Blockage Of Voltage-dependent Anion Channel Research Articles

Mitochondrial VDAC as an Electromechanical Probe of Lipid-Dependent Membrane Binding of Alpha-Synuclein

Alpha-synuclein (α-syn) is a small, intrinsically disordered cytoplasmic protein highly expressed in the central nervous system and implicated in Parkinson disease. α-Syn was found to specifically bind to native mitochondrial membranes in live cells and is known to be involved in neurodegenerative mitochondrial dysfunction and in neuroapoptosis. Thus, understanding the molecular mechanisms of α-syn interaction with model membranes mimicking the mitochondrial outer membrane (MOM) is of particular importance. Here we study the α-syn-membrane interaction on a single-molecule level using the voltage dependent anion channel (VDAC) to probe the kinetic and structural rearrangements of α-syn at the lipid membrane. We find that both the frequency and duration of the VDAC blockage by α-syn depend strongly on lipid composition. The highest interaction frequency of α-syn with VDAC is observed in anionic lipids; it drops by 100-fold in cationic lipids. The interaction frequency also increases with nonlamellar phosphatidylethanolamine content in neutral membranes. The duration of blockage is also affected by lipid composition, particularly the onset of α-syn translocation through the VDAC pore. These results support a model in which α-syn's N-terminus binding to the membrane is followed by C-terminal blockage of the VDAC pore. Comparison of electrophysiological data with macroscopic measurements of α-syn-lipid binding using Fluorescence Correlation Spectroscopy and Bilayer Overtone Analysis methods suggests that three domains of α-syn play different roles in membrane interactions and, consequently, with VDAC pore. Our results establish VDAC as a suitable electromechanical probe to report on monomeric α-syn membrane binding kinetics and dynamic conformational adaptations to the lipid charges and lipid packing stress. This system will facilitate further exploration of the well-recognized, but not well understood, regulatory roles of mitochondrial lipids in bioenergetics and cell metabolism.

Using Engineered Recombinant Tubulin Sotypes to Study VDAC Regulation

Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated in regulation of permeability of the mitochondrial voltage-dependent anion channel (VDAC). Tubulin, a structural protein of microtubules, is a heterodimer comprising α/β subunits. In cells tubulin possesses significant variability due to the expression of several tubulin genes. Seven α- and eight β-tubulin isoforms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous tubulin CTTs hindered the analysis of their interaction with VDAC reconstituted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with peptide-protein chimeras evaluated the frequency of the VDAC blockage events to demonstrate that the on-rate is highly dependent on the tail chemical composition. In that study CTT-like peptides were grafted to albumin, a protein that does not bind to the membrane surface, though it is now conclusively established that membrane binding of the tubulin body is an essential step in tubulin/VDAC interaction. Here we took an advantage of recently generated recombinant yeast α/β-tubulin constructs with CTTs from various human tubulin isotypes (Sirajuddin et al., 2014). The effects of each of the engineered constructs on the reconstituted VDAC were compared to those of w.t. yeast tubulin and recombinant tubulin without CTTs. Single-molecule studies of the tubulin-VDAC interaction showed that length and total anionic charge of the individual CTTs correlate strongly with the average duration of the blockage. In particular, at 30-mV applied voltage the characteristic time of VDAC blockage by the longer and more highly charged β3-CTT is up to hundredfold greater than that observed for the α1-CTT, with the apparent gating charge doubled. These results suggest novel implications in VDAC regulation by the diversity of tubulin isotypes found in vertebrates.

Membrane Lipid Composition Regulates Alpha-Synuclein Blockage of and Translocation through the Mitochondrial Voltage-Dependent Anion Channel

Alpha-synuclein (α-syn), an intrinsically disordered protein, is a major component of Lewy bodies, a hallmark of Parkinson's disease (PD). α-Syn has been found to interact with both inner and outer mitochondrial membranes (MOM), impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel reconstitution technique, we demonstrated that monomeric α-syn both reversibly blocks and translocates through the voltage-dependent anion channel (VDAC) at nanomolar concentrations. Considering VDAC's major role in regulating metabolite fluxes across the MOM, a functional interaction between α-syn and VDAC could be essential for physiological adaptation of mitochondria and dysfunction in PD. Here we show that the α-syn-VDAC interaction is modulated by membrane lipid composition relevant to MOM. We have found that the on-rate of VDAC blockage by α-syn increases up to 10-fold with increase of phosphoethanolamine (PE) content in phosphocholine (PC) membranes. Remarkably, the off-rate was also lipid-dependent, as seen by a 5 mV increase of the turnover potential separating regimes of blockage and translocation with the increase of PE content. We have also found that at physiologically low salt concentrations, the on-rates increase more than 10-fold compared to experiments performed in high salt, while translocation of α-syn through VDAC is impeded. α-Syn differential binding to lipid membranes was also tested using independent methods. These results suggest that the blockage of VDAC by α-syn involves α-syn interaction with the membrane and is governed by both hydrophobic and electrostatic components. Such evidence provides further support for our hypothesis that α-syn needs to bind to the membrane prior to blocking and translocating through the VDAC pore. We propose a new regulatory role of mitochondrial lipids in the (patho-)physiology of monomeric α-syn interaction with mitochondria.

Membrane-Binding Peptide Inhibits Blockage of Mitochondrial VDAC by Alpha-Synuclein: In Search of A-Synuclein Toxicity Prevention

Involvement of the intrinsically disordered protein alpha-synuclein (a-syn) in Parkinson's disease (PD) and other synucleinopathies is complicated and difficult to trace on cellular and molecular levels. Recently we established that a-syn can compromise mitochondrial function by affecting the outer membrane channel VDAC (Voltage Dependent Anion Channel). In planar lipid membranes, a-syn blocks the VDAC pore, thus reducing transport of ions and metabolites mediated by this channel. Our results suggest that a-syn is able to use VDAC as an entryway to mitochondria, where it can target specific enzymes. We proposed that binding of synuclein molecules to the lipid membrane is crucial for both a-syn blocking and translocating through VDAC. Here we report an inhibitory effect of a small membrane-binding peptide (MBP) on the a-syn interaction with VDAC. Additions of MBP in micromolar concentrations to the same side of the membrane as a-syn results in a progressive reduction of the frequency of VDAC blockage events; at 50 µM of MBP the a-syn-induced blockages of the channel are virtually abolished. Furthermore, the presence of MBP affects only the frequency of blockage events, but not their duration, suggesting a competition for membrane binding of MBP and a-syn. Similarly, MBP reduces a-syn blockage of another large beta-barrel - the a-hemolysin channel. Using the method of bilayer overtone analysis, we found that MBP, similar to a-syn, has a propensity for membrane binding, but with a much lower affinity of Kd ∼ 3 μM, compared with a-syn-membrane binding with Kd ∼ 30 nM. Our results demonstrate that it is possible to regulate a-syn blockage and translocation through VDAC by a rationally designed peptide, thus suggesting new venues in the search for small-molecule inhibitors of a-syn mitochondrial toxicity in PD and other synucleinopathies.

Alpha-Synuclein Blocks VDAC Suggesting Mechanism of Mitochondrial Regulation and Toxicity in Parkinson Disease

Alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is implicated in the etiology of Parkinson disease (PD) and a number of other neurodegenerative dementias. Though recent research demonstrates the involvement of α-syn in a variety of mitochondrial dysfunction in neurodegeneration, the molecular mechanism of α-syn toxicity and its effect on neuronal mitochondria remain vague. We demonstrate a functional interaction between α-syn and the voltage-dependent anion channel (VDAC), the major conduit for ATP and other bioenergetics metabolites in the mitochondrial outer membrane. We found that at nanomolar concentrations, the full-length α-syn, its 45 amino acid C-terminal truncated mutant, as well as β- and α-synuclein isoforms induce reversible and highly voltage-dependent blockage of VDAC reconstituted into planar lipid bilayers. Binding parameters varied with each isoform, revealing the key role of the negatively charged C-terminal of synucleins in blocking a positively charged pore of VDAC. Synuclein-blocked states of VDAC differ in ionic selectivity, implying the existence of several conformation states of synuclein molecules in the channel pore. We propose a model of α-syn interaction with VDAC, in which the negatively charged C-terminus of α-syn enters the net-positive channel pore, providing a steric block for ATP flux. Experiments with a yeast strain deficient in VDAC1 demonstrate that α-syn toxicity in yeast depends on VDAC, revealing α-syn interaction with the channel in living cells. Thus, our findings show the long-sought physiological and pathophysiological roles for monomeric α-syn, which reconcile previous observations of various synuclein effects on mitochondrial bioenergetics.

High-Affinity Interaction with VDAC Links Cytosolic Proteins to Mitochondrial Regulation in Health, Cancer, and Neurodegeneration

We address the question of how mitochondria control cell survival and cell death by studying the voltage-dependent anion channel (VDAC). VDAC, the major channel of the mitochondrial outer membrane (MOM), is a well-recognized key conduit for ATP and other bioenergetics metabolites fluxes across MOM. We have found that dimeric tubulin, the subunit of microtubule, induces highly efficient reversible blockage of VDAC reconstituted into planar lipid membranes. Although the tubulin-blocked state still conducts small ions, it is virtually impermeable to ATP. We propose that by modulating VDAC permeability for ATP and other respiratory substrates, tubulin controls mitochondrial respiration. These findings are supported by experiments with isolated mitochondria and human hepatoma cells, thus uncovering a mechanism of regulation of mitochondrial energetics by free tubulin and also suggesting how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (the Warburg effect).We also found a functional interaction between VDAC and α-synuclein (α-syn), an intrinsically disordered neuronal protein intimately associated with Parkinson disease (PD) pathogenesis. Importantly, in addition to regulation of VDAC permeability by α-syn, our data indicate that VDAC facilitates translocation of α-syn across MOM where it could target complexes of the mitochondrial respiratory chain in the inner membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. Considering that VDAC is a major conduit for respiratory substrates across the mitochondrial outer membrane, we conclude that the α-syn/VDAC functional interaction reveals the elusive physiological and pathophysiological roles for monomeric α-syn in PD and also in general neurodegeneration.

ATP transport through VDAC and the VDAC-tubulin complex probed by equilibrium and nonequilibrium MD simulations.

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Using umbrella-sampling simulations, we established the thermodynamic and kinetic components governing ATP transport across the VDAC1 channel. We found that there are several low-affinity binding sites for ATP along the translocation pathway and that the main barrier for ATP transport is located around the center of the channel and is formed predominantly by residues in the N-terminus. The binding affinity of ATP to an open channel was found to be in the millimolar to micromolar range. However, we show that this weak binding increases the ATP translocation probability by about 10-fold compared with the VDAC pore in which attractive interactions were artificially removed. Recently, it was found that free dimeric tubulin induces a highly efficient, reversible blockage of VDAC reconstituted into planar lipid membranes. It was proposed that by blocking VDAC permeability for ATP/ADP and other mitochondrial respiratory substrates tubulin controls mitochondrial respiration. Using the Rosetta protein-protein docking algorithm, we established a tentative structure of the VDAC-tubulin complex. An extensive set of equilibrium and nonequilibrium (under applied electric field) molecular dynamics (MD) simulations was used to establish the conductance of the open and blocked channel. It was found that the presence of the unstructured C-terminal tail of tubulin in the VDAC pore decreases its conductance by more than 40% and switches its selectivity from anionic to cationic. The subsequent 1D potential of mean force (PMF) computations for the VDAC-tubulin complex show that the state renders ATP transport virtually impossible. A number of residues pivotal for tubulin binding to the channel were identified that help to clarify the molecular details of VDAC-tubulin interaction and to provide new insight into the mechanism of the control of mitochondria respiration by VDAC.

S.27.01 Mitochondria and Aβ metabolism

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm, and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. For example, one of the long-standing puzzles was that in permeabilized cells, adenine nucleotide translocase is less accessible to cytosolic ADP than in isolated mitochondria. Still another puzzle was that, according to channel-reconstitution experiments, voltage regulation of VDAC is limited to potentials exceeding 30 mV, which are believed to be much too high for MOM. We have solved these puzzles and uncovered multiple new functional links by identifying a missing player in the regulation of VDAC and, hence, MOM permeability — the cytoskeletal protein tubulin. We have shown that, depending on VDAC phosphorylation state and applied voltage, nanomolar to micromolar concentrations of dimeric tubulin induce functionally important reversible blockage of VDAC reconstituted into planar phospholipid membranes. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective “gating charge” of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology are. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity. The findings, obtained in a channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Taken together, these results suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC interaction with tubulin at the mitochondria–cytosol interface. Immediate physiological implications include new insights into serine/threonine kinase signaling pathways, Ca2 + homeostasis, and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. In the present review, we speculate how these findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells: REGULATION BY FREE TUBULIN AND ERASTIN

Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms--VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state. Cells expressing predominantly VDAC3 were least sensitive to depolarization induced by increased free tubulin. In planar lipid bilayers, free tubulin inhibited VDAC1 and VDAC2 but not VDAC3. Erastin, a compound that interacts with VDAC, blocked and reversed mitochondrial depolarization after microtubule destabilizers in intact cells and antagonized tubulin-induced VDAC blockage in planar bilayers. In conclusion, free tubulin inhibits VDAC1/2 and limits mitochondrial metabolism in HepG2 cells, contributing to the Warburg phenomenon. Reversal of tubulin-VDAC interaction by erastin antagonizes Warburg metabolism and restores oxidative mitochondrial metabolism.

Current Insights into the Molecular Mechanisms of VDAC-Tubulin Interaction

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Recently, we found that free dimeric tubulin induces highly efficient reversible blockage of VDAC reconstituted into planar lipid membranes. Although the tubulin-blocked state is still conductive for small ions, it has reduced dimensions compared with open state, reversed ionic selectivity, and most importantly is virtually impermeable for ATP. We proposed that by blocking VDAC permeability, tubulin controls mitochondrial respiration. We suggested a model of VDAC-tubulin interaction where the negatively charged C-terminal tail (CTT) of tubulin partially blocks VDAC pore. The corresponding effective “gating charge” of the blockage is impressively high of about 10-14 elementary charges, which compares well with the gating charge of the most voltage-sensitive channels. Surprisingly, this gating charge only mildly depends on salt concentration and significantly exceeds the total number of negative charges of tubulin CTT. This indicates the applied field interacts not only with CTT but also with VDAC charges and dipoles, with all of them contributing to the effective gating charge. Our results also demonstrate that additional steps are involved in the blockage process. We found that lipid-dependent tubulin binding to the membrane greatly impacts VDAC blockage by tubulin. At 100 mM KCl, the charged lipids significantly affect the conductance of the tubulin-blocked state and the on-rate the blockage, but even at 1 M KCl the lipids with different non-lamellar tendencies were shown to change the on-rate by two orders of magnitude. These data allowed us to conclude that hydrophobic interactions between the tubulin and the membrane represent an essential component of the multistep process of VDAC blockage by tubulin, providing a new instructive example of lipid-controlled protein-protein interactions.

Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding

The voltage dependent anion channel (VDAC) is involved in regulation of metabolite flux across the mitochondrial outer membrane (MOM). Hexokinse II (HK2) is known to bind the MOM where it phosphorylates glucose into glucose-6-phosphate (G6P). High expression of HXK2 is a common phenotype of many cancers, where its concentration can be 200 times of that in noncancerous cells, and is implicated in the Warburg effect. It is believed that VDAC serves as a HXK2 binding site in the MOM. The 15 amino acid N-terminal sequence of HXK2 is responsible for mitochondrial binding and, when conjugated to TAT (TAT-HK2), binds to mitochondria with higher affinity than native HXK2, causing HXK2 detachment. We have previously found that dimeric tubulin reversibly binds and partially blocks VDAC inhibiting metabolite flux across the MOM. Now we show that this binding can be attenuated by TAT-HXK2 peptide as well as by full length HXK2. We have found that TAT-HXK2 and recombinant full length HXK2 inhibit tubulin blockage of VDAC reconstituted into planar lipid bilayers without altering characteristic channel properties such as single channel conductance and selectivity. Binding of HXK2 to VDAC is verified by the generation of high-frequency excess current noise without channel closure. HXK2 bound to VDAC prevents subsequent tubulin binding, but only when added before tubulin, and inhibits tubulin-induced VDAC blockage in a dose dependent manner. Moreover, G6P, which is known to cause HXK2 detachment from the MOM, fully reverses the inhibition of tubulin-VDAC binding. This suggests that HXK2 detachment from VDAC (and hence the MOM) is caused by a HXK2 conformational change upon G6P binding. Thus we propose a novel mechanism of mitochondrial respiration control in cancer cells through the competition between HXK2 and tubulin for VDAC binding.

Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel

Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding the many cellular processes. Recently, we have found that dimeric αβ-tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of mitochondrial outer membrane. Here, we show that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude.

VDAC Phosphorylation in Control of Mitochondrial Respiration

Tubulin has been recently identified as a powerful regulator of voltage dependent anion channel (VDAC). Dimeric tubulin induces reversible blockage of VDAC reconstituted into planar lipid membranes and blocks the flux of ATP/ADP across the mitochondrial outer membrane. Here we show that phosphorylation of VDAC by cytosolic kinases protein kinase A (PKA) and glycogen synthase kinase-3β (GSK3β) increases the on-rate of tubulin binding by orders of magnitude, but only for tubulin at the cis (or cytosolic) side of the membrane. This and the fact that the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 are in agreement with our findings that phosphorylation state of VDAC determines its blockage by tubulin. When HepG2 cells were treated with the microtubule-destabilizing agent colchicine that is known to increase free tubulin, mitochondrial potential, as measured by tetramethylrhodamine methyester (TMRM) uptake, decreased. Inhibition of PKA activity with H89 increased mitochondrial potential and, furthermore, reversed mitochondrial depolarization induced by colchicine. To further explore a role VDAC-tubulin interaction in cell proliferation and survival, we studied the effect of erastin, the selective anti-tumour agent involved in the RAS-RAF-MEK signalling pathway, on VDAC blockage by tubulin. We found that erastin reverses tubulin blockage of VDAC isolated from HepG2 cells, promoting the unblocked state of the channel. Our findings suggest a novel functional link between serine/threonine kinase signalling pathways, mitochondrial respiration, and highly dynamic microtubule network which is characteristic of carcinogenesis and cell proliferation.

VDAC Regulation by Tubulin and its Physiological Implications

A significant portion of the mitochondria outer membrane (MOM) control functions is realized through the voltage-dependent anion channel (VDAC) that constitutes a major pathway for ATP/ADP across MOM. We have recently found that dimeric tubulin induces reversible blockage of VDAC reconstituted into planar lipid membrane and dramatically reduces mitochondrial respiration. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective “gating charge” of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity. The findings, obtained in the channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Thus, regulation of VDAC by tubulin that was first discovered in vitro, was then related to the action of this protein in vivo. The mechanism of VDAC-tubulin interaction appears to be more complex than was initially thought; in particular, it is very sensitive to the membrane lipid composition. Tubulin adsorption on the membrane affects the on-rate of tubulin-VDAC binding and depends on the lipid packing stress as well as on the membrane surface charge. Immediate physiological implications include new insights into the function of serine/threonine kinase signaling pathways, microtubule activity in the case of the highly dynamic microtubule network in cancerogenesis and cell proliferation, and the role of lipids in mitochondrial dynamics and MOM permeabilization characteristic of apoptosis.

A Putative Role of Voltage-Dependent Anion Channel in Ischemia

Ischemia is characterized by the inhibition of ATP production via oxidative phosphorylation due to oxygen deprivation, which leads to mitochondria depolarization. Impaired mitochondrial respiration involves the shift to an anaerobic metabolism and ionic imbalance that leads to a decrease of cytosolic pH and generation of reactive oxygen species (ROS). The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane, which regulates the passage of ATP, ADP, and other mitochondrial respiratory substrates across this membrane. Motivated by these facts, we are interested in establishing a possible functional link between the physiological features of ischemia and VDAC. It has been reported that cytosolic acidification protects cells from death during early ischemic stress. However, the mechanism of this protection is still unclear. Ischemia is followed by a reperfusion stage in which pH is restored to neutral leading to cell death. In experiments with VDAC reconstituted into planar lipid membranes we show that VDAC voltage-induced closure increases at low pH. Interestingly, the channel fully re-opens after the pH is restored to neutral. The cytoskeleton protein, dimeric tubulin regulates mitochondrial membrane potential by blocking VDAC permeability for ATP (Sheldon et al., 2011; Gurnev et. al, 2011). We found that, synergistically with the effect on gating, acidification significantly promotes VDAC blockage by tubulin, and speculate here that acidic pH-induced VDAC inhibition could potentially be involved in the protective effect of acidosis and also in the mitochondria depolarization usually detected during ischemia. A possible effect of ROS on VDAC permeability was assessed by probing VDAC functioning in planar membranes containing oxidized lipids. We have found that oxidized lipids affect VDAC interaction with tubulin. The relevance of these observations for the mechanisms of ischemia and apoptosis is discussed.

Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.

Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.

Governing Respiration: Tubulin's C-Terminus Interaction with VDAC

Mitochondria have long been known to localize within and move along the tubulin-microtubule network. It was shown that tubulin binds to isolated mitochondria with high-affinity and specifically associates with the mitochondrial voltage-dependent anion channel (VDAC). We found that nanomolar concentrations of dimeric tubulin vastly increase VDAC sensitivity to voltage allowing for VDAC blockage at low transmembrane potentials. Tubulin interaction with VDAC requires the presence of anionic C-terminal tails (CTT) on the intact protein. Tubulin with proteolytically cleaved CTTs does not block the channel. Actin, also an acidic protein but lacking CTTs, does not induce VDAC blockage. Two synthetic peptides with the sequences of mammalian α and β brain tubulin CTT do not induce detectable channel closure up to micromolar concentrations. These results suggest a completely new role for dimeric tubulin and its charged CTT. We propose a model for tubulin-VDAC channel interaction in which the tubulin CTT penetrates into the channel lumen, potentially reaching through the channel and interacting with a positively charged domain of VDAC. We found that tubulin/VDAC interaction is greatly dependent on the state of VDAC phosphorylation. Remarkably, phosphorylated VDAC is more than an order of magnitude more sensitive to tubulin-induced closure than dephosphorylated VDAC.Tubulin addition to isolated mitochondria increases Km for ADP and lowers oxygen consumption, most likely by restricting flux of ADP through VDAC. Thus interaction of tubulin CTT with VDAC blocks nucleotide passage into and out of mitochondria, thereby regulating oxidative phosphorylation. Examination of the evolution of CTT sequences in mitochondria-containing cells reveals high conservation of charge and length of the tails. Considering the known conservation of VDAC folding pattern throughout mitochondria-containing eukaryotes, we propose that this interaction is widespread and ancient.