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.

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.

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.

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.

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.

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.

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.

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 envelope protein ion channel activity promotes virus fitness and pathogenesis.

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1ß were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1ß was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.

Acidification asymmetrically affects voltage-dependent anion channel implicating the involvement of salt bridges.

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to "gate" between an open and several "closed" states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.

Inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment.

The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.

Analysis of SARS-CoV E protein ion channel activity by tuning the protein and lipid charge.

A partial characterization of the ion channels formed by the SARS coronavirus (CoV) envelope (E) protein was previously reported (C. Verdiá-Báguena et al., 2012 [12]). Here, we provide new significant insights on the involvement of lipids in the structure and function of the CoV E protein channel on the basis of three series of experiments. First, reversal potential measurements over a wide range of pH allow the dissection of the contributions to channel selectivity coming from ionizable residues of the protein transmembrane domain and also from the negatively charged groups of diphytanoyl phosphatidylserine (DPhPS) lipid. The corresponding effective pKas are consistent with the model pKas of the acidic residue candidates for titration. Second, the change of channel conductance with salt concentration reveals two distinct regimes (Donnan-controlled electrodiffusion and bulk-like electrodiffusion) fully compatible with the outcomes of selectivity experiments. Third, by measuring channel conductance in mixtures of neutral diphytanoyl phosphatidylcholine (DPhPC) lipids and negatively charged DPhPS lipids in low and high salt concentrations we conclude that the protein-lipid conformation in the channel is likely the same in charged and neutral lipids. Overall, the whole set of experiments supports the proteolipidic structure of SARS-CoV E channels and explains the large difference in channel conductance observed between neutral and charged membranes.

Lipid charge regulation of non-specific biological ion channels.

Ion channels are specialized proteins that enable the movement of charges through otherwise impermeable lipidic membranes. Their action is essential in living organisms facilitating electric signaling, muscle contraction or osmotic stress response among other effects. The protein and the lipid charges configure a polarized interface that yields local ionic concentrations and electric potentials that are very different from those of the bulk electrolyte. The combined effect of gradients of ionic concentration and electric potential causes the transport of ions through channels. Here we analyze charge regulation effects in different protein-lipid conformations, stressing how important is the role of electrostatic interactions in the ion channel function that traditionally has been rationalized paying attention mainly to changes in pore size. Tuning lipid charge combined with conductance and selectivity measurements is shown to be a complementary method to evidence lipid involvement in the structure of a biological ion channel.

Hydrophobic pulmonary surfactant proteins SP-B and SP-C induce pore formation in planar lipid membranes: evidence for proteolipid pores.

Pulmonary surfactant is a complex mixture of lipids and specific surfactant proteins, including the hydrophobic proteins SP-B and SP-C, in charge of stabilizing the respiratory surface of mammalian lungs. The combined action of both proteins is responsible for the proper structure and dynamics of membrane arrays in the pulmonary surfactant network that covers the respiratory surface. In this study, we explore the possibility that proteins SP-B and SP-C induce the permeabilization of phospholipid membranes via pore formation. To this end, electrophysiological measurements have been carried out in planar lipid membranes prepared with different lipid/protein mixtures. Our main result is that channel-like structures are detected in the presence of SP-B, SP-C, or the native mixture of both proteins. Current traces show a high variety of conductance states (from pS to nS) that are dependent both on the lipid composition and the applied potential. We also show that the type of host lipid crucially determines the ionic selectivity of the observed pores: the anionic selectivity observed in zwitterionic membranes is inverted to cationic selectivity in the presence of negatively charged lipids. All those results suggest that SP-B and SP-C proteins promote the formation of proteolipid channels in which lipid molecules are functionally involved. We propose that proteolipidic membrane-permeabilizing structures may have an important role to tune ionic and lipidic flows through the pulmonary surfactant membrane network at the alveolar surfaces.

Increased salt concentration promotes competitive block of OmpF channel by protons.

Porins are channel-forming proteins that are located in the outer membranes (OM) of Gram-negative bacteria and allow the influx of hydrophilic nutrients and the extrusion of waste products. The fine regulation of the ion transport through these wide channels could play an important role in the survival of the bacteria in acidic media. We investigate here the mechanism responsible for the pH sensitivity of the trimeric porin OmpF, of Escherichia coli. Planar lipid bilayer electrophysiology and site-directed mutagenesis were used to study the effect of pH on the ion conductive properties of the OmpF channel in its fully open, "nongated" conformation. At low pH we observe a large drop in the OmpF open channel conductance that is accompanied by a substantial increase of the current noise. These channel features are strongly dependent on the salt concentration and we propose that they are originated by competitive binding of cations and protons occurring in the narrow central constriction of the channel. This subtle mechanism reveals to be capital for the channel function because it not only drives the channel sensitivity to pH but is also indispensable for the particularly efficient permeation mechanism of the channel at physiological conditions (~neutral pH).

Electrostatic interactions drive the nonsteric directional block of OmpF channel by La3+.

Ion channels regulate the transport of molecules and the electric signal transduction in living cells by means of complex and even highly sophisticated mechanisms. We focus here on the crucial role that polyvalent ions, well-known modulators of many biological nanosystems, play in ion channel function. In particular, we show that trace amounts of lanthanum are able to block the bacterial porin OmpF, a large biological pore of Escherichia coli wide enough to exchange antibiotics and other larger molecules. The underlying mechanism has a strong directional character: it is sensitive to the sign of the applied voltage and to the side of the blocker addition. We explore these channel features by combining planar lipid bilayer electrophysiology at the single channel level, site-directed mutagenesis, and inductively coupled plasma mass spectrometry (ICP-MS). In contrast to other well-described channel blockers, which seem to occlude the narrower part of the pore, we envisage a nonsteric mechanism based on electrostatic interactions.

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.