VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function.

The mitochondrial channel, VDAC, forms large (3 nm in diameter) aqueous pores through membranes. We measured ATP flow (using the luciferin/luciferase method) through these channels after reconstitution into planar phospholipid membranes. In the open state of VDAC, as many as 2 x 10(6) ATP molecules can flow through one channel per second. The half-maximum rate occurs at approximately 75 mM ATP. The permeability of a single channel for ATP is 1.1 x 10(-14) cm3/s (about 1 cm/s after correcting for cross-sectional area), which is 100 times less than the permeability for chloride and 10 times less than that for succinate. Channel closure results in a 50% reduction in conductance, showing that monovalent ions are still quite permeable, yet ATP flux is almost totally blocked. This is consistent with an electrostatic barrier that results in inversion of the selectivity of the channel and could be an example of how large channels selectively control the flow of charged metabolites. Thus VDAC is ideally suited to controlling the flow of ATP between the cytosol and the mitochondrial spaces.     

Probing tubulin-blocked state of VDAC by varying membrane surface charge

Reversible blockage of the voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane by dimeric tubulin is being recognized as a potent regulator of mitochondrial respiration. The tubulin-blocked state of VDAC is impermeant for ATP but only partially closed for small ions. This residual conductance allows studying the nature of the tubulin-blocked state in single-channel reconstitution experiments. Here we probe this state by changing lipid bilayer charge from positive to neutral to negative. We find that voltage sensitivity of the tubulin-VDAC blockage practically does not depend on the lipid charge and salt concentration with the effective gating charge staying within the range of 10-14 elementary charges. At physiologically relevant low salt concentrations, the conductance of the tubulin-blocked state is decreased by positive and increased by negative charge of the lipids, whereas the conductance of the open channel is much less sensitive to this parameter. Such a behavior supports the model in which tubulin's negatively charged tail enters the VDAC pore, inverting its anionic selectivity to cationic and increasing proximity of ion pathways to the nearest lipid charges as compared with the open state of the channel.     

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     

The Electrostatics of VDAC: Implications for Selectivity and Gating

The voltage-dependent anion channel (VDAC) is the major pathway mediating the transfer of metabolites and ions across the mitochondrial outer membrane. Two hallmarks of the channel in the open state are high metabolite flux and anion selectivity, while the partially closed state blocks metabolites and is cation selective. Here we report the results from electrostatics calculations carried out on the recently determined high-resolution structure of murine VDAC1 (mVDAC1). Poisson-Boltzmann calculations show that the ion transfer free energy through the channel is favorable for anions, suggesting that mVDAC1 represents the open state. This claim is buttressed by Poisson-Nernst-Planck calculations that predict a high single-channel conductance indicative of the open state and an anion selectivity of 1.75—nearly a twofold selectivity for anions over cations. These calculations were repeated on mutant channels and gave selectivity changes in accord with experimental observations. We were then able to engineer an in silico mutant channel with three point mutations that converted mVDAC1 into a channel with a preference for cations. Finally, we investigated two proposals for how the channel gates between the open and the closed state. Both models involve the movement of the N-terminal helix, but neither motion produced the observed voltage sensitivity, nor did either model result in a cation-selective channel, which is observed experimentally. Thus, we were able to rule out certain models for channel gating, but the true motion has yet to be determined.     

Tubulin-blocked state of VDAC studied by polymer and ATP partitioning

Recently reported functional interaction between voltage-dependent anion channel of the outer mitochondrial membrane, VDAC, and dimeric tubulin is observed as a reversible channel blockage. Using partitioning of poly-(ethylene glycol)s of different molecular weights and reversal potential measurements, we probe the size and ion selectivity of the fully open and tubulin-blocked states of VDAC reconstituted into planar lipid bilayers. While the effective radius of the channel decreases by only a factor of 1.34±0.15, the selectivity reverses from initially anionic to cationic. Directly measuring ATP partitioning we demonstrate that these changes prohibit ATP from entering the channel in its tubulin-blocked state.     

Channels in Mitochondrial Membranes: Knowns,Unknowns, and Prospects for the Future

Abstract

Rapid diffusion of hydrophilic molecules across the outer membrane of mitochondria has been related to the presence of a protein of 29 to 37 kDa, called voltage-dependent anion channel (VDAC), able to generate large aqueous pores when integrated in planar lipid bilayers. Functional properties of VDAC from different origins appear highly conserved in artificial membranes: at low transmembrane potentials, the channel is in a highly conducting state, but a raise of the potential (both positive and negative) reduces drastically the current and changes the ionic selectivity from slightly anionic to cationic. It has thus been suggested that VDAC is not a mere molecular sieve but that it may control mitochondrial physiology by restricting the access of metabolites of different valence in response to voltage and/or by interacting with a soluble protein of the intermembrane space. The latest application of the patch clamp and tip-dip techniques, however, has indicated both a different electric behavior of the outer membrane and that other proteins may play a role in the permeation of molecules. Biochemical studies, use of site-directed mutants, and electron microscopy of two-dimensional crystal arrays of VDAC have contributed to propose a monomelic β barrel as the structural model of the channel. An important insight into the physiology of the inner membrane of mammalian mitochondria has come from the direct observation of the membrane with the patch clamp. A slightly anionic., voltage-dependent conductance of 107 pS and one of 9.7 pS, K⁺-selective and ATP-sensitive, are the best characterized at the single channel level. Under certain conditions, however, the inner membrane can also show unselective nS peak transitions, possibly arising from a cooperative assembly of multiple substates.     

Dual Mechanism of Ion Permeation through VDAC Revealed with Inorganic Phosphate Ions and Phosphate Metabolites

In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a “charged brush” which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.     

The voltage-dependent anion channel

Recently, it has been recognized that there is a metabolic coupling between the cytosol and mitochondria, where the outer mitochondrial membrane (OMM), the boundary between these compartments, has important functions. In this crosstalk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of ions and metabolites across the OMM is the voltage-dependent anion channel (VDAC). The interaction of VDAC with Ca2+, ATP glutamate, NADH, and different proteins was demonstrated, and these interactions may regulate OMM permeability. This review includes information on VDAC purification methods, characterization of its channel activity (selectivity, voltage-dependence, conductance), and the regulation of VDAC channel by ligands, such as Ca2+, glutamate and ATP and touches on many aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis.     

VDAC closure increases calcium ion flux

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca(2+) flux across the outer membrane occurs mainly through VDAC. Since both Ca(2+) fluxes and VDAC are involved in apoptosis, we examined whether Ca(2+) is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca(2+) and it functions normally with or without Ca(2+). Additionally, VDAC generally shows a higher permeability to Ca(2+) in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca(2+) flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.     

VDAC channels differentiate between natural metabolites and synthetic molecules

VDAC provides the major permeability pathway through the mitochondrial outer membrane by forming voltage-gated channels with pore radius of 1.2-1.5 nm. We find that VDAC can select among comparably-charged molecules with a much smaller effective radius, 0.4-0.5 nm. The molecules studied were the nucleotides, ATP, UTP, NADH and synthetic anions, tetraglutamate (T-Glu) and 1-hydroxypyrene-3,6,8-trisulfonate (HPTS). VDAC channels were reconstituted into planar phospholipid membranes bathed in 1.0 M NaCl (buffered to pH 8.0). The nucleotides decreased the conductance of VDAC for NaCl demonstrating that they could permeate into the channel. In contrast, T-Glu and HPTS did not change the single-channel conductance, indicating exclusion from the channel. Reversal potential measurements report near ideal selectivity of Na + over T-Glu. The nucleotides increased single-channel noise as they penetrated into the channel, while T-Glu had no effect. HPTS increased noise, but unlike NADH, this was not voltage-dependent when HPTS was added asymmetrically, indicating no penetration into the channel. The differences in effective size and charge cannot explain the difference in permeation characteristics. Thus VDAC must select among these based on shape and charge distribution. We propose that the electrostatic environment within the channel has been evolutionarily selected to favor the passage of adenine nucleotides.     

Bacterial Expression,Purification and Characterization of a Rice Voltage-Dependent,Anion-Selective Channel Isoform,OsVDAC4

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 β-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies—spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in β-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large β-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.     

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum

Summary The mitochondrial outer membrane contains voltagegated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect of either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.     

VDAC closure increases calcium ion flux

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca²⁺ flux across the outer membrane occurs mainly through VDAC. Since both Ca²⁺ fluxes and VDAC are involved in apoptosis, we examined whether Ca²⁺ is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca²⁺ and it functions normally with or without Ca²⁺. Additionally, VDAC generally shows a higher permeability to Ca²⁺ in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca²⁺ flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.     

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria

Summary We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained fromParamecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0m salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl^– than to K⁺ and is impermeable to Ca⁺⁺. The procedure for obtaining VDAC and the properties of the channel are highly reproducible.VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl^– permeability suggest that there may indeed be a voltage-dependent Cl^– permeability in mitochondria.The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.     

The Physiological Properties of a Novel Family of VDAC-Like Proteins from Drosophila melanogaster

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (β-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.     

Multiple conductance channel activity of wild-type and voltage-dependent anion-selective channel (VDAC)-less yeast mitochondria.

Yeast mitoplasts (mitochondria with the outer membrane stripped away) exhibit multiple conductance channel activity (MCC) in patch-clamp experiments that is very similar to the activity previously described in mammalian mitoplasts. The possible involvement of the voltage-dependent anion-selective channel (VDAC) of the outer membrane in MCC activity was explored by comparing the channel activity in wild-type yeast mitoplasts with that of a VDAC-deletion mutant. The channel activity recorded from the mutant is essentially the same as that of the wild-type in the voltage range of -40 to 30 mV. These observations indicate that VDAC is not required for MCC activity. Interestingly, the channel activity of the VDAC-less yeast mitoplasts exhibits altered gating properties at transmembrane potentials above and below this range. We conclude that the deletion of VDAC somehow results in a modification of MCC's voltage dependence. In fact, the voltage profile recorded from the VDAC-less mutant resembles that of VDAC.     

Ca2+-dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC)

Cell function depends on the distribution of cytosolic and mitochondrial factors across the outer mitochondrial membrane (OMM). Passage of metabolites through the OMM has been attributed to the voltage-dependent anion-selective channel (VDAC), which can form a large conductance and permanently open a channel in lipid bilayers. However, recent data indicate that the transport of metabolites through the OMM is controlled in the cells. Recognizing that the bilayer studies had been commonly conducted at supraphysiological [Ca2+] and [K+], we determined the effect of Ca2+ on VDAC activity. In liposomes, the purified VDAC displays Ca2+-dependent control of the molecular cut-off size and shows Ca2+-regulated Ca2+ permeability in the physiological [Ca2+] range. In bilayer experiments, at submicromolar [Ca2+], the purified VDAC or isolated OMM does not show sustained large conductance but rather exhibits gating between a nonconducting state and various subconductance states. Ca2+ addition causes a reversible increase in the conductance and may evoke channel opening to full conductance. Furthermore, single cell imaging data indicate that Ca2+ may facilitate the cation and ATP transport across the OMM. Thus, the VDAC gating is dependent on the physiological concentrations of cations, allowing the OMM to control the passage of ions and some small molecules. The OMM barrier is likely to decrease during the calcium signal.     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999     

An N-terminal nucleotide-binding site in VDAC1: involvement in regulating mitochondrial function

In a previous study, we presented evidence for the existence of a nucleotide-binding site (NBS) in the N-terminal region of the voltage-dependent anion channel (VDAC1). In this study, further localization and possible roles of the proposed VDAC1-NBS were investigated using site-directed mutagenesis. The predicated NBS of murine VDAC1 (mVDAC1) was mutated by replacing two glycine residues with alanines or a conserved lysine residue with a serine. Expression of the G21A,G23A- and K20S-mVDAC1s in human T-REx-293 cells in which endogenous VDAC1 expression had been silenced affected cell growth and cytosolic ATP levels. While G21A,G23A-mVDAC1-expressing cells displayed growth rates similar to native-mVDAC1-expressing cells, the K20S-mVDAC1-expressing cells displayed significantly retarded growth and increased resistance to cell death. Cells expressing either mVDAC1 mutant also displayed significantly reduced cellular ATP levels. When K20S-mutant mVDAC1 was expressed in porin1-less yeast, the transformed cells grew slower on non-fermentable carbon sources, while isolated mitochondria expressing either mVDAC1 mutant showed significant reduction in ATP synthesis. Purified K20S-mVDAC1 displayed a significant decrease in [alpha-(32)P]BzATP-binding and altered channel properties, that is, reduced ion selectivity, while the G21A,G23A-mutant protein displayed only a mild reduction in channel selectivity. These results suggest that mutations in the proposed VDAC1-NBS, particularly the K20S, altered channel activity, thereby interfering with VDAC function as the major pathway for the transport of metabolites and adenine nucleotides across the outer mitochondrial membrane. Finally, involvement of the VDAC1-NBS in the control of mitochondrial ATP synthesis, cell growth and viability is discussed.     

The influence of depletion of voltage dependent anion selective channel on protein import into the yeast Saccharomyces cerevisiae mitochondria

The supply of substrates to the respiratory chain as well as of other metabolites (e.g. ATP) into inner compartments of mitochondria is crucial to preprotein import into these organelles. Transport of the compounds across the outer mitochondrial membrane is enabled by mitochondrial porin, also known as the voltage-dependent anion-selective channel (VDAC). Our previous studies led to the conclusion that the transport of metabolites through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria missing VDAC (now termed YVDAC1) is considerably restricted. Therefore we expected that depletion of YVDAC1 should also hamper protein import into the mutant mitochondria. We report here that YVDAC1-depleted mitochondria are able to import a fusion protein termed pSu9-DHFR in the amount comparable to that of wild type mitochondria, although over a considerably longer time. The rate of import of the fusion protein into YVDAC1-depleted mitochondria is dis- tinctly lower than into wild type mitochondria probably due to restricted ATP access to the intermembrane space and is additionally influenced by the way the supporting respiratory substrates are transported through the outer membrane. In the presence of ethanol, diffusing freely through lipid membranes, YVDAC1-depleted mitochondria are able to import the fusion protein at a higher rate than in the presence of external NADH which is, like ATP, transported through the outer membrane by facilitated diffusion. It has been shown that transport of external NADH across the outer membrane of YVDAC1-depleted mitochondria is supported by the protein import machinery, i.e. the TOM complex (Kmita & Budzińska, 2000, Biochim. Biophys. Acta 1509, 86-94.). Since the TOM complex might also contribute to the permeability of the membrane to ATP, it seems possible that external NADH and ATP as well as the imported preprotein could compete with one another for the passage through the outer membrane in YVDAC1-depleted mitochondria.