Voltage-dependent Anion Channel Isoforms Research Articles

Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

Synergism of small molecules targeting VDAC with sorafenib, regorafenib or lenvatinib on hepatocarcinoma cell proliferation and survival

Voltage dependent anion channels (VDAC) in the outer mitochondrial membrane regulate the influx of metabolites that sustain mitochondrial metabolism and the efflux of ATP to the cytosol. Free tubulin and NADH close VDAC. The VDAC-binding small molecules X1 and SC18 modulate mitochondrial metabolism. X1 antagonizes the inhibitory effect of tubulin on VDAC. SC18 occupies an NADH-binding pocket in the inner wall of all VDAC isoforms. Here, we hypothesized that X1 and SC18 have a synergistic effect with sorafenib, regorafenib or lenvatinib to arrest proliferation and induce death in hepatocarcinoma cells. We used colony formation assays to determine cell proliferation, and a combination of calcein/propidium iodide, and trypan blue exclusion to assess cell death in the well differentiated Huh7 and the poorly differentiated SNU-449 cells. Synergism was assessed using the Chou-Talalay method. The inhibitory effect of X1, SC18, sorafenib, regorafenib and lenvatinib was concentration and time dependent. IC50s calculated from the inhibition of clonogenic capacity were lower than those determined from cell survival. At IC50s that inhibited cell proliferation, SC18 arrested cells in G0/G1. SC18 at 0.25–2 IC50s had a synergistic effect with sorafenib on clonogenic inhibition in Huh7 and SNU-449 cells, and with regorafenib or lenvatinib in SNU-449 cells. X1 or SC18 also had synergistic effects with sorafenib on promoting cell death at 0.5–2 IC50s for SC18 in Huh7 and SNU-449 cells. These results suggest that small molecules targeting VDAC represent a potential new class of drugs to treat liver cancer.

Photoradical-Mediated Catalyst-Independent Protein Cross-Link with Unusual Fluorescence Properties.

Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp-Phe/Tyr cross-links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λex-max =280 nm→360 nm) and emission (λem-max =330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.

Defining the roles and regulation of the mitochondrial VDAC isoforms one molecule at a time.

The voltage-dependent anion channel (VDAC) is recognized as the key metabolite conduit in the mitochondrial outer membrane and regulator for fluxes of water-soluble metabolites, ions, and polypeptides. The uniqueness of VDAC arises from its position at the interface between the mitochondria and the cytosol making VDAC an important cellular communication hub. VDAC has three isoforms in mammals: VDAC1, 2, and 3 which share high sequence similarity, indicative of a similar pore-forming structure. All three VDAC isoforms form similar highly conductive, anion-selective, voltage-gated channels when reconstituted into planar lipid membranes implying that they can concurrently facilitate the transport of the negatively charged respiratory metabolites. However, cellular model systems and isoform-specific knock-out mice demonstrate distinctive functional roles for each VDAC isoform. We explored the biophysical basis for these differences by reconstituting human, mouse, and zebrafish VDAC isoforms in planar lipid bilayers and performing single channel electrophysiology. We have uncovered that interactions with cytosolic regulators, such as the Parkinson's disease associated alpha-synuclein, vary in their on-rates by two-orders of magnitude between each isoform. Furthermore, each isoform varies in their ability translocate synuclein across the lipid bilayer. Exploring the calcium transport properties of each isoform, we show that VDAC3 has the highest calcium permeability. We demonstrate that VDAC2 can dynamically switch between channel properties of VDAC1 and VDAC3, and explored the structural basis for its unique plasticity using mutational and structural analysis and molecular dynamics simulations. We find that both VDAC2's N-terminal extension and cysteine residues contribute to, but are not sufficient to explain VDAC2's striking behavior. Our results suggest that tuning of beta-barrel and loop flexibility between the isoforms could define their biophysical properties and help to explain distinct physiological roles.

Role of VDAC isoforms in bioenergetics and metabolism.

Voltage-dependent anion channel (VDAC) is a beta-barrel channel on the mitochondrial outer membrane (MOM) known to transport water-soluble metabolites such as ATP, ADP, and pyruvate. It is central to the regulation of cellular metabolism and serves as a cellular hub given its location at the interface between cytosol and mitochondria. There are three mammalian isoforms of VDAC: VDAC1, 2, and 3. They show high sequence similarity and form similar highly conductive voltage-gated channels in vitro. However, cellular and mice knockout (KO) studies of VDAC isoforms suggest a unique physiological role for each isoform. While VDAC1 KO only leads to mild bioenergetic defects, VDAC3 KO results in male infertility and VDAC2 KO causes developmental impairment in mice. To investigate the role of VDAC isoforms in cellular function, we generated HeLa cells with CRISPR-Cas9 KO of each VDAC isoform. We found isoform-specific differences in mitochondria respiration such as basal and maximal respiration rates for the different isoform KO cells using the Seahorse Metabolic Flux Analyzer. VDAC1 and VDAC3 KO cells showed a significant loss in respiration compared to WT while there was no significant difference detected for VDAC2 KO cells. Interestingly, only VDAC3 KO resulted in complete loss of spare respiratory capacity which could be attributed to defects in the fuel uptake pathway as the VDAC3 KO cells show increased glutamine dependence and loss of flexibility to utilize other metabolites compared to WT HeLa cells. We also detected isoform-dependent differences in calcium signal in mitochondrial intermembrane space (IMS) using IMS targeted calcium sensor (IMS-RGeco1). These results suggest different metabolite and calcium transport functionality for the VDAC isoforms which may contribute to their distinct physiological functions in vivo through isoform-specific regulation of metabolism and calcium signaling.

VDAC interactions with lipids and their effect on isoform specificity.

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel, the most abundant protein of the mitochondrial outer membrane, allowing passage of ions and metabolites (such as ATP, ADP, NADH⋯) in and out of the mitochondria. Besides its transport function, VDAC is also implicated in mitochondrial regulation with roles in apoptosis, calcium homeostasis or neurodegeneration. However, its role in those different pathways remains to be understood at the molecular level. VDAC possesses 3 isoforms in mammals (VDAC1, 2 and 3), sharing a high sequence identity, the same architecture and an overlapping tissue distribution. They display very similar transport properties such as conductance, selectivity and voltage-induced closure while their isoform specificity originates from their different interaction with partner proteins. This may determine their unique role in mitochondrial regulation, where each isoform seems to have different functions and a different set of partners: VDAC1 is implicated in neurodegeneration (Alzheimer and Parkinson disease), VDAC2 in apoptosis and calcium homeostasis, and VDAC3 probably in spermatogenesis and oxidative stress. Using a combination of biochemistry, molecular dynamics and electrophysiology, we investigated the lipid organization around each VDAC isoform and their impact on VDAC function. Our data suggest that the three isoforms have different affinities for lipids, and that could contribute to the selection of interacting partners.

Parkin Insufficiency Accentuates High-Fat Diet–Induced Cardiac Remodeling and Contractile Dysfunction Through VDAC1-Mediated Mitochondrial Ca2+ Overload

Mitochondrial Ca2+ overload contributes to obesity cardiomyopathy, yet mechanisms that directly regulate it remain elusive. The authors investigated the role of Parkin on obesity-induced cardiac remodeling and dysfunction in human hearts and a mouse model of 24-week high-fat diet (HFD) feeding. Parkin knockout aggravated HFD-induced cardiac remodeling and dysfunction, mitochondrial Ca2+ overload, and apoptosis without affecting global metabolism, blood pressure, and aortic stiffness. Parkin deficiency unmasked HFD-induced decline in voltage-dependent anion channel (VDAC) type 1 degradation through the ubiquitin-proteasome system but not other VDAC isoforms or mitochondrial Ca2+ uniporter complex. These data suggest that Parkin-mediated proteolysis of VDAC type 1 is a promising therapeutic target for obesity cardiomyopathy.

Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

Infection cycles of viruses are highly dependent on membrane-associated host factors. To uncover the infection cycle of Bamboo mosaic virus (BaMV) in detail, we purified the membrane-associated viral complexes from infected Nicotiana benthamiana plants and analyzed the involved host factors. Four isoforms of voltage-dependent anion channel (VDAC) proteins on the outer membrane of mitochondria were identified due to their upregulated expression in the BaMV complex-enriched membranous fraction. Results from loss- and gain-of-function experiments indicated that NbVDAC2, -3, and -4 are essential for efficient BaMV accumulation. During BaMV infection, all NbVDACs concentrated into larger aggregates, which overlapped and trafficked with BaMV virions to the structure designated as the "dynamic BaMV-induced complex." Besides the endoplasmic reticulum and mitochondria, BaMV replicase and double-stranded RNAs were also found in this complex, suggesting the dynamic BaMV-induced complex is a replication complex. Yeast two-hybrid and pull-down assays confirmed that BaMV triple gene block protein 1 (TGBp1) could interact with NbVDACs. Confocal microscopy revealed that TGBp1 is sufficient to induce NbVDAC aggregates, which suggests that TGBp1 may play a pivotal role in the NbVDAC-virion complex. Collectively, these findings indicate that NbVDACs may associate with the dynamic BaMV-induced complex via TGBp1 and NbVDAC2, -3, or -4 and can promote BaMV accumulation. This study reveals the involvement of mitochondrial proteins in a viral complex and virus infection.

Glutamate 73 Promotes Anti-arrhythmic Effects of Voltage-Dependent Anion Channel Through Regulation of Mitochondrial Ca2+ Uptake.

Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca2+ signaling. The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes.

Plant mitochondria import DNA via alternative membrane complexes involving various VDAC isoforms

Mitochondria possess transport mechanisms for import of RNA and DNA. Based on import into isolated Solanum tuberosum mitochondria in the presence of competitors, inhibitors or effectors, we show that DNA fragments of different size classes are taken up into plant organelles through distinct channels. Alternative channels can also be activated according to the amount of DNA substrate of a given size class. Analyses of Arabidopsis thaliana knockout lines pointed out a differential involvement of individual voltage-dependent anion channel (VDAC) isoforms in the formation of alternative channels. We propose several outer and inner membrane proteins as VDAC partners in these pathways.

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport

Voltage-dependent anion channel (VDAC) is the most ubiquitous channel at the mitochondrial outer membrane, and is believed to be the pathway for calcium entering or leaving the mitochondria. Therefore, understanding the molecular mechanisms of how VDAC regulates calcium influx and efflux from the mitochondria is of particular interest for mitochondrial physiology. When the Parkinson’s disease (PD) related neuronal protein, alpha-synuclein (αSyn), is added to the reconstituted VDAC, it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule VDAC electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, αSyn reverses the channel’s selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by αSyn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. -Our findings with higher calcium concentrations also demonstrate that the phenomenon of “charge inversion” is taking place at the level of a single polypeptide chain. Measurements of ion selectivity of three VDAC isoforms in CaCl2 gradient show that VDAC3 exhibits the highest calcium permeability among them, followed by VDAC2 and VDAC1, thus pointing to isoform-dependent physiological function. Mutation of the E73 residue – VDAC1 purported calcium binding site – shows that there is no measurable effect of the mutation in either open or αSyn-blocked VDAC1 states. Our results confirm VDACs involvement in calcium signaling and reveal a new regulatory role of αSyn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction.

Role of VDAC Isoforms in Alpha-Synuclein Entry into Mitochondria

Parkinson's disease (PD) patients show loss of neurons, mitochondrial dysfunction, and the presence of Lewy bodies. Lewy bodies are composed of misfolded α-synuclein, which is a risk factor in both familial and sporadic PD. Studies have shown that α-synuclein can cause mitochondrial dysfunction and increase reactive oxygen species production. This has been attributed to the interaction of α-synuclein with complexes of the electron transport chain in the inner mitochondrial membrane, but it is not clear how α-synuclein crosses the outer membrane to enter the mitochondria. In neurons, α-synuclein has been shown to bind Voltage-Dependent Anion Channel (VDAC), an outer mitochondrial membrane protein that shuttles metabolites between the cytosol and the mitochondria. Also, single-channel electrophysiology experiments show that monomeric α-synuclein can transiently block and translocate through all VDAC isoforms. However, the three VDAC isoforms in mammals — VDAC1, VDAC2, and VDAC3 that form channels of almost identical conductance and anion selectivity —show distinctly different interaction with α-synuclein in experiments with reconstituted VDACs. To understand the mechanism of α-synuclein translocation into mitochondria in live cells, we performed Förster Resonance Energy Transfer (FRET) microscopy using Alexa488-α-synuclein and VDAC1, 2 or 3 -GFP. The change in FRET signal as α-synuclein enters VDAC pore and translocates through it was used to visualize α-synuclein entry into the mitochondria. The contribution of each isoform in α-synuclein translocation was studied by knocking down individual VDAC isoforms in live cells. Elucidating the mechanism of α-synuclein entry into mitochondria and, in particular, the role of each VDAC isoform in α-synuclein translocation is crucial for understanding of the impact of α-synuclein in mitochondrial dysfunction and the ways to ameliorate mitochondrial function at the early stages of neurodegeneration.

VDAC regulation of mitochondrial calcium flux: From channel biophysics to disease

Voltage-dependent anion channel (VDAC), the most abundant mitochondrial outer membrane protein, is important for a variety of mitochondrial functions including metabolite exchange, calcium transport, and apoptosis. While VDAC’s role in shuttling metabolites between the cytosol and mitochondria is well established, there is a growing interest in understanding the mechanisms of its regulation of mitochondrial calcium transport. Here we review the current literature on VDAC’s role in calcium signaling, its biophysical properties, physiological function, and pathology focusing on its importance in cardiac diseases. We discuss the specific biophysical properties of the three VDAC isoforms in mammalian cells—VDAC 1, 2, and 3—in relationship to calcium transport and their distinct roles in cell physiology and disease. Highlighting the emerging evidence that cytosolic proteins interact with VDAC and regulate its calcium permeability, we advocate for continued investigation into the VDAC interactome at the contact sites between mitochondria and organelles and its role in mitochondrial calcium transport.

A finite element iterative solver for a PNP ion channel model with Neumann boundary condition and membrane surface charge

In this paper, an effective finite element iterative algorithm is presented for solving a Poisson-Nernst-Planck ion channel (PNPic) model with Neumann boundary value condition and a membrane surface charge density. It is constructed by a solution decomposition scheme to avoid singularity problems caused by atomic charges, an alternating block iterative scheme to sharply reduce computation complexity and computer memory requirement, and a Slotboom variable transformation scheme to significantly enhance numerical stability, as well as a modified Newton iterative scheme to efficiently solve each related nonlinear finite element equation. This PNPic finite element solver is then implemented as a software package that works for an ion channel protein with a crystallographic structure in a mixture solution of multiple ionic species. Furthermore, a finite element scheme is presented to compute a volume integral of a potential/concentration function over a block of a solvent region. This work can greatly improve the accuracy of a visualization tool for depicting the distribution pattern of a three-dimensional potential/concentration function across membrane in a simple two-dimensional curve. Numerical results for a mouse voltage-dependent anion-channel isoform (mVDAC1) in a solution of up to four ionic species are reported. They demonstrate the convergence of the PNPic iterative solver, the performance of the software package, and the valuable usage of the visualization tool in the comparison study of different potential and concentration functions. They also validate that this PNPic model can well retain the anion selectivity property of mVDAC1.

The Study of HIV-1 Vpr-Membrane and Vpr-hVDAC-1 Interactions by Graphene Field-Effect Transistor Biosensors.

The viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1) is involved in many cellular processes during the viral life cycle; however, its associated mechanisms remain unclear. Here, we designed an Escherichia coli expression construct to achieve a milligram yield of recombinant Vpr. In addition, we fabricated a graphene field-effect transistor (G-FET) biosensor, with the modification of a supported lipid bilayer (SLB), to study the interaction between Vpr and its interaction partners. The Dirac point of the SLB/G-FET was observed to shift in response to the binding of Vpr to the SLB. By fitting the normalized shift of the Dirac point as a function of Vpr concentration to the Langmuir adsorption isotherm equation, we could extract the dissociation constant (Kd) to quantify the Vpr binding affinity. When the 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) membrane was used as the SLB, the dissociation constant was determined to be 9.6 ± 2.1 μM. In contrast, only a slight shift of the Dirac point was observed in response to the addition of Vpr when the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane was used as the SLB. Taking advantage of the much weaker binding of Vpr to the DOPC membrane, we prepared a human voltage-dependent anion channel isoform 1 (hVDAC-1)-embedded DOPC membrane as the SLB for the G-FET and used it to determine the dissociation constant to be 5.1 ± 0.9 μM. In summary, using the clinically relevant Vpr protein as an example, we demonstrated that an SLB/G-FET biosensor is a suitable tool for studying the interaction between a membrane-associated protein and its interaction partners.

Do folding elements trade‐off with function in the human mitochondrial metabolite transporter?

In humans, transmembrane β‐barrels are found exclusively in the outer mitochondrial membrane (OMM). They comprise metabolite transporters, translocases, and chaperones, which together control cellular homeostasis. The metabolite transporters — known as voltage‐dependent anion channels (VDACs) – are the most abundant OMM barrels. They are 19‐stranded β‐barrel structures that also form important pharmacological targets due to their roles in metabolite flux, steroidogenesis, gametogenesis, and ROS regulation. Despite long‐standing efforts, molecular factors that regulate VDAC function and folding at the atomistic level are not known. Here, we map the folding nucleus and the assembly pathway of human VDAC isoform 2 in phosphocholine vesicles. We carry out a comprehensive measurement of hVDAC2 folding kinetics, and equilibrium thermodynamic contribution, of each of the 270 non‐Ala residues of the 294‐residue barrel. Interestingly, we obtain a two‐state thermodynamic equilibrium that is attained through two detectable on‐pathway early folding intermediates, which are assembled in milliseconds – seconds. These intermediates undergo slow conformational restructuring to form the folded VDAC2 barrel. We find that specific charged residues that form a part of these early intermediates also positively regulate the gating characteristics of VDAC2. This observation is unprecedented, as directed evolution of protein sequences in favor of function usually place a stability burden on its structure that additionally impedes protein assembly. Our findings provide molecular insight on the assembly pathway of human VDAC2, and directly link these folding elements with VDAC2 function as the metabolite transporter of human mitochondria.Support or Funding InformationThis work was supported by the Wellcome Trust/DBT India Alliance Fellowship [grant number IA/I/14/1/501305] awarded to RM.

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology.

Voltage-dependent anion channel (VDAC) is the major pathway for the transport of ions and metabolites across the mitochondrial outer membrane. Among the three known mammalian VDAC isoforms, VDAC3 is the least characterized, but unique functional roles have been proposed in cellular and animal models. Yet, a high-sequence similarity between VDAC1 and VDAC3 is indicative of a similar pore-forming structure. Here, we conclusively show that VDAC3 forms stable, highly conductive voltage-gated channels that, much like VDAC1, are weakly anion selective and facilitate metabolite exchange, but exhibit unique properties when interacting with the cytosolic proteins α-synuclein and tubulin. These two proteins are known to be potent regulators of VDAC1 and induce similar characteristic blockages (on the millisecond time scale) of VDAC3, but with 10- to 100-fold reduced on-rates and altered α-synuclein blocking times, indicative of an isoform-specific function. Through cysteine scanning mutagenesis, we found that VDAC3's cysteine residues regulate its interaction with α-synuclein, demonstrating VDAC3-unique functional properties and further highlighting a general molecular mechanism for VDAC isoform-specific regulation of mitochondrial bioenergetics.

Structural and functional characterization of Solanum tuberosum VDAC36.

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum, stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.