus in a reduced form by a reaction that uses NADH and ATP and the energy-linked transhydrogenase.     

Inhibition by divalent cations and sulphydryl reagents of the passive Ca2+ transport in human red blood cells observed in the presence of vanadate

The uptake of 45Ca2+ by human red blood cells induced by vanadate was found to be inhibited by a number of divalent cations. The following order of potencies was determined (in parentheses, IC50 in mmol/l): Cu2+ (0.006), Zn2+ (0.014), Cd2+ (0.030), Co2+ (0.20), Ni2+ (0.25), Mn2+ (8.0), Ba2+ (9.0), Sr2+ (14.0). The effects of Cu2+, Zn2+ and Cd2+ were biphasic--over a critical concentration their inhibitory potencies decreased, and finally, were lost. Besides Ca2+, Sr2+, Ba2+ and Mn2+ were also taken up, but only Ca2+ and Sr2+ were capable of eliciting the Gárdos effect. Ni2+ was not taken up. Several HS reagents also inhibited 45Ca2+ uptake. The following order of potencies was determined (in parentheses, IC50 in mmol/l): mersalyl (0.0025), 5,5'-dithiobis(2,2'-dinitrobenzoic acid) (0.011), p-chloromercuric acid (0.042), N-ethylmaleimide (2.0). The effects of all HS reagents except N-ethylmaleimide were biphasic. The biphasicity of the actions of the indicated agents was caused by the opening of a new pathway for 45Ca2+ entry which is different from that observed in the presence of vanadate alone, and is inhibited by low concentrations of these agents. The modified form of the anion channel seems to be identical with the former pathway. The last one is mediated by a transport protein which has an ionic specificity similar to Ca2+ channels in excitable tissues, and contains an HS group which is essential for the transport function.     

Temperature dependence of the mitochondrial inner membrane anion channel: the relationship between temperature and inhibition by magnesium

The mitochondrial inner membrane anion channel (IMAC) carries a wide variety of anions and is postulated to be involved in mitochondrial volume homeostasis in conjunction with the K+/H+ antiporter, thus allowing the respiratory chain proton pumps to drive salt efflux. How it is regulated is uncertain; however, it is inhibited by matrix Mg2+ and matrix protons. Previously determined values for the IC50 suggested that the channel would be closed under physiological conditions. In a previous study (Liu, G., Hinch, B., Davatol-Hag, H., Lu, Y., Powers, M., and Beavis, A. D. (1996) J. Biol. Chem. 271, 19717-19723), it was demonstrated that the channel is highly temperature-dependent, and that a large component of this sensitivity resulted from an effect on the pIC50 for protons. We have now investigated the effect of temperature on the inhibition by Mg2+ and have found that it too is temperature-dependent. When the temperature is raised from 20 degrees C to 45 degrees C, the IC50 increases from 22 to 350 microm at pH 7.4 and from 80 to 1.5 mm at pH 8.4, respectively. The Arrhenius plot for the IC50 is linear with a slope = -80 kJ/mol. The IC50 is also strongly pH-dependent, and at 37 degrees C increases from 90 microm at pH 7.4 to 1230 microm at pH 8.4. In view of the extremely rapid fluxes that IMAC is capable of conducting at 37 degrees C, we conclude that inhibition by matrix Mg2+ and protons is necessary to limit its activity under physiological conditions. We conclude that the primary role of Mg2+ is to ensure IMAC is poised to allow regulation by small changes in pH in the physiological range. This control is mediated by a direct effect of H+ on the activity, in addition to an indirect effect mediated by a change in the Mg2+ IC50. The question that remains is not whether IMAC can be active at physiological concentrations of Mg2+ and H+, but what other factors might increase its sensitivity to changes in mitochondrial volume.     

Phosphate transport and proteins with SH groups in rat liver mitochondria

Phosphate transport in rat liver mitochondria was studied by following [32P] phosphate uptake within physiological concentrations. Transport inhibition due to mersalyl and protection by mersalyl against N-ethylmaleimide measured in those conditions corresponded to earlier results obtained by the swelling technique. When mitochondria were incubated with [3H] N-ethylmaleimide in the presence of mersalyl, the radioactive labeling in proteins of particles obtained after sonication was decreased in all fractions, but three proteins were both highly alkylated and also highly protected by mersalyl (M.W. 48,000 - 36,000 - 31,000). Two of these (M.W. 36,000 and 31,000) were partially purified by ultrogel chromatography in the presence of sodium dodecyl sulfate. Furthermore, it was shown that both phosphate and nigericin diminished labeling by N-ethylmaleimide in the final supernatant fraction. Two proteins (M.W. 98,000 and 31,000) were significantly alkylated by [3H] N-ethylmaleimide and protected by phosphate and nigericin.     

Localization and Characterization of Binding Sites with High Affinity for [3H]Ouabain in Cerebral Cortex of Rabbit Brain Using Quantitative Autoradiography

[3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8-12 nM for specific high-affinity binding of [3H]ouabain were found by both methods. High-affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1, 3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50 = 0.7-0.9 mM), N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+-ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.     

On the inhibition of the mitochondrial inner membrane anion uniporter by cationic amphiphiles and other drugs

Depleting the mitochondrial matrix of divalent cations with the ionophore A23187 activates a pH-sensitive, anion uniport pathway which can transport many anions normally regarded as impermeant (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). Addition of valinomycin to respiring mitochondria can also induce the uptake of a wide variety of anions; however, the mechanism of anion transport during this "respiration-induced" swelling is less certain. In this paper, I demonstrate that both of these processes are inhibited by a variety of cationic amphiphiles including propranolol, quinine, amiodarone, imipramine and amitriptyline, and the benzodiazepine R05-4864. Although the IC50 values for the two processes are not equal, the ratio of IC50 values for the two processes appears to be the same for all drugs. Measurements of net transmembrane proton fluxes that occur during the assays reveal that respiration-induced swelling is associated with extensive proton ejection, the peak of which coincides with the maximum rate of anion transport. Moreover, from measurements of matrix buffering power, it is estimated that the matrix pH is 3 units more alkaline during respiration-induced swelling than during A23187-induced swelling. It is also shown that the IC50 for A23187-induced transport is pH-dependent in a manner consistent with modulation of drug binding by protonation of two sites. These findings allow the difference in IC50 values for the two types of assay to be explained by the pH dependence of the binding constant for the drug. Furthermore, the pH gradient generated during respiration-induced swelling is so large that the electrical component of the proton-motive force will be negligible. Thus, despite the fact that the mitochondria are "energized," rapid electrophoretic anion influx is possible. These data provide evidence that the transport of anions in these two types of assay occurs via the same pathway.     

Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria. Distinction between sulfhydryl groups of the H+ channel and the nucleotide binding site

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.     

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.     

Induction of mitochondrial Ca2+ uptake by mersalyl

1. The addition of mersalyl to aged mitochondria from rat kidneys, is followed by induction of an ATP-driven Ca2+ uptake which is sensitive to Ruthenium Red. 2. This Ca2+ influx requires Mg2+, albumin, and is accomplished by membrane energization. 3. The activation of Ca2+ uptake by the mercurial in the presence of ATP can be explained if it is assumed that the inorganic phosphate generated by ATPase activity, and trapped in the matrix by the thiol reagent, provides the negative potential which results in an electrophoresis cation influx.     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

NMDA and glycine regulate the affinity of the Mg2+-block site in NR1-1a/NR2A NMDA receptor channels expressed in Xenopus oocytes

NMDA receptors are glutamate-regulated ion channels of critical importance for many neurophysiological and neuropathological processes. Mg2+ blocks the NMDA receptor by binding to the channel pore with an apparent affinity that depends on the membrane potential. We have investigated the effect of NMDA and the required co-agonist glycine on the affinity of the Mg2+ block site in NR1-1a/NR2A NMDA receptors expressed in Xenopus oocytes. We found that NMDA and glycine increase the IC50 value of the Mg2+-block site at pH 7.4 and in the presence of physiological concentration of Ca2+. The increase the IC50 value may correspond to a decrease in Mg2+-block affinity. This effect may result in an increased influx of Ca2+, and this influx may constitute up to a third of the total Ca2+ influx induced by NMDA. At high pH, or at low concentrations of Ca2+, NMDA and glycine have an opposite effect and instead decreased the IC50 value of the Mg2+-block. These results indicate that glutamate and glycine can regulate the affinity of the Mg2+-block site. This effect may have implications for the understanding the role of NMDA receptors both under physiological and pathophysiological conditions.     

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+.     

Action of spermine on phosphate transport in liver mitochondria

Spermine, at concentrations similar to those normally present in the cytosol of liver cells, facilitates the transport of phosphate into mitochondria and thus its accumulation within the matrix space. Both mersalyl and N-ethylmaleimide (NEM) inhibit phosphate influx either in the absence or in the presence of spermine. These inhibitors also inhibit, but only partially, the efflux from mitochondria of phosphate generated within the matrix space by the hydrolysis of ATP induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or the valinomycin-K+ system. The inhibition of phosphate efflux by both mersalyl and NEM is almost completely removed, unlike that of phosphate influx, by spermine. The possibility that spermine may induce phosphate efflux by damaging mitochondrial membranes and consequently inducing an unspecific permeability to phosphate is excluded by the full restoration of transmembrane potential once FCCP has been removed by albumin. Since spermine does not react with either thiol groups or thiol group reagents, the simplest explanation of the reported results is that the pathway of phosphate efflux is distinct from that of phosphate influx.     

Permeability of yeast mitochondrial internal membrane: structure-activity relationship]

In order to investigate the possible relations between the anionic permeability and the functions (or the structure ) of the inner mitochondrial membrane, three types of organelles isolated from S. cerevisiae were tested: mitochondria (aerobic culture), promitochondria (anaerobic culture) and CAP-mitochondria (aerobic culture with chloramphenicol added). By using the technique of swelling in isoosmotic potassium salts, after a derermination of the isotonic conditions, it was possible to discriminate between an electrogenic (valinomycin induced) or an electroneutral (both valinomycin and uncoupler induced) translocation. 1) Mitochondria: The permeability properties of mitochondria are energy dependent: a) Respiring mitochondria are permeable to Cl-; Mg2+, however, inhibits this translocation. Phosphate transport seems to be exclusively electrogenic and mersalyl sensitive, but swelling inhibition by that thiol reagent is restored by Mg2+. b) Non respiring mitochondria are impermeable to Cl-, but ATP addition restores the permeability. Thiocyanate permeates as the anionic form and acetate as the undissociated form. The phosphate transport, sensitive to mersalyl, seems to be partially electrogenic. 2) Promitochondria: Deficient of respiratory enzymes but containing an oligomycin sensitive ATPase, they are impermeable to Cl- only when Mg2+ is added. In these conditions, an electrogenic phosphate transport, sensitive to mersalyl, is observed. 3) CAP-mitochondria: Although CAP-mitochondria are cytochrome deficient and contain an oligomycin insensitive ATPase, they are also impermeable to Cl- in presence of Mg2+. As in fully differenciated mitochondria, an electroneutral phosphate entry is observed; Mg2+ is required for mersalyl sensitivity.     

Transmembrane Ca2+ exchange in depolarized rat myometrium mitochondria

Polarization of the inner membrane is the key factor in maintenance of the physiologically significant cations accumulation, in particular Ca2+, in the mitochondria. It has been well established that mitochondria accumulate calcium through the uniporter, driven by the mitochondrial membrane potential. Nevertheless, it has been shown that depolarized mitochondria also accumulate Ca2+. The aim of this paper is to investigate free Ca level in depolarized myometrium mitochondria. As we have shown previously Ca2+ addition to the incubation medium, that did not contain K-phosphate, ATP and Mg2+, led to inner mitochondrial membrane depolarization. Nevertheless Ca2+ addition to such medium led to the concentration-dependent accumulation of this cation in the matrix. RuR or Mg addition to the incubation medium led to the higher elevation of mitochondrial Ca2+ level in depolarized mitochondria. Mitochondrial Ca2+ level was not affected by 5 microM cyclosporine A. It was suggested that H+/Ca2+ exchanger could provide calcium accumulation in depolarized mitochondria. The elevation of mitochondrial Ca2+ level after addition of Mg2+ and RuR may be due to inhibition of Ca2+- efflux through Ca2+ uniporter.