The voltage-dependent anion channel as a biological transistor: theoretical considerations

The voltage-dependent anion channel (VDAC) is a porin of the mitochondrial outer membrane with a bell-shaped permeability-voltage characteristic. This porin restricts the flow of negatively charged metabolites at certain non-zero voltages, and thus might regulate their flux across the mitochondrial outer membrane. Here, we have developed a mathematical model illustrating the possibility of interaction between two steady-state fluxes of negatively charged metabolites circulating across the VDAC in a membrane. The fluxes interact by contributing to generation of the membrane electrical potential with subsequent closure of the VDAC. The model predicts that the VDAC might function as a single-molecule biological transistor and amplifier, because according to the obtained calculations a small change in the flux of one pair of different negatively charged metabolites causes a significant modulation of a more powerful flux of another pair of negatively charged metabolites circulating across the same membrane with the VDAC. Such transistor-like behavior of the VDAC in the mitochondrial outer membrane might be an important principle of the cell energy metabolism regulation under some physiological conditions.     

Structure of the mitochondrial outer membrane channel derived from electron microscopy of 2D crystals

A structural model for the channel in the mitochondrial outer membrane is presented, derived from electron microscopic studies of two-dimensional crystals and inferences from the primary structure of the 30-kDa polypeptide which forms the channel. The channel is represented as a cylindrical beta-barrel, with a carbon backbone diameter of 3.8 nm. The axial projection of the cylinder is divided radially into four sectors by four interchannel contact points. These sectors are characterized in terms of their interactions with lipid and macromolecular ligands, and in terms of the presence or absence of exposed basic amino acids.     

Ultrastructure of Nitrosospira sp. X101 with crystalline outer membrane protein (COMP)

Abstract The outer membrane (OM) structure of Nitrosospira sp. X101 was studied by different electron microscopic techniques and SDS-PAGE. A crystalline outer membrane protein was visible in freeze-etched cells, occasionally seen also in the thin sectioned cells, but was difficult to see in a negatively-stained preparation. The lattice probably consists of large globular protein subunits with a hexagonal arrangement. The molecular weights of the major proteins in the cell envelope are 35 kDa, 40 kDa and 42 kDa.     

Channels in the mitochondrial outer membrane: Evidence from patch clamp studies

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane ofNeurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.     

Importance of the mitochondrial outer membrane channel as a model biological channel

The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.     

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast

Summary The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.     

Electron microscopy and image analysis of the mitochondrial outer membrane channel,VDAC

The channel protein in the outer membrane ofNeurospora crassa mitochondria, VDAC, forms extended planar crystals on the membrane. The arrays, which are induced by phospholipase A₂, are polymorphic, varying from parallelogram (P) to near-rectangular (R) geometry with increased phospholipase treatment. Computer-based analysis of projection images of negatively stained VDAC arrays indicates that the protein forms a transmembrane channel in the P array. Comparison of average images of arrays embedded in different negative stains suggests that the bore of the channel is 2–2.5 nm. The locations of functionally important lysine clusters on VDAC are inferred from the effects of succinylation on projection images of arrays negatively stained with phosphotungstate. Projection images of unstained frozen-hydrated arrays indicate the general shape of the channel and suggest each channel is formed by one 31-kDa VDAC polypeptide.     

Properties of channels in the mitochondrial outer membrane

Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6±1 subunits.     

Evidence for small pores in the outer membrane of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various ³H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [¹⁴C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.     

Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of M_r 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.     

Purification and properties of the voltage-dependent anion channel of the outer mitochondrial membrane

The methods for the purification of functionally active mitochondrial porin or voltage-dependent anion channel of the outer mitochondrial membrane are critically evaluated. Two rapid and efficient methods are now available. Both make use of a hydroxyapatite/celite column as a single chromatographic step. However, in one method with long polar head-group detergents, porin passes through the column, whereas in the other method, with shorter polar headgroup detergents, porin is first bound to the column and then eluted by the addition of salts. On the basis of these results, a model for the arrangement of porin in the detergent-protein micelles is proposed.     

Regulation of the mitochondrial outer membrane channel,VDAC

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channelin vitro raises the possibility that VDAC may perform a regulatory rolein vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.     

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).     

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.     

AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria

To date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. Using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (OM) of chloroplasts and mitochondria and exposed its N-terminal enzymatic domain to the cytosol. It was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. Mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.        

An anion-selective channel from the Pseudomonas aeruginosa outer membrane

Protein P from Pseudomonas aeruginosa outer membrane was reconstituted in lipid bilayer membranes from diphytanoylphosphatidylcholine. The reconstitution resulted in the formation of anion-selective channels with a conductance of 160 pS for 0.1 M chloride solution. The channels were at least 100-times more selective for anions than for cations as judged from zero-current membrane potentials. The single-channel conductance was dependent on the size of the different anions and saturated at higher salt concentrations suggesting single ion occupancy of the protein P channel.     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.     

Characterization and function of the mitochondrial outer membrane peptide-sensitive channel

The PSC (peptide-sensitive Channel), a cationic channel of large conductance, has been characterized in yeast and mammalian mitochondria by three different methods, tip-dip, patch clamp of giant liposomes, and planar bilayers. The yeast and mammalian PSC share the common property to be blocked by basic peptides such as pCyt OX IV (1–12)Y which contains the first 12 residues of the presequence of cytochromec oxidase subunit IV. The electrophysiological data are consistent with a translocation of the peptide through the pore. Analysis of the frequency of observation of the PSC in different fractions indicates that the channel is located in the outer mitochondrial membrane. Uptake measurements of iodinated peptides by intact mitochondria from a porin-less mutant show that the peptides are translocated through the outer membrane, presumably at the level of PSC. Among the peptides active on PSC, several, such as pCyt OX IV (1–22) and the reduced form of the mast cell degranulating peptide, induce an alteration of the voltage dependence or of the inactivation rate subsisting after washing and which is eliminated only by proteolysis of the interacting peptide. These irreversible effects may account for the variability of the properties of the PSC which would interact with cytosolic or intermembrane cations, peptides, or proteins, thus modulating the channel permeability. Finally, several lines of evidence suggest the participation of the PSC in protein translocation and some interaction with the general insertion pore of the outer membrane translocation machinery.     

Structure of the outer mitochondrial membrane analysis of x-ray diffraction from the plant membrane

X-ray diffraction from centrifugally oriented specimens of plant outer mitochondrial membranes suggests that these membranes contain prominent in-plane subunits. The short lamellar repeat which these specimens display (as low as 5.1 nm) points to a predominantly internal localization of the protein components of these membranes. The simplest model for the putative in-plane subunit consistent with autocorrelation analysis of the normal-incidence diffraction data consists of two concentric rings of electron density with diameters of (approx.) 2 and 4 nm. These rings could represent the planar projections of concentric cylindrical shells, aligned normal to the membrane surface.