Nucleotide Interactions of the Human Voltage-dependent Anion Channel

The voltage-dependent anion channel (VDAC) mediates and gates the flux of metabolites and ions across the outer mitochondrial membrane and is a key player in cellular metabolism and apoptosis. Here we characterized the binding of nucleotides to human VDAC1 (hVDAC1) on a single-residue level using NMR spectroscopy and site-directed mutagenesis. We find that hVDAC1 possesses one major binding region for ATP, UTP, and GTP that partially overlaps with a previously determined NADH binding site. This nucleotide binding region is formed by the N-terminal α-helix, the linker connecting the helix to the first β-strand and adjacent barrel residues. hVDAC1 preferentially binds the charged forms of ATP, providing support for a mechanism of metabolite transport in which direct binding to the charged form exerts selectivity while at the same time permeation of the Mg²⁺-complexed ATP form is possible.     

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.     

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.     

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     

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.     

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.     

VDAC structure, selectivity, and dynamics

VDAC channels exist in the mitochondrial outer membrane of all eukaryotic organisms. Of the different isoforms present in one organism, it seems that one of these is the canonical VDAC whose properties and 3D structure are highly conserved. The fundamental role of these channels is to control the flux of metabolites between the cytosol and mitochondrial spaces. Based on many functional studies, the fundamental structure of the pore wall consists of one α helix and 13 β strands tilted at a 46° angle. This results in a pore with an estimated internal diameter of 2.5nm. This structure has not yet been resolved. The published 3D structure consists of 19 β strands and is different from the functional structure that forms voltage-gated channels. The selectivity of the channel is exquisite, being able to select for ATP over molecules of the same size and charge. Voltage gating involves two separate gating processes. The mechanism involves the translocation of a positively charged portion of the wall of the channel to the membrane surface resulting in a reduction in pore diameter and volume and an inversion in ion selectivity. This mechanism is consistent with experiments probing changes in selectivity, voltage gating, kinetics and energetics. Other published mechanisms are in conflict with experimental results. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

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。     

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.     

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.     

Pyridoxal 5-phosphate, Phenyl Phosphate and Acetyl Phosphate, as Inhibitors of Photophosphorylation Competitive with Phosphate

Pyridoxal 5-phosphate, phenyl phosphate and acetyl phosphate,as well as rß-naphthyl monophosphate, inhibited photophosphorylationof spinach chloroplasts competitively with P_i and noncompetitivelywith ADP. The apparent dissociation constant of the inhibitor-enzymecomplex (K_i) values of pyridoxal 5-phosphate, phenyl phosphateand acetyl phosphate for the P_i site were 1.1, 3.8 and 2.4 _mM,respectively. These organic phosphates inhibited Ca²⁺-ATPaseof the isolated coupling factor 1 (CF₁) (EC 3.6.1.3 [EC] ) noncompetitivelywith ATP. AMP, creatine phosphate, fructose 1,6-bisphosphate,glucose 6-phosphate, 3-phosphoglyceric acid, ribose 5-phosphateand PP_i did not significantly inhibit photophosphorylation.Like rß-naphthyl monophosphate, pyridoxal 5-phosphateand phenyl phosphate inhibited photophosphorylation and thecoupled electron transport, but were almost without effect onthe basal electron transport. On the other hand, acetyl phosphateconsiderably inhibited photophosphorylation, but had almostno effect on the coupled electron transport rate and the basalrate. The results suggest that these organic phosphates inhibitphotophosphorylation by binding at the P_i site on the activecenter of CF₁ and that their binding inhibits the ATPase activityof isolated CF₁. These four organic phosphates which inhibited photophosphorylationcompetitively with Pi could not substitute for ADP or ATP ininhibiting ferricyanide photoreduction by decreasing H⁺-permeabilitythrough CF₁ and in protecting the ATPase of isolated CF₁ againstcold-anion inactivation. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H.S. (Received May 25, 1981; Accepted September 28, 1981)     

Inhibition of the mitochondrial inner membrane anion channel by dicyclohexylcarbodiimide. Evidence for a specific transport pathway

Electrophoretic uniport of anions through the inner mitochondrial membrane can be activated by alkaline pH or by depleting the matrix of divalent cations. It has also been suggested that, in the presence of valinomycin and potassium, respiration can also activate anion uniport. We have proposed that a single pathway is responsible for all three of these transport processes (Garlid, K. D., and Beavis, A. D. (1986) Biochim. Biophys. Acta 853, 187-204). We now present evidence that like the "pH-dependent" pore the divalent cation-regulated pore and the "respiration-induced" pore are blocked by N,N'-dicyclohexylcarbodiimide (DCCD). Moreover, the kinetics of inhibition of the latter two pathways are identical and exhibit a second order rate constant of 2.6 X 10(-3) (nmol DCCD/mg)-1.min-1. DCCD inhibits the uniport of Cl-, phosphate, malate, and other lipophobic anions completely, but it has no effect on the classical electroneutral phosphate and dicarboxylate carriers. In Mg2+-depleted mitochondria DCCD partially inhibits the transport of SCN-; however, in Mg2+-containing mitochondria and at low pH, no inhibition is observed. Furthermore, in DCCD-treated mitochondria, even following depletion of Mg2+, the transport of SCN- is independent of pH. These results lead us to conclude that two pathways for anion uniport exist: a specific, regulated pathway which can conduct a wide variety of anions and a nonregulated pathway through the lipid bilayer which only conducts lipid-soluble ions.     

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.     

Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores

Voltage-gated ion channels sense voltage by shuttling arginine residues located in the S4 segment across the membrane electric field. The molecular pathway for this arginine permeation is not understood, nor is the filtering mechanism that permits passage of charged arginines but excludes solution ions. We find that substituting the first S4 arginine with smaller amino acids opens a high-conductance pathway for solution cations in the Shaker K(+) channel at rest. The cationic current does not flow through the central K(+) pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain. The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane arginine permeation. We propose that when S4 moves it ratchets between conformations in which one arginine after another occupies and occludes to ions the narrowest part of this pathway.     

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.     

Associative mechanism for phosphoryl transfer: a molecular dynamics simulation of Escherichia coli adenylate kinase complexed with its substrates

The ternary complex of Escherichia coli adenylate kinase (ECAK) with its substrates adenosine monophosphate (AMP) and Mg-ATP, which catalyzes the reversible transfer of a phosphoryl group between adenosine triphosphate (ATP) and AMP, was studied using molecular dynamics. The starting structure for the simulation was assembled from the crystal structures of ECAK complexed with the bisubstrate analog diadenosine pentaphosphate (AP(5)A) and of Bacillus stearothermophilus adenylate kinase complexed with AP(5)A, Mg(2+), and 4 coordinated water molecules, and by deleting 1 phosphate group from AP(5)A. The interactions of ECAK residues with the various moieties of ATP and AMP were compared to those inferred from NMR, X-ray crystallography, site-directed mutagenesis, and enzyme kinetic studies. The simulation supports the hypothesis that hydrogen bonds between AMP's adenine and the protein are at the origin of the high nucleoside monophosphate (NMP) specificity of AK. The ATP adenine and ribose moieties are only loosely bound to the protein, while the ATP phosphates are strongly bound to surrounding residues. The coordination sphere of Mg(2+), consisting of 4 waters and oxygens of the ATP beta- and gamma-phosphates, stays approximately octahedral during the simulation. The important role of the conserved Lys13 in the P loop in stabilizing the active site by bridging the ATP and AMP phosphates is evident. The influence of Mg(2+), of its coordination waters, and of surrounding charged residues in maintaining the geometry and distances of the AMP alpha-phosphate and ATP beta- and gamma-phosphates is sufficient to support an associative reaction mechanism for phosphoryl transfer.     

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.     

Bax increases the pore size of rat brain mitochondrial voltage-dependent anion channel in the presence of tBid

Voltage-dependent anion channel (VDAC), Bax, and tBid play a central role in apoptosis regulation but their functioning is still very controversial. VDAC forms voltage gated pore in planar lipid bilayers, and acts as the pathway for the movement of substances in and out of the mitochondria by passive diffusion. Here we report that there is increase in the pore size of VDAC in the presence of Bax and tBid through bilayer electrophysiological experiments. We hereby hypothesize that this increase in pore size might cause swelling in the mitochondria, leading to the rupture of mitochondrial outer membrane and release of cytochrome c causing brain cell death.     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.