The cation-selective substate of the mitochondrial outer membrane pore: Single-channel conductance and influence on intermembrane and peripheral kinases

The polyanion-induced substate of the outer mitochondrial membrane was studiedin vivo andin vitro. Study of the substate in artificial bilayers showed that it is highly cation selective. The induction of the substate in intact mitochondria leads to a complete inhibition of the intermembrane kinases, such as creatine kinase and adenylate kinase, which were excluded from the external ATP pool. Peripheral kinases, such as hexokinase, were blocked when they utilized internal ATP. The results with intact mitochondria suggested the existence of two regions of the outer membrane containing channels of different states, which may be involved in the regulation of intermembrane and peripheral kinases.     

Further characterization of contact sites from mitochondria of different tissues: topology of peripheral kinases

A membrane fraction of intermediate density between inner and outer membrane was isolated by density gradient centrifugation from osmotically disrupted mitochondria of rat liver, brain, and kidney. The fraction was hexokinase rich and could therefore be further purified using specific antibodies against hexokinase and immunogold labelling techniques. In agreement with recent findings the gradient fraction which cosedimented with hexokinase contained the boundary membrane contact sites because it was composed of outer and inner membrane components and beside hexokinase, was enriched also by activity of creatine kinase and nucleoside diphosphate kinase. In contrast the activity of adenylate kinase appeared to be concentrated beyond the contact sites in the outer membrane fraction. By employing surface proteolysis analysis and specific blockers of the outer membrane pore we observed that the location of the kinases relative to the membrane components in the contact fraction resembled that of intact mitochondria. This specific organization of some peripheral kinases in the contact sites suggested an important role of the voltage dependence of the outer membrane pore, in that the pore may become limiting in anion exchange because of influence of the inner membrane potential on the closely attached outer membrane. Such control of anion exchange would lead to a dynamic compartmentation at the mitochondrial surface by the formation of contact sites, which may explain the preferential utilization of cytosolic creatine by the mitochondrial creatine kinase, as postulated in the phosphocreatine shuttle.     

The role of contact sites between inner and outer mitochondrial membrane in energy transfer

Three functions have been suggested to be localized in contact sites between the inner and the outer membrane of mitochondria from mammalian cells: (i) transfer of energy from matrix to cytosol through the action of peripheral kinases; (ii) import of mitochondrial precursor proteins; and (iii) transfer of lipids between outer and inner membrane. In the contact site-related energy transfer a number of kinases localized in the periphery of the mitochondrion play a crucial role. Two examples of such kinases are relevant here: (i) hexokinase isoenzyme I which is capable of binding to the outer aspect of the outer membrane; and (ii) the mitochondrial isoenzyme of creatine kinase which is localized in the intermembrane space. Recently, evidence was presented that both hexokinase and creatine kinase are preferentially localized in contact sites (Adams, V. et al. (1989) Biochim. Biophys. Acta 981, 213-225). The aim of the present experiments was two-fold. First, to establish methods which enable the bioenergetic aspects of energy transfer mediated by kinases in contact sites to be measured. In these experiments emphasis was on hexokinase, while 31P-NMR was the major experimental technique. Second, we wanted to develop methods which can give insight into factors playing a role in the formation of contact sites involved in energy transfer. In the latter approach, mitochondrial creatine kinase was studied using monolayer techniques.     

Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases

The mitochondrial outer membrane separates the intermembrane space from the cytosol. The whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. Experimental evidence is reviewed supporting the hypothesis of dynamic ADP compartmentation within the intermembrane space. The outer membrane creates a diffusion barrier for small molecules (adenine nucleotides, creatine phosphate, creatine etc.) causing rate-dependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the outer membrane becomes leaky, cytochrome c and apoptosis-inducing factor can be released, leading to apoptosis, and as a bioenergetic consequence the cytosolic phosphorylation potential decreases. Leaky outer membranes can be detected in saponin-skinned fibres with spectrophotometric and oxygraphic methods. This is of special interest in respect to acute impairment of mitochondria during ischaemia/reperfusion.     

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.     

Dual Electron Microscopic Localization of Mitochondrial Creatine Kinase in Brain Mitochondria

Mitochondrial creatine kinase in brain mitochondria appears to be located at two different intramitochondrial sites. By using immunogold-labeling techniques, a peripheral immunoreactivity was localized between the two boundary membranes, while an additional, central immunoreactivity was found at the crista surface. The peripheral enzyme was accessible to the antibodies after treatment of the brain mitochondria with 100-300 μg digitonin/mg mitochondrial protein, which left 75% of the activity bound to the membranes. Electron microscopic analyses revealed that 43% of the labeled, peripheral creatine kinase was bound at those places where outer membrane vesicles remained attached to the inner envelope membrane, suggesting that the enzyme is in involved in contact formation between outer and inner mitochondrial membranes. Postembedding staining of mitochondria on thin sections of brain tissue or in the isolated state led to the observation of a second location of creatine kinase inside the mitochondria, along the cristae, which was not accessible to the antibodies in isolated, digitonin-treated mitochondria.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).     

Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation

To define more clearly the interactions between mitochondrial creatine kinase and the adenine nucleotide translocase, the outer membrane of rat heart mitochondria was removed by digitonin, producing an inner membrane-matrix (mitoplast) preparation. This mitoplast fracton was well-coupled and contained a high specific activity of mitochondrial creatine kinase. Outer membrane permeabilization was documented by the loss of adenylate kinase, a soluble intermembrane enzyme, and by direct antibody inhibition of mitochondrial creatine kinase activity. With this preparation, we documented four important aspects of functional coupling. Kinetic studies showed that oxidative phosphorylation decreased the value of the ternary enzyme-substrate complex dissociation constant for MgATP from 140 to 16 microM. Two approaches were used to document the adenine nucleotide translocase specificity for ADP generated by mitochondrial creatine kinase. Exogenous pyruvate kinase (20 IU/ml) could not readily phosphorylate ADP produced by creatine kinase, since added pyruvate kinase did not markedly inhibit creatine + ATP-stimulated respiration. Additionally, when ADP was produced by mitochondrial creatine kinase, the inhibition of the translocase required 2 nmol of atractyloside/mg of mitoplast protein, while only 1 nmol/mg was necessary when exogenous ADP was added. Finally, the mass action ratio of the mitochondrial creatine kinase reaction exceeded the apparent equilibrium constant when ATP was supplied to the creatine kinase reaction by oxidative phosphorylation. Overall, these results are consistent with much data from intact rat heart mitochondria, and suggest that the outer membrane plays a minor role in the compartmentation of adenine nucleotides. Furthermore, since the removal of the outer membrane does not alter the unique coupling between oxidative phosphorylation and mitochondrial creatine kinase, we suggest that this cooperation is the result of protein-protein proximity at the inner membrane surface.     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.     

Cross-linking analysis of yeast mitochondrial outer membrane

By enrichment of contact sites between the two mitochondrial boundary membranes it has been shown that this fraction contained a high activity of glutathione transferase and hexokinase which was bound to the outer membrane pore protein (Ohlendieck, K. et al. (1986) Biochim. Biophys. Acta 860, 672-689). Therefore, an interaction between the three proteins in the contact sites has been suggested. Cross-linking experiments with isolated outer membrane of yeast mitochondria show that glutathione transferase and the pore protein are already associated in the free outer membrane. Porin appeared to adopt four different oligomeric complexes in the membrane, including interactions with a 14 kDa polypeptide, which has glutathione transferase activity. The latter polypeptide could be phosphorylated by intrinsic or extrinsic protein kinases, while the porin itself was not phosphorylated. Yeast hexokinase, when bound to the outer membrane, was able to cross-link to the pore protein.     

Role of phosphorylation of porin (VDAC) in regulation of mitochondrial outer membrane under normal conditions and alcohol intoxication

The present work is an overview of the factors regulating permeability of the outer membrane of mitochondria and the state of the channels formed by porin (voltage-dependent anion channels, VDAC). According to the accumulated data, modulation of the outer membrane permeability can be induced by endogenous phosphorylation of VDAC channels. Different protein kinases, such as protein kinase A, protein kinase C, tyrosine protein kinase, hexokinase, glycogen synthetase kinase-3β (GSK-3β), Akt and p38 kinases, were shown to be involved in VDAC phosphorylation. Among these protein kinases, alcohol-induced stress-kinases, GSK-3β, Akt, and p38 identified in mitochondria may participate in phosphorylation of porin, modulation of VDAC conductance, and regulation of the outer membrane permeability.     

Oligomeric C-terminal truncated Bax preferentially releases cytochrome c but not adenylate kinase from mitochondria, outer membrane vesicles and proteoliposomes

The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C-terminal truncated oligomeric Bax (BaxDeltaC). Micromolar concentrations of BaxDeltaC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxDeltaC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxDeltaC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxDeltaC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase-porin-adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxDeltaC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxDeltaC. BaxDeltaC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria. CONCLUSIONS: (1) BaxDeltaC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxDeltaC action. (3) A limited pool of cytochrome c becomes the first target for BaxDeltaC.     

Perspectives on the mitochondrial multiple conductance channel

A multiple conductance channel (MCC) with a peak conductance of over 1 nS is recorded from mitoplasts (mitochondria with the inner membrane exposed) using patch-clamp techniques. MCC shares many general characteristics with other intracellular megachannels, many of which are weakly selective, voltage-dependent, and calcium sensitive. A role in protein import is suggested by the transient blockade of MCC by peptides responsible for targeting mitochondrial precursor proteins. MCC is compared with the peptide-sensitive channel of the outer membrane because of similarities in targeting peptide blockade. The pharmacology and regulation of MCC by physiological effectors are reviewed and compared with the properties of the pore hypothesized to be responsible for the mitochondrial inner membrane permeability transition.     

The mitochondrial channel VDAC has a cation-selective open state

The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance "open" state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.     

Voltage-dependent anion channel (VDAC) as mitochondrial governator--thinking outside the box

Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease.     

Voltage-dependent anion channel (VDAC) as mitochondrial governator—Thinking outside the box

Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease.     

Regulation of Metabolite Flux through Voltage-Gating of VDAC Channels

The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, ``closed' state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5–11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically. Received: 4 November 1996/Revised: 8 January 1997