Charged Biological Membranes Repel Large Neutral Molecules by Surface Dielectrophoresis and Counterion Pressure.

Macromolecular crowding is the usual condition of cells. The implications of the crowded cellular environment for protein stability and folding, protein-protein interactions, and intracellular transport drive a growing interest in quantifying the effects of crowding. While the properties of crowded solutions have been extensively studied, less attention has been paid to the interaction of crowders with the cellular boundaries, i.e., membranes. However, membranes are key components of cells and most subcellular organelles, playing a central role in regulating protein channel and receptor functions by recruiting and binding charged and neutral solutes. While membrane interactions with charged solutes are dominated by electrostatic forces, here we show that significant charge-induced forces also exist between membranes and neutral solutes. Using neutron reflectometry measurements and molecular dynamics simulations of poly(ethylene glycol) (PEG) polymers of different molecular weights near charged and neutral membranes, we demonstrate the roles of surface dielectrophoresis and counterion pressure in repelling PEG from charged membrane surfaces. The resulting depletion zone is expected to have consequences for drug design and delivery, the activity of proteins near membrane surfaces, and the transport of small molecules along the membrane surface.

The Complex Proteolipidic Behavior of the SARS-CoV-2 Envelope Protein Channel: Weak Selectivity and Heterogeneous Oligomerization.

The envelope (E) protein is a small polypeptide that can form ion channels in coronaviruses. In SARS coronavirus 2 (SARS-CoV-2), the agent that caused the recent COVID-19 pandemic, and its predecessor SARS-CoV-1, E protein is found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where virion budding takes place. Several reports claim that E protein promotes the formation of "cation-selective channels". However, whether this term represents specificity to certain ions (e.g., potassium or calcium) or the partial or total exclusion of anions is debatable. Herein, we discuss this claim based on the available data for SARS-CoV-1 and -2 E and on new experiments performed using the untagged full-length E protein from SARS-CoV-2 in planar lipid membranes of different types, including those that closely mimic the ERGIC membrane composition. We provide evidence that the selectivity of the E-induced channels is very mild and depends strongly on lipid environment. Thus, despite past and recent claims, we found no indication that the E protein forms cation-selective channels that prevent anion transport, and even less that E protein forms bona fide specific calcium channels. In fact, the E channel maintains its multi-ionic non-specific neutral character even in concentrated solutions of Ca2+ ions. Also, in contrast to previous studies, we found no evidence that SARS-CoV-2 E channel activation requires a particular voltage, high calcium concentrations or low pH, in agreement with available data from SARS-CoV-1 E. In addition, sedimentation velocity experiments suggest that the E channel population is mostly pentameric, but very dynamic and probably heterogeneous, consistent with the broad distribution of conductance values typically found in electrophysiological experiments. The latter has been explained by the presence of proteolipidic channel structures.

Biphasic concentration patterns in ionic transport under nanoconfinement revealed in steady-state and time-dependent properties.

Ion permeation across nanoscopic structures differs considerably from microfluidics because of strong steric constraints, transformed solvent properties, and charge-regulation effects revealed mostly in diluted solutions. However, little is known about nanofluidics in moderately concentrated solutions, which are critically important for industrial applications and living systems. Here, we show that nanoconfinement triggers general biphasic concentration patterns in a myriad of ion transport properties by using two contrasting systems: a biological ion channel and a much larger synthetic nanopore. Our findings show a low-concentration regime ruled by classical Debye screening and another one where ion-ion correlations and enhanced ion-surface interactions contribute differently to each electrophysiological property. Thus, different quantities (e.g., conductance vs noise) measured under the same conditions may appear contradictory because they belong to different concentration regimes. In addition, non-linear effects that are barely visible in bulk conductivity only in extremely concentrated solutions become apparent in nanochannels around physiological conditions.

SARS-CoV-2 accessory protein 7b forms homotetramers in detergent.

A global pandemic is underway caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b, with only 44 (SARS-CoV) and 43 (SARS-CoV-2) residues. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells, and the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that in both SARS viruses 7b is almost completely α-helical and has a single transmembrane domain. In SDS, 7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants. Combination of SDS gel electrophoresis and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, but a monomer-dimer-tetramer equilibrium in the presence of reductant. This data suggests that although disulfide-linked dimers may be present, they are not essential to form tetramers. Inclusion of pentamers or higher oligomers in the SARS-2 7b model were detrimental to fit quality. Preliminary models of this association was generated with AlphaFold2, and two alternative models were exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provided any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b to model membranes produced occasional membrane permeabilization, but this was not consistent with bona fide ion channels made of a tetrameric assembly of α-helices.

Triplin: Functional Probing of Its Structure and the Dynamics of the Voltage-Gating Process.

Gram-negative bacteria have a large variety of channel-forming proteins in their outer membrane, generally referred to as porins. Some display weak voltage dependence. A similar trimeric channel former, named Triplin, displays very steep voltage dependence, rivaling that responsible for the electrical excitability of mammals, and high inter-subunit cooperativity. We report detailed insights into the molecular basis for these very unusual properties explored at the single-molecule level. By using chemical modification to reduce the charge on the voltage sensors, they were shown to be positively charged structures. Trypsin cleavage of the sensor eliminates voltage gating by cleaving the sensor. From asymmetrical addition of these reagents, the positively charged voltage sensors translocate across the membrane and are, thus, responsible energetically for the steep voltage dependence. A mechanism underlying the cooperativity was also identified. Theoretical calculations indicate that the charge on the voltage sensor can explain the rectification of the current flowing through the open pores if it is located near the pore mouth in the open state. All results support the hypothesis that one of the three subunits is oriented in a direction opposite to that of the other two. These properties make Triplin perhaps the most complex pore-forming molecular machine described to date.

Boron clusters (ferrabisdicarbollides) shaping the future as radiosensitizers for multimodal (chemo/radio/PBFR) therapy of glioblastoma.

Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, and is highly resistant to conventional radiotherapy and chemotherapy. Therefore, the development of multidrug resistance and tumor recurrence are frequent. Given the poor survival with the current treatments, new therapeutic strategies are urgently needed. Radiotherapy (RT) is a common cancer treatment modality for GBM. However, there is still a need to improve RT efficiency, while reducing the severe side effects. Radiosensitizers can enhance the killing effect on tumor cells with less side effects on healthy tissues. Herein, we present our pioneering study on the highly stable and amphiphilic metallacarboranes, ferrabis(dicarbollides) ([o-FESAN]- and [8,8'-I2-o-FESAN]-), as potential radiosensitizers for GBM radiotherapy. We propose radiation methodologies that utilize secondary radiation emissions from iodine and iron, using ferrabis(dicarbollides) as iodine/iron donors, aiming to achieve a greater therapeutic effect than that of a conventional radiotherapy. As a proof-of-concept, we show that using 2D and 3D models of U87 cells, the cellular viability and survival were reduced using this treatment approach. We also tested for the first time the proton boron fusion reaction (PBFR) with ferrabis(dicarbollides), taking advantage of their high boron (11B) content. The results from the cellular damage response obtained suggest that proton boron fusion radiation therapy, when combined with boron-rich compounds, is a promising modality to fight against resistant tumors. Although these results are encouraging, more developments are needed to further explore ferrabis(dicarbollides) as radiosensitizers towards a positive impact on the therapeutic strategies for GBM.

Transport mechanisms of SARS-CoV-E viroporin in calcium solutions: Lipid-dependent Anomalous Mole Fraction Effect and regulation of pore conductance.

The envelope protein E of the SARS-CoV coronavirus is an archetype of viroporin. It is a small hydrophobic protein displaying ion channel activity that has proven highly relevant in virus-host interaction and virulence. Ion transport through E channel was shown to alter Ca2+ homeostasis in the cell and trigger inflammation processes. Here, we study transport properties of the E viroporin in mixed solutions of potassium and calcium chloride that contain a fixed total concentration (mole fraction experiments). The channel is reconstituted in planar membranes of different lipid compositions, including a lipid mixture that mimics the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane where the virus localizes within the cell. We find that the E ion conductance changes non-monotonically with the total ionic concentration displaying an Anomalous Mole Fraction Effect (AMFE) only when charged lipids are present in the membrane. We also observe that E channel insertion in ERGIC-mimic membranes - including lipid with intrinsic negative curvature - enhances ion permeation at physiological concentrations of pure CaCl2 or KCl solutions, with a preferential transport of Ca2+ in mixed KCl-CaCl2 solutions. Altogether, our findings demonstrate that the presence of calcium modulates the transport properties of the E channel by interacting preferentially with charged lipids through different mechanisms including direct Coulombic interactions and possibly inducing changes in membrane morphology.

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.

PEG Equilibrium Partitioning in the α-Hemolysin Channel: Neutral Polymer Interaction with Channel Charges.

We study the interaction of neutral polyethylene glycol (PEG) molecules of different molecular weights (MWs) with the charged residues of the α-hemolysin channel secreted by Staphylococcus aureus. Previously reported experiments of PEG equilibrium partitioning into this nanopore show that the charge state of the channel changes the ability of PEG entry in an MW-dependent manner. We explain such an effect by parameter-free calculations of the PEG self-energy from the channel 3D atomic structure that include repulsive dielectrophoretic and hydrostatic forces on the polymer. We found that the pH-induced shift in the measured free energy of partitioning ΔΔGexp from single-channel conductance measurements agrees with calculated energy changes ΔΔEcalc. Our results show that the PEG-sizing technique may need corrections in the case of charged biological pores.

Access resistance in protein nanopores. A structure-based computational approach.

Single-channel conductance measurements in biological pores have demonstrated the importance of interfacial effects in nanopores, particularly in protein channels with low aspect ratio (length over aperture radius). Access resistance (AR), the contribution to the total measured resistance arising from the electrodiffusive limitation that ions experience in passing from bulk solution to confinement within the pore, becomes essential in the description of ionic transport across these biological channels. Common analytical estimates of AR are based on idealized nanopore models, cylindrical in shape, electrically neutral and embedded in a neutral substrate. Here we calculate the AR of five protein channels by using their atomic structure and a mean-field approach based on solving 3D Poisson and Nernst-Planck equations. Our approach accounts for the influence of the protein charged ionizable residues, the geometry of the pore mouth and the ion concentration gradients near the pore. We compare numerical calculations with the few available AR measurements and show for several protein channels that analytical predictions tend to overestimate AR for physiological concentrations and below. We also discuss the relationship between AR and the size of the channel aperture in single-pore channels and three-pore channels and demonstrate that in the latter case, there is an enhancement of AR.

Scaling Behavior of Ionic Transport in Membrane Nanochannels.

Ionic conductance in membrane channels exhibits a power-law dependence on electrolyte concentration ( G ∼ cα). The many scaling exponents, α, reported in the literature usually require detailed interpretations concerning each particular system under study. Here, we critically evaluate the predictive power of scaling exponents by analyzing conductance measurements in four biological channels with contrasting architectures. We show that scaling behavior depends on several interconnected effects whose contributions change with concentration so that the use of oversimplified models missing critical factors could be misleading. In fact, the presence of interfacial effects could give rise to an apparent universal scaling that hides the channel distinctive features. We complement our study with 3D structure-based Poisson-Nernst-Planck (PNP) calculations, giving results in line with experiments and validating scaling arguments. Our findings not only provide a unified framework for the study of ion transport in confined geometries but also highlight that scaling arguments are powerful and simple tools with which to offer a comprehensive perspective of complex systems, especially those in which the actual structure is unknown.

Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice.IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses.

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses.

Effect of endosomal acidification on small ion transport through the anthrax toxin PA63 channel.

Tight regulation of pH is critical for the structure and function of cells and organelles. The pH environment changes dramatically along the endocytic pathway, an internalization transport process that is 'hijacked' by many intracellularly active bacterial exotoxins, including the anthrax toxin. Here, we investigate the role of pH on single-channel properties of the anthrax toxin protective antigen (PA63 ). Using conductance and current noise analysis, blocker binding, ion selectivity, and poly(ethylene glycol) partitioning measurements, we show that the channel exists in two different open states ('maximum' and 'main') at pH ≥ 5.5, while only a maximum conductance state is detected at pH < 5.5. We describe two substantially distinct patterns of PA63 conductance dependence on KCl concentration uncovered at pH 6.5 and 4.5.

Ion Transport in Confined Geometries below the Nanoscale: Access Resistance Dominates Protein Channel Conductance in Diluted Solutions.

Synthetic nanopores and mesoscopic protein channels have common traits like the importance of electrostatic interactions between the permeating ions and the nanochannel. Ion transport at the nanoscale occurs under confinement conditions so that the usual assumptions made in microfluidics are challenged, among others, by interfacial effects such as access resistance (AR). Here, we show that a sound interpretation of electrophysiological measurements in terms of channel ion selective properties requires the consideration of interfacial effects, up to the point that they dominate protein channel conductance in diluted solutions. We measure AR in a large ion channel, the bacterial porin OmpF, by means of single-channel conductance measurements in electrolyte solutions containing varying concentrations of high molecular weight PEG, sterically excluded from the pore. Comparison of experiments performed in charged and neutral planar membranes shows that lipid surface charges modify the ion distribution and determine the value of AR, indicating that lipid molecules are more than passive scaffolds even in the case of large transmembrane proteins. We also found that AR may reach up to 80% of the total channel conductance in diluted solutions, where electrophysiological recordings register essentially the AR of the system and depend marginally on the pore characteristics. These findings may have implications for several low aspect ratio biological channels that perform their physiological function in a low ionic strength and macromolecule crowded environment, just the two conditions enhancing the AR contribution.

Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus.

It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to in vivo attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein.IMPORTANCE The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.

Ion channel activity of the CSFV p7 viroporin in surrogates of the ER lipid bilayer.

Viroporins comprise a family of non-structural proteins that play significant and diverse roles during the replication cycle of many animal viruses. Consequently, they have become promising targets for inhibitory drug and vaccine development. Structure­function traits common to all members of the family are their small size (ca. 60­120 aa), high hydrophobicity, and the presence of helical domains that transverse the membrane and assemble into oligomeric-permeating structures therein. The possibility that viroporins show in particular conditions any kind of specificity in the transport of ions and small solutes remains a point of contention in the field. Here we have approached this issue using the Classical Swine Fever Virus (CSFV) protein p7 viroporin as a model. We have previously reported that CSFV-p7 induces release of ANTS (MW: 427.33) from lipid vesicles that emulate the Endoplasmic Reticulum (ER) membrane, and that this process is dependent on pH, modulated by the lipid composition, and recreated by a C-terminal transmembrane helix. Here we have assayed CSFV-p7 for its capacity to form ion-conducting channels in ER-like planar lipid membranes, and established whether this activity is subject to regulation by the same factors. The analysis of electrophysiological recordings in ER membrane surrogates suggests that CSFV-p7 forms pores wide enough to allow ANTS release. Moreover, we were able to discriminate between two pore structures with slightly different sizes and opposite ion selectivities. The fact that the relative abundances of each pore type depend crucially on membrane composition strengthens the view that the physicochemical properties of the lipid bilayers present in the cell endomembrane system modulate viroporin activity.

Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome.

Severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a viroporin involved in virulence. E protein ion channel (IC) activity is specifically correlated with enhanced pulmonary damage, edema accumulation and death. IL-1ß driven proinflammation is associated with those pathological signatures, however its link to IC activity remains unknown. In this report, we demonstrate that SARS-CoV E protein forms protein-lipid channels in ERGIC/Golgi membranes that are permeable to calcium ions, a highly relevant feature never reported before. Calcium ions together with pH modulated E protein pore charge and selectivity. Interestingly, E protein IC activity boosted the activation of the NLRP3 inflammasome, leading to IL-1ß overproduction. Calcium transport through the E protein IC was the main trigger of this process. These findings strikingly link SARS-CoV E protein IC induced ionic disturbances at the cell level to immunopathological consequences and disease worsening in the infected organism.