Restricting α-synuclein transport into mitochondria by inhibition of α-synuclein-VDAC complexation as a potential therapeutic target for Parkinson's disease treatment.

Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

Voltage-activated complexation of α-synuclein with three diverse ß-barrel channels: VDAC, MspA, and α-hemolysin.

Voltage-activated complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or α-synuclein (αSyn). However, the roles played by VDAC's characteristic attributes-its anion selectivity and voltage gating behavior-have remained unclear. Here, we compare in vitro measurements of voltage-activated complexation of αSyn with three well-characterized ß-barrel channels-VDAC, MspA, and α-hemolysin-that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels are observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play a role. The reaction free energy landscapes thus calculated reveal a non-trivial dependence of the αSyn/channel complex stability on the surface density of αSyn.

Correction to: Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC.

In the published article, an error was noticed and this has been corrected with this erratum publication.

Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC.

An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC ß-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.

Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel.

The microtubule protein tubulin is a heterodimer comprising α/ß subunits, in which each subunit features multiple isotypes in vertebrates. For example, seven α-tubulin and eight ß-tubulin isotypes in the human tubulin gene family vary mostly in the length and primary sequence of the disordered anionic carboxyl-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. Here, we demonstrate that it may be a key feature of tubulin's role in regulation of the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC). Using recombinant yeast α/ß-tubulin constructs with α-CTTs, ß-CTTs, or both from various human tubulin isotypes, we probed their interactions with VDAC reconstituted into planar lipid bilayers. A comparative study of the blockage kinetics revealed that either α-CTTs or ß-CTTs block the VDAC pore and that the efficiency of blockage by individual CTTs spans 2 orders of magnitude, depending on the CTT isotype. ß-Tubulin constructs, notably ß3, blocked VDAC most effectively. We quantitatively described these experimental results using a physical model that accounted only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. Based on these results, we speculate that the effectiveness of VDAC regulation by tubulin depends on the predominant tubulin isotype in a cell. Consequently, the fluxes of ATP/ADP through the channel could vary significantly, depending on the isotype, thus suggesting an intriguing link between VDAC regulation and the diversity of tubulin isotypes present in vertebrates.

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin.

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different ß-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of "polymers pushing polymers" is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.

Real-Time Nanopore-Based Recognition of Protein Translocation Success.

A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here, we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two "selectivity tags"-regions of different but uniform charge density-at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease.

Cation-Selective Channel Regulated by Anions According to Their Hofmeister Ranking.

Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H2 PO4-

Tubulin tail sequences and post-translational modifications regulate closure of mitochondrial voltage-dependent anion channel (VDAC).

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and ß-tubulin subunits. It was unclear whether the α- and ß-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the ß-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40.

Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning ß-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function.

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

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.

Alpha-synuclein lipid-dependent membrane binding and translocation through the α-hemolysin channel.

Gauging the interactions of a natively unfolded Parkinson disease-related protein, alpha-synuclein (α-syn) with membranes and its pathways between and within cells is important for understanding its pathogenesis. Here, to address these questions, we use a robust ß-barrel channel, α-hemolysin, reconstituted into planar lipid bilayers. Transient, ~95% blockage of the channel current by α-syn was observed when 1), α-syn was added from the membrane side where the shorter (stem) part of the channel is exposed; and 2), the applied potential was lower on the side of α-syn addition. While the on-rate of α-syn binding to the channel strongly increased with the applied field, the off-rate displayed a turnover behavior. Statistical analysis suggests that at voltages >50 mV, a significant fraction of the α-syn molecules bound to the channel undergoes subsequent translocation. The observed on-rate varied by >100 times depending on the bilayer lipid composition. Removal of the last 25 amino acids from the highly negatively charged C-terminal of α-syn resulted in a significant decrease in the binding rates. Taken together, these results demonstrate that ß-barrel channels may serve as sensitive probes of α-syn interactions with membranes as well as model systems for studies of channel-assisted protein transport.

Cationic cell-penetrating peptide binds to planar lipid bilayers containing negatively charged lipids but does not induce conductive pores.

Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 µM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 µM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes.

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 αß-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.

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

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

Inversion of membrane surface charge by trivalent cations probed with a cation-selective channel.

We demonstrate that the cation-selective channel formed by gramicidin A can be used as a reliable sensor for studying the multivalent ion accumulation at the surfaces of charged lipid membranes and the "charge inversion" phenomenon. In asymmetrically charged membranes with the individual leaflets formed from pure negative and positive lipids bathed by 0.1 M CsCl solutions the channel exhibits current rectification, which is comparable to that of a typical n/p semiconductor diode. We show that even at these highly asymmetrical conditions the channel conductance can be satisfactorily described by the electrodiffusion equation in the constant field approximation but, due to predictable limitations, only when the applied voltages do not exceed 50 mV. Analysis of the changes in the voltage-dependent channel conductance upon addition of trivalent cations allows us to gauge their interactions with the membrane surface. The inversion of the sign of the effective surface charge takes place at the concentrations, which correlate with the cation size. Specifically, these concentrations are close to 0.05 mM for lanthanum, 0.25 mM for hexaamminecobalt, and 4 mM for spermidine.

Altered plasmodial surface anion channel activity and in vitro resistance to permeating antimalarial compounds.

Erythrocytes infected with malaria parasites have increased permeability to various solutes. These changes may be mediated by an unusual small conductance ion channel known as the plasmodial surface anion channel (PSAC). While channel activity benefits the parasite by permitting nutrient acquisition, it can also be detrimental because water-soluble antimalarials may more readily access their parasite targets via this channel. Recently, two such toxins, blasticidin S and leupeptin, were used to select mutant parasites with altered PSAC activities, suggesting acquired resistance via reduced channel-mediated toxin uptake. Surprisingly, although these toxins have similar structures and charge, we now show that reduced permeability of one does not protect the intracellular parasite from the other. Leupeptin accumulation in the blasticidin S-resistant mutant was relatively preserved, consistent with retained in vitro susceptibility to leupeptin. Subsequent in vitro selection with both toxins generated a double mutant parasite having additional changes in PSAC, implicating an antimalarial resistance mechanism for water-soluble drugs requiring channel-mediated uptake at the erythrocyte membrane. Characterization of these mutants revealed a single conserved channel on each mutant, albeit with distinct gating properties. These findings are consistent with a shared channel that mediates uptake of ions, nutrients and toxins. This channel's gating and selectivity properties can be modified in response to in vitro selective pressure.

Tunable Electromechanical Nanopore Trap Reveals Populations of Peripheral Membrane Protein Binding Conformations.

We demonstrate that a naturally occurring nanopore, the voltage-dependent anion channel (VDAC) of the mitochondrion, can be used to electromechanically trap and interrogate proteins bound to a lipid surface at the single-molecule level. Electromechanically probing α-synuclein (αSyn), an intrinsically disordered neuronal protein intimately associated with Parkinson's pathology, reveals wide variation in the time required for individual proteins to unbind from the same membrane surface. The observed distributions of unbinding times span up to 3 orders of magnitude and depend strongly on the lipid composition of the membrane; surprisingly, lipid membranes to which αSyn binds weakly are most likely to contain subpopulations in which electromechanically driven unbinding is very slow. We conclude that unbinding of αSyn from the membrane surface depends not only on membrane binding affinity but also on the conformation adopted by an individual αSyn molecule on the membrane surface.

Effect of a post-translational modification mimic on protein translocation through a nanopore.

Post-translational modifications (PTMs) of proteins are recognized as crucial components of cell signaling pathways through modulating folding, altering stability, changing interactions with ligands, and, therefore, serving multiple regulatory functions. PTMs occur as covalent modifications of the protein's amino acid side chains or the length and composition of their termini. Here we study the functional consequences of PTMs for α-synuclein (αSyn) interactions with the nanopore of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. PTMs were mimicked by a divalent Alexa Fluor 488 sidechain attached separately at two positions on the αSyn C-terminus. Using single-channel reconstitution into planar lipid membranes, we find that such modifications change interactions drastically in both efficiency of VDAC inhibition by αSyn and its translocation through the VDAC nanopore. Analysis of the on/off kinetics in terms of an interaction "quasipotential" allows the positions of the C-terminal modifications to be determined with an accuracy of about three residues. Moreover, our results uncover a previously unobserved mechanism by which cytosolic proteins control ß-barrel channels and thus a new regulatory function for PTMs.

The antiarrhythmic compound efsevin directly modulates voltage-dependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake.

BACKGROUND AND PURPOSE: The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevin-VDAC2 interaction. EXPERIMENTAL APPROACH: To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligand-protein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevin-induced modulation of VDAC2 were analysed in HL-1 cardiomyocytes. KEY RESULTS: In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anion-selective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the pore-lining N-terminal α-helix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevin-responsiveness. Upon heterologous expression in HL-1 cardiomyocytes, both channels, wild-type VDAC2 and the efsevin-insensitive VDAC2AAA restored mitochondrial Ca2+ uptake, but only wild-type VDAC2 was sensitive to efsevin. CONCLUSION AND IMPLICATIONS: In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SR-mitochondria Ca2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure-aided chemical optimization of efsevin.