Voltage activation of heart inner mitochondrial membrane channels

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above ±60mV [Kinnallyet al., Biochem. Biophys. Res. Commun. 176, 1183–1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin Aare likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.     

Regulation of mitochondrial proteolysis Selective degradation of inner membrane polypeptides

The in vitro degradation of respiratory chain polypeptide components by a proteinase associated with the intermembrane space fraction was studied in rat liver mitochondria. Differences in susceptibility to proteolysis were detected by gel analysis after electrophoretic separation of the degraded polypeptides. A 55 kDa subunit is protected from proteolysis by the ATP molecule.     

Selective solubilization of the components of the mitochondrial inner membrane by lysolecithin.

1. Of various phospholipids tested, lysolecithin was the most efficient in the solubilization of the components of beef heart submitochondrial particles. Lysolecithin solubilized selectively nicotinamide nucleotide transhydrogenase, succinate dehydrogenase, NADH dehydrogenase and oligomycin-sensitive ATPase. Various cytochromes other than cytochrome c were only slightly solubilized. 2. The effect of various parameters, e.g. ionic strength, pH, time of centrifugation, and concentrations of lysolecithin and protein was investigated. Increasing times of centrifugation led to a partial sedimentation of NADH dehydrogenase, and a complete sedimentation of oligomycin-sensitive ATPase and cytochrome oxidase. 3. Further fractionation of the lysolecithin extract by centrifugation in the presence of low concentrations of cholate gave a complete separation of NADH dehydrogenase and transhydrogenase, indicating that these enzymes are not related functionally. 4. With the lysolecithin fractionation procedure a more than 10-fold purification of transhydrogenase was achieved. Polyacrylamide gel electrophoresis of the partially purified transhydrogenase in the presence of sodium dodecyl sulphate showed major increases in protein-stained bands corresponding to between 70 000 and 54 000 daltons. 5. A possible mechanism for the detergent action of lysolecithin involving a specific exchange of bound phospholipids for lysolecithin is discussed.     

Selective solubilization of nicotinamide nucleotide transhydrogenase from the mitochondrial inner membrane

Nicotinamide nucleotide transhydrogenase was solubilized from beef heart submitochondrial particles employing Triton X-100 or lysolecithin. Lysolecithin was considerably more efficient and selective and released over 80 % of the transhydrogenase acdtivity from the membrane together with succinate dehydrogenase. Solubilization of NADH dehydrogenase and cytochrome oxidase was more efficiently accomplished with Triton than with lysolecithin. Both detergents released ATPase to various extents. Transhydrogenase remaining bound to particles after treatment with lysolecithin still catalyzed energy-linked transhydrogenation.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

Mitochondrial respiratory chain inhibitors modulate the metal-induced inner mitochondrial membrane permeabilization

To elucidate the molecular mechanisms of the protective action of stigmatellin (an inhibitor of complex III of mitochondrial electron transport chain, mtETC) against the heavy metal-induced cytotoxicity, we tested its effectiveness against mitochondrial membrane permeabilization produced by heavy metal ions Cd2(+), Hg2(+), Cu2(+) and Zn2(+), as well as by Ca2(+) (in the presence of P(i)) or Se (in form of Na?SeO?) using isolated rat liver mitochondria. It was shown that stigmatellin modulated mitochondrial swelling produced by these metals/metalloids in the isotonic sucrose medium in the presence of ascorbate plus tetramethyl-p-phenylenediamine (complex IV substrates added for energization of the mitochondria). It was found that stigmatellin and other mtETC inhibitors enhanced the mitochondrial swelling induced by selenite. However, in the same medium, all the mtETC inhibitors tested as well as cyclosporin A and bongkrekic acid did not significantly affect Cu2(+)-induced swelling. In contrast, the high-amplitude swelling produced by Cd2(+), Hg2(+), Zn2(+), or Ca2(+) plus P(i) was significantly depressed by these inhibitors. Significant differences in the action of these metals/metalloids on the redox status of pyridine nucleotides, transmembrane potential and mitochondrial respiration were also observed. In the light of these results as well as the data from the recent literature, our hypothesis on a possible involvement of the respiratory supercomplex, formed by complex I (P-site) and complex III (S-site) in the mitochondrial permeabilization mediated by the mitochondrial transition pore, is updated.     

The effect of antimycin A on mouse liver inner mitochondrial membrane channel activity.

In a patch-clamp study, we found antimycin A in low (1-2) microM concentrations decreased the open probability of the multiple conductance channel activity and the approximately 110 picosiemens channel of the inner mitochondrial membrane (for a review of mitochondrial channels see Kinnally, K. W., Antonenko, Yu. N., and Zorov, D. B. (1992) J. Bioenerg. Biomembr. 24, 99-110). Higher antimycin A concentrations (e.g. 10 microM) facilitated multiple conductance channel opening. These effects were reversible, and the binding site(s) are probably distinct from those responsible for the inhibition of the electron transport chain, since the latter are virtually irreversible. A model with two closed and two open states is presented for the approximately 110-picosiemens activity.     

Effect of visible light on the mitochondrial inner membrane.

Exposure of mitochondria to visible light in the presence of riboflavin resulted in the initial release of respiratory control, followed by inhibition of electron transport and dissolution of structural integrity. Under these conditions, however, cytochrome c oxidase activity remained unchanged. ATPase activity was stimulated initially and remained in this activitated state even under continued illumination. In submitochondrial preparations, both electron transport and ATPase declined as a function of illumination time; cytochrome c oxidase was not sensitive to light. Enzyme inactivation also occurred to a lesser extent in the absence of riboflavin.     

The inner mitochondrial membrane contains ion-conducting channels similar to those found in bacteria

Patch-clamp experiments were performed on rat liver mitochondria inner membranes. Application of voltage gradients of either polarity revealed the presence of several different conductances, ranging up to 1.3 nS in symmetrical 150 mM KCl. Evidence is presented that at least those higher than 0.3 nS are substates of the highest conductance channel. Increasing matrix-side-positive (unphysiological) transmembrane voltage gradients favored the switch of the 1.3 nS channel to operation in lower conductance states. The size of these conductances, the presence of substates and the channel behavior are strongly reminiscent on one hand of the observations on the membrane of protoplasts from the gram-positive bacterium Streptococcus faecalis, [Zoratti, M. and Petronilli, V. (1988) FEBS Lett. 240, 105-109], and on the other of some properties of previously described channels of mitochondrial origin.     

Calcium modulation of mitochondrial inner membrane channel activity.

Protocols were defined that enable patch-clamp studies of the approximately 107 pS voltage dependent channel and a class of activity we refer to as MCC (multiconductance channel) which is characterized by multiple levels and transitions as high as 1 to 1.5 nS. If free calcium was kept at 10(-7) M or lower during mitochondrial isolation, no activity was observed at low voltage (+/- 60 mV). If free calcium levels were higher, MCC activity was observed in about 96% of the patches. The observation of approximately 107 pS channel was enhanced from 2% to 68% of patches by washing isolated mitoplasts (mitochondria stripped of outer membrane) with EGTA. Increasing matrix calcium from 10(-9) to 10(-5) M decreased the probability of opening for the MCC and approximately 107 pS activities.     

Bilirubin increases mitochondrial inner membrane conductance

Bilirubin accumulates within, and induces loose coupling in, rat liver mitochondria. This state, characterized by a normal protonmotive force, but increased oxygen consumption and inner membrane conductance, could impair cellular energy metabolism. Loose coupling is observed at bilirubin concentrations (12-24 microM) attained in tissues of kernicteric animals.     

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.     

Reducibility of cytochromeb in mitochondrial inner membrane

When mitochondrial inner membrane was disintegrated into Complex I-III, IV, and oligomycin-sensitive ATPase, about 50% of cytochromeb in Complex I-III was readily reduced with NADH, as judged by the appearance of a peak at 562 nm, while in whole mitochondria less than 25% of cytochromeb was reduced by succinate. On addition of antimycin to the substrate-reduced Complex I-III, cytochromeb was further reduced to 71% of the total, and the peak at 562 nm was red-shifted to 564 nm as in the case of dithionite reduction. These results indicate that the 562 nm and 564 nm peaks, at 29°C correspond, respectively, tob ₅₆₀ andb _562.5 at 77°K of Davis et al. [7] and to b _K and b _T of Chance et al. [2]. When Complex I-III and oligomycin-sensitive ATPase were reconstituted to form a membrane, about 60% of cytochromeb in Complex I-III was readily reduced with NADH. In this case the 562 nm peak was not red-shifted. However, the difference spectrum of NADH-reduced membraneminus that in the presence of deoxycholate showed a peak at 565 nm. A mirror image of the difference spectrum was obtained on addition of an uncoupler,m-chlorocarbonyl cyanide phenylhydrazone. This is characteristic for b _T. These results support the idea that the occurrence of spectral peaks of b _T and b _K is not due to two species but to single speciesAbbreviations OS-ATPase oligomycin sensitive ATPase - CCCP m-chlorocarbonyl cyanide phenylhydrazone - F₁ coupling factor one - OSCP oligomycinsensitivity-conferring protein     

Dynamic subcompartmentalization of the mitochondrial inner membrane

The inner membrane of mitochondria is organized in two morphologically distinct domains, the inner boundary membrane (IBM) and the cristae membrane (CM), which are connected by narrow, tubular cristae junctions. The protein composition of these domains, their dynamics, and their biogenesis and maintenance are poorly understood at the molecular level. We have used quantitative immunoelectron microscopy to determine the distribution of a collection of representative proteins in yeast mitochondria belonging to seven major processes: oxidative phosphorylation, protein translocation, metabolite exchange, mitochondrial morphology, protein translation, iron-sulfur biogenesis, and protein degradation. We show that proteins are distributed in an uneven, yet not exclusive, manner between IBM and CM. The individual distributions reflect the physiological functions of proteins. Moreover, proteins can redistribute between the domains upon changes of the physiological state of the cell. Impairing assembly of complex III affects the distribution of partially assembled subunits. We propose a model for the generation of this dynamic subcompartmentalization of the mitochondrial inner membrane.