Tatiana K. Rostovtseva

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.

Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration

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, dependent on the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent Km for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria–cytosol interface. This tubulin–VDAC interaction requires tubulin anionic C-terminal tail (CTT) peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in CTT throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character are only found in cells that do not have mitochondria.

ATP Flux Is Controlled by a Voltage-gated Channel from the Mitochondrial Outer Membrane

A voltage-gated channel, called VDAC (mitochondrial porin) is known to be responsible for most of the metabolite flux across the mitochondrial outer membrane. Here, direct measurements of ATP flux through VDAC channels reconstituted into planar phospholipid membranes establish that VDAC is sufficient to provide passage for ATP efflux from mitochondria. Further, the gating of the channel can shut down ATP flux completely while, simultaneously, allowing the flow of small ions. Thus, these channels are ideally suited to control ATP flux through the mitochondrial outer membrane and, consequently, mitochondrial function. The block to ATP flow through the closed state is likely to be not steric but electrostatic.

Residue Ionization and Ion Transport through OmpF Channels

Single trimeric channels of the general bacterial porin, OmpF, were reconstituted into planar lipid membranes and their conductance, selectivity, and open-channel noise were studied over a wide range of proton concentrations. From pH 1 to pH 12, channel transport properties displayed three characteristic regimes. First, in acidic solutions, channel conductance is a strong function of pH; it increases by approximately threefold as the proton concentration decreases from pH 1 to pH 5. This rise in conductance is accompanied by a sharp increase in cation transport number and by pronounced open-channel low-frequency current noise with a peak at approximately pH 2.5. Random stepwise transients with amplitudes at approximately 1/5 of the monomer conductance are major contributors to this noise. Second, over the middle range (pH 5/pH 9), channel conductance and selectivity stay virtually constant; open channel noise is at its minimum. Third, over the basic range (pH 9/pH 12), channel conductance and cation selectivity start to grow again with an onset of a higher frequency open-channel noise. We attribute these effects to the reversible protonation of channel residues whose pH-dependent charge influences transport by direct interactions with ions passing through the channel.

Voltage Gating of VDAC Is Regulated by Nonlamellar Lipids of Mitochondrial Membranes

Evidence is accumulating that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and nonlamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another nonlamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of the voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two nonlamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.

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

Background: Metabolites generating mitochondrial membrane potential (ΔΨ) enter through voltage-dependent anion channels (VDAC). Results: VDAC3 contributed to ΔΨ formation more than VDAC1/2. VDAC3 knockdown decreased ATP and NADH/NAD+. Tubulin decreased VDAC1/2 not VDAC3 conductance, an effect antagonized by erastin. Conclusion: Tubulin negatively modulates mitochondrial metabolism by closing VDAC1/2. Significance: Antagonism of tubulin-dependent VDAC closure reverses mitochondrial suppression in Warburg metabolism. 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.

Dynamics of Nucleotides in VDAC Channels: Structure-Specific Noise Generation

Nucleotide penetration into the voltage-dependent mitochondrial ion channel (VDAC) reduces single-channel conductance and generates excess current noise through a fully open channel. VDAC channels were reconstituted into planar phospholipid membranes bathed in 1.0 M NaCl. At a given nucleotide concentration, the average decrease in small-ion channel conductance induced by mononucleotides ATP, ADP, AMP, and UTP and dinucleotides beta- and alpha-NADH, NAD, and NADPH are very close. However, the excess current noise is about seven times higher in the presence of NADPH than in the presence of ATP and is about 40 times higher than in the presence of UTP. The nucleotide-generated low-frequency noise obeys the following sequence: beta-NADPH > beta-NADH = alpha-NADH > ATP > ADP > beta-NAD > or = AMP > UTP. Measurements of bulk-phase diffusion coefficients and of the effective charge of the nucleotides in 1.0 M NaCl suggest that differences in size and charge cannot be the major factors responsible for the ability to generate current noise. Thus, although the ability of nucleotides to partition into the channels pore, as assessed by the reduction in conductance, is very similar, the ability to generate current noise involves a detailed recognition of the three-dimensional structure of the nucleotide by the VDAC channel. A possible mechanism for this selectivity is two noise-generating processes operating in parallel.

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.

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.

α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease

Background: The intrinsically disordered protein α-synuclein, a hallmark of Parkinson disease, is involved in mitochondrial dysfunction in neurodegeneration and directly interacts with mitochondria. Results: α-Synuclein regulates VDAC permeability; α-synuclein toxicity in yeast depends on VDAC. Conclusion: α-Synuclein both blocks VDAC and translocates via this channel across the mitochondrial outer membrane. Significance: (Patho)physiological roles of monomeric α-synuclein may originate from its interaction with VDAC. Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.