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.     

Modulation of the Voltage-Dependent Anion Channel (VDAC) by Glutamate1

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 ± 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na⁺, acetylcholine⁺, dopamine,⁺ and glutamate^–, relative to Cl^–, were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate^– was impermeable, while the relative permeability to acetylcholine⁺ increased and to dopamine⁺ remained unchanged. At the high concentrations (0.1–0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (±10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20–60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.     

Assessing the role of residue E73 and lipid headgroup charge in VDAC1 voltage gating

The voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane (MOM) where it regulates transport of ions and metabolites in and out of the organelle. VDAC function is extensively studied in a lipid bilayer system that allows conductance monitoring of reconstituted channels under applied voltage. The process of switching from a high-conductance state, open to metabolites, to a variety of low-conducting states, which excludes metabolite transport, is termed voltage gating and the mechanism remains poorly understood. Recent studies have implicated the involvement of the membrane-solvated residue E73 in the gating process through β-barrel destabilization. However, there has been no direct experimental evidence of E73 involvement in VDAC1 voltage gating. Here, using electrophysiology measurements, we exclude the involvement of E73 in murine VDAC1 (mVDAC1) voltage gating process. With an established protocol of assessing voltage gating of VDACs reconstituted into planar lipid membranes, we definitively show that mVDAC1 gating properties do not change when E73 is replaced by either a glutamine or an alanine. We further demonstrate that cholesterol has no effect on mVDAC1 gating characteristics, though it was shown that E73 is coordinating residue in the cholesterol binding site. In contrast, we found a pronounced gating effect based on the charge of the phospholipid headgroup, where the positive charge stimulates and negative charge suppresses gating. These findings call for critical evaluation of the existing models of VDAC gating and contribute to our understanding of VDAC's role in control of MOM permeability and regulation of mitochondrial respiration and metabolism.     

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     

New insights into the mechanism of permeation through large channels

The mitochondrial channel, VDAC, regulates metabolite flux across the outer membrane. The open conformation has a higher conductance and anionic selectivity, whereas closed states prefer cations and exclude metabolites. In this study five mutations were introduced into mouse VDAC2 to neutralize the voltage sensor. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity, and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for wild-type VDAC2. The outer membranes of mitochondria containing the mutant are permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from an anionic to a cationic preference does not substantially influence the flux of negatively charged metabolites. This finding supports our previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall.     

Key regions of VDAC1 functioning in apoptosis induction and regulation by hexokinase

The voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, and thus controls cross-talk between mitochondria and the cytosol. VDAC also serves as a site for the docking of cytosolic proteins, such as hexokinase, and is recognized as a key protein in mitochondria-mediated apoptosis. The role of VDAC in apoptosis has emerged from various studies showing its involvement in cytochrome c release and apoptotic cell death as well as its interaction with proteins regulating apoptosis, including the mitochondria-bound isoforms of hexokinase (HK-I, HK-II). Recently, the functional HK-VDAC association has shifted from being considered in a predominantly metabolic light to the recognition of its major impact on the regulation of apoptotic responsiveness of the cell. Here, we demonstrate that the HK-VDAC1 interaction can be disrupted by mutating VDAC1 and by VDAC1-based peptides, consequently leading to diminished HK anti-apoptotic activity, suggesting that disruption of HK binding to VDAC1 can decrease tumor cell survival. Indeed, understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of differing functions, all important for cell life and death. By expressing VDAC1 mutants and VDAC1-based peptides, we have identified VDAC1 amino acid residues and domains important for interaction with HK and protection against apoptosis. These include negatively- and positively-charged residues, some of which are located within β-strands of the protein. The N-terminal region of VDAC1 binds HK-I and prevents HK-mediated protection against apoptosis induced by STS, while expression of a VDAC N-terminal peptide detaches HK-I-GFP from mitochondria. These findings indicate that the interaction of HK with VDAC1 involves charged residues in several β-strands and in the N-terminal domain. Displacing HK, serving as the ‘guardian of the mitochondrion’, from its binding site on VDAC1 may thus be exploited as an approach to cancer therapy.     

Elimination and restoration of voltage dependence in the mitochondrial channel,VDAC, by graded modification with succinic anhydride

Summary The major permeability pathways of the outer mitochondrial membrane are the voltage-gated channels called VDAC. It is known that the conductance of these channels decreases as the transmembrane voltage is increased in the positive or negative direction. These channels are known to display a preference for anions over cations of similar size and valence. It was proposed (Doring & Colombini, 1985b) that a set of positive charges lining the channel may be responsible for both voltage dependence and selectivity. A prediction of this proposal is that progressive replacement of the positive charges with negative charges should at first diminish, and then restore, voltage dependence. At the same time, the channel's preference for anions over cations should diminish then reverse. Succinic anhydride was used to perform these experiments as it replaces positively charged amino groups with negatively charged carboxyl groups. When channels, which had been inserted into phospholipid membranes, were treated with moderate amounts of the anhydride, they lost their voltage dependence and preference for anions. With further succinylation, voltage dependence was regenerated while the channels became cation selective. The voltage needed to close one-half of the channels increased in those treatments in which voltage dependence was diminished. As voltage dependence was restored, the voltage needed to close half of the channels decreased. The energy difference between the open and closed state in the absence of an applied field changed little with succinylation, indicating that the procedure did not cause large changes in VDAC's structure but specifically altered those charges responsible for voltage gating and selectivity.     

Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel.

The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane. However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating. We have probed the VDAC channel by subjecting the cloned yeast (S. cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties. This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel. Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites). We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure. The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure. This process would make a natural coupling between motion of the sensor and channel gating.     

Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1

VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.     

The key role of the energized state of Saccharomyces cerevisiae mitochondria in modulations of the outer membrane channels by the intermembrane space proteins

Mitochondria of the yeast Saccharomyces cerevisiae constitute a perfect model to study the outer membrane channel modulation as besides the TOM complex channel they contain only a single isoform of the VDAC channel and it is possible to obtain viable mutants devoid of the channel. Here, we report that the fraction of the intermembrane space isolated from wild type and the VDAC channel-depleted yeast mitochondria, except of the well-known VDAC channel modulator activity, displays also the TOM complex channel modulating activity as measured in the reconstituted system and with intact mitochondria. The important factor influencing the action of both modulating activities is the energized state of mitochondria. Moreover, the presence of the VDAC channel itself seems to be crucial to properties of the intermembrane space protein (s) able to modulate the outer membrane channels because in the case of intact mitochondria quantitative differences are observed between modulating capabilities of the fractions isolated from wild type and mutant mitochondria.     

Concentration dependent ion selectivity in VDAC: a molecular dynamics simulation study

The voltage-dependent anion channel (VDAC) forms the major pore in the outer mitochondrial membrane. Its high conducting open state features a moderate anion selectivity. There is some evidence indicating that the electrophysiological properties of VDAC vary with the salt concentration. Using a theoretical approach the molecular basis for this concentration dependence was investigated. Molecular dynamics simulations and continuum electrostatic calculations performed on the mouse VDAC1 isoform clearly demonstrate that the distribution of fixed charges in the channel creates an electric field, which determines the anion preference of VDAC at low salt concentration. Increasing the salt concentration in the bulk results in a higher concentration of ions in the VDAC wide pore. This event induces a large electrostatic screening of the charged residues promoting a less anion selective channel. Residues that are responsible for the electrostatic pattern of the channel were identified using the molecular dynamics trajectories. Some of these residues are found to be conserved suggesting that ion permeation between different VDAC species occurs through a common mechanism. This inference is buttressed by electrophysiological experiments performed on bean VDAC32 protein akin to mouse VDAC.     

Mapping the ruthenium red-binding site of the voltage-dependent anion channel-1

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.     

Acidification Asymmetrically Affects Voltage-dependent Anion Channel Implicating the Involvement of Salt Bridges

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to “gate” between an open and several “closed” states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pK_a values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.     

Probing the orientation of yeast VDAC1 in vivo

Voltage dependent anion channel (VDAC) is a vital ion channel in mitochondrial outer membranes and its structure was recently shown to be a 19 stranded β-barrel. However the orientation of VDAC in the membrane remains unclear. We probe here the topology and membrane orientation of yeast Saccharomyces cerevisiae in vivo. Five FLAG-epitopes were independently inserted into scVDAC1 and their surface exposure in intact and disrupted mitochondria detected by immunoprecipitation. Functionality was confirmed by measurements of respiration. Two epitopes suggest that VDAC (scVDAC) has its C-terminus exposed to the cytoplasm whilst two others are more equivocal and, when combined with published data, suggest a dynamic behavior.     

Hexokinase-I protection against apoptotic cell death is mediated via interaction with the voltage-dependent anion channel-1: mapping the site of binding

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents.     

The mitochondrial outer membrane channel, VDAC, is regulated by a synthetic polyanion

A synthetic polyanion has been found to modulate the properties of the mitochondrial outer membrane channel, VDAC. This 10 kDa polyanion, first synthesized and described by Konig and co-workers, is a 1:2:3 copolymer of methacrylate, maleate, and styrene. It had been shown to interfere with the access of metabolites to the mitochondrial inner spaces. Here we show that, at nanomolar levels, the polyanion increases the voltage dependence of VDAC channels over 5-fold. Some channels seem to be totally blocked while others display the higher voltage dependence and are able to close at very low membrane potentials (5 mV). At 27 micrograms/ml polyanion, VDAC channels are closed while inserted into liposomes in the absence of any applied potential. The closed state of VDAC induced by the polyanion has similar properties to the closed state induced by elevated membrane potentials. The physical size of the polyanion-induced closed state (in VDAC-containing liposomes) is about 0.9 nm in radius. How this estimate fits with estimates of the channel's open state and estimated volume changes between the open and closed states, is discussed.     

线粒体电压依赖性阴离子通道及其调控功能

电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)是存在于线粒体外膜上的31kDa膜蛋白,能在膜上形成亲水性通道,调控阴离子、阳离子、ATP以及其他代谢物进出线粒体,在调节细胞代谢、维持胞内钙稳态,调节细胞凋亡和坏死等过程中发挥重要功能。现就VDAC的结构、特性、活性调节及对细胞功能的调控作一综述。     

Affixing N-terminal α-helix to the wall of the voltage-dependent anion channel does not prevent its voltage gating

The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 β-strands and an N-terminal α-helix located near the midpoint of the pore. The position of this α-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal α-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the α-helix to the wall of the β-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with β-strand 11 of the pore wall during voltage gating.     

VacA from Helicobacter pylori: a hexameric chloride channel.

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self-associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (P(Cl):P(Na) = 4.2:1), inhibition by the blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and a permeability sequence, SCN- > I- > Br- > Cl- > F, consistent with a 'weak field strength' binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s(-1)) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane-bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.