Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis

Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology.     

Photogating of ionic currents across a lipid bilayer.

Photoformation of metalloporphyrin cations in a lipid bilayer increases the ionic currents of negative and decreases those of positive hydrophobic ions. At low concentrations of the mobile hydrophobic ion, a 30% change in conductivity is observed that decreases with increasing concentration of positive tetraphenylphosphonium ion and increases drastically with increasing concentration of negative tetraphenylboride ion. In the region of saturated conductance of boride ion, the increase in conductivity is 3.6-fold. A 15-fold increase is observed with the protonophore carbonyl cyanide 3-chlorophenylhydrazone. In this case the net charge gated is 300 times greater than the photogenerated charge in the bilayer membrane. Thus there is a net gain in this organic field effect phototransistor. The gating can also be accomplished by continuous light or chemical oxidants. Photogating is explained as space charge effects inside the bilayer.     

Extreme accumulation of nucleotides in simulated hydrothermal pore systems

We simulate molecular transport in elongated hydrothermal pore systems influenced by a thermal gradient. We find extreme accumulation of molecules in a wide variety of plugged pores. The mechanism is able to provide highly concentrated single nucleotides, suitable for operations of an RNA world at the origin of life. It is driven solely by the thermal gradient across a pore. On the one hand, the fluid is shuttled by thermal convection along the pore, whereas on the other hand, the molecules drift across the pore, driven by thermodiffusion. As a result, millimeter-sized pores accumulate even single nucleotides more than 10(8)-fold into micrometer-sized regions. The enhanced concentration of molecules is found in the bulk water near the closed bottom end of the pore. Because the accumulation depends exponentially on the pore length and temperature difference, it is considerably robust with respect to changes in the cleft geometry and the molecular dimensions. Whereas thin pores can concentrate only long polynucleotides, thicker pores accumulate short and long polynucleotides equally well and allow various molecular compositions. This setting also provides a temperature oscillation, shown previously to exponentially replicate DNA in the protein-assisted PCR. Our results indicate that, for life to evolve, complicated active membrane transport is not required for the initial steps. We find that interlinked mineral pores in a thermal gradient provide a compelling high-concentration starting point for the molecular evolution of life.     

Ion exclusion by sub-2-nm carbon nanotube pores

Biological pores regulate the cellular traffic of a large variety of solutes, often with high selectivity and fast flow rates. These pores share several common structural features: the inner surface of the pore is frequently lined with hydrophobic residues, and the selectivity filter regions often contain charged functional groups. Hydrophobic, narrow-diameter carbon nanotubes can provide a simplified model of membrane channels by reproducing these critical features in a simpler and more robust platform. Previous studies demonstrated that carbon nanotube pores can support a water flux comparable to natural aquaporin channels. Here, we investigate ion transport through these pores using a sub-2-nm, aligned carbon nanotube membrane nanofluidic platform. To mimic the charged groups at the selectivity region, we introduce negatively charged groups at the opening of the carbon nanotubes by plasma treatment. Pressure-driven filtration experiments, coupled with capillary electrophoresis analysis of the permeate and feed, are used to quantify ion exclusion in these membranes as a function of solution ionic strength, pH, and ion valence. We show that carbon nanotube membranes exhibit significant ion exclusion that can be as high as 98% under certain conditions. Our results strongly support a Donnan-type rejection mechanism, dominated by electrostatic interactions between fixed membrane charges and mobile ions, whereas steric and hydrodynamic effects appear to be less important.     

Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import.

Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca2+ homeostasis.     

A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.     

Effects of histamine and some other substances on molecular selectivity of the capillary wall to plasma proteins and dextran

Lymph/plasma concentration ratios (R) were measured for Dextran-110 and for several plasma protein components resolved by disc electrophoresis or gel filtration of dog leg lymph before and after injection of histamine, bradykinin, serotonin (5-HT), or vasodilators into the paw. Most of the molecules studied were larger than can be transported by the hypothetical small pore system (radius 40 Å). Control R for plasma proteins showed a general decline with increasing molecular size. Transport could be accounted for equally well by ultrafiltration through a small number of large pores ($\text{r ≧ 1600}$Å) or by turnover of endothelial vesicles (internal radius 250 Å). Secondary deviations from a strict permeability-molecular size relation suggest that for proteins, permeability is decreased by negative charge. Dextran molecules are transported less readily than proteins of the same effective size. This cannot be attributed to charge, but may be due to differences in molecular configuration or to chemical interactions. Histamine or bradykinin (but not 5-HT or vasodilators) diminish the selectivity of the blood-lymph barrier with respect to molecular size. The effects of electrical charge are also diminished, as is the difference between Dextrans and proteins. These changes in permeability are due entirely to alteration of the capillary pathway for large molecule transport (large pores or vesicles). The data cannot readily be fitted to theoretical predictions for an increase in size or number of large pores, but can be accounted for by an increase in size and volume turnover of endothelial vesicles (cytopempsis).     

Liquid-vapor oscillations of water in hydrophobic nanopores

Water plays a key role in biological membrane transport. In ion channels and water-conducting pores (aquaporins), one-dimensional confinement in conjunction with strong surface effects changes the physical behavior of water. In molecular dynamics simulations of water in short (0.8 nm) hydrophobic pores the water density in the pore fluctuates on a nanosecond time scale. In long simulations (460 ns in total) at pore radii ranging from 0.35 to 1.0 nm we quantify the kinetics of oscillations between a liquid-filled and a vapor-filled pore. This behavior can be explained as capillary evaporation alternating with capillary condensation, driven by pressure fluctuations in the water outside the pore. The free-energy difference between the two states depends linearly on the radius. The free-energy landscape shows how a metastable liquid state gradually develops with increasing radius. For radii > approximately 0.55 nm it becomes the globally stable state and the vapor state vanishes. One-dimensional confinement affects the dynamic behavior of the water molecules and increases the self diffusion by a factor of 2-3 compared with bulk water. Permeabilities for the narrow pores are of the same order of magnitude as for biological water pores. Water flow is not continuous but occurs in bursts. Our results suggest that simulations aimed at collective phenomena such as hydrophobic effects may require simulation times >50 ns. For water in confined geometries, it is not possible to extrapolate from bulk or short time behavior to longer time scales.     

A simple light-driven transmembrane proton pump.

Light-induced lipophilic porphyrin/aqueous acceptor charge separation across a single lipid-water interface can pump protons across the lipid bilayer when the hydrophobic weak acids, carbonylcyanide m-chlorophenylhydrazone and its p-trifluoromethoxyphenyl analogue, are present. These compounds act as proton carriers across lipid bilayers. In their symmetric presence across the bilayer, the positive currents and voltages produced by the photogeneration of porphyrin cations are replaced by larger negative currents and voltages. The maximum negative current and voltage occur at the pH of maximum dark conductance. The reversed larger current and voltage show a positive ionic charge transport in the same direction as the electron transfer. This transport can form an ion concentration gradient. The movement of protons is verified by an unusual D2O isotope effect that increases the negative ionic current by 2- to 3-fold. These effects suggest that an interfacial pK shift of the weak acid caused by the local electric field of photoformed porphyrin cations/acceptor anions functions as the driving force. The estimated pumping efficiency is 10-30%. Time-resolved results show that proton pumping across the bilayer occurs on the millisecond time scale, similar to that of biological pumps. This light-driven proteinless pump offers a simple model for a prebiological energy transducer.     

Selective and asymmetric molecular transport across electroporated cell membranes.

Transport of a divalent cation (Ca2+) and three DNA indicators [ethidium bromide (EB), propidium iodide (PI), and ethidium homodimer (EthD-1)] across electroporated membranes of several mammalian cell lines was found to be selective and asymmetrical. In low salt medium, Ca2+ and EB were preferentially transported across the anodefacing cell membrane while PI and EthD-1 predominately entered at the site facing the cathode. In high salt medium, the entry site for Ca2+ and EB was reversed to the cathode-facing hemisphere while it remained unchanged for PI and EthD-1. In all these experiments, the observed transport patterns remained unaffected whether the dyes (or ion) were present during or added after the electroporating pulse. The data suggest that asymmetric pores are created on both sides of the membrane facing the electrodes, with smaller pore size (but greater in number) on the anode side and larger pores (with a lower population) on the cathode side. Furthermore, the rate of resealing of the membrane pores is significantly enhanced in high ionic strength medium, thus affecting the entry site. The asymmetric transport pattern is neither caused by electrophoresis induced by the externally applied electric field nor due to one-sided membrane breakdown as previously believed.     

From the Cover: Bioelectronic silicon nanowire devices using functional membrane proteins

Modern means of communication rely on electric fields and currents to carry the flow of information. In contrast, biological systems follow a different paradigm that uses ion gradients and currents, flows of small molecules, and membrane electric potentials. Living organisms use a sophisticated arsenal of membrane receptors, channels, and pumps to control signal transduction to a degree that is unmatched by manmade devices. Electronic circuits that use such biological components could achieve drastically increased functionality; however, this approach requires nearly seamless integration of biological and manmade structures. We present a versatile hybrid platform for such integration that uses shielded nanowires (NWs) that are coated with a continuous lipid bilayer. We show that when shielded silicon NW transistors incorporate transmembrane peptide pores gramicidin A and alamethicin in the lipid bilayer they can achieve ionic to electronic signal transduction by using voltage-gated or chemically gated ion transport through the membrane pores.     

Flow-dependent channel formation in clots by an erythrocyte-bound fibrinolytic agent

Studies in animal models have shown that plasminogen activators bound to erythrocytes (RBC-PA) have an extended lifetime in the circulation and are safer than free PAs. RBC-PAs incorporate into nascent thrombi, which are focally lysed from within, an attractive thromboprophylactic option. In static systems, RBC-PAs cleave surrounding fibrin fibers, forming pores larger than the cells themselves, and move around the pore edges, enlarging them until eventual clot dissolution. We hypothesized that under flow in blood vessels, RBC-PAs form functional patent channels before clot dissolution. Here we used perfusion chambers to study clot lysis by RBC-PAs under static versus arterial and venous flow conditions. We found that flow decelerates bulk clot lysis but quickly generates patent channels filled with passing RBCs, via pore enlargement and merging in the direction of flow. Formation of such channels by RBC-PAs may help rescue ischemic tissue before bulk dissolution of potentially occlusive clots.     

A helical-dipole model describes the single-channel current rectification of an uncharged peptide ion channel.

We are designing simple peptide ion channels as model systems for the study of the physical principles controlling conduction through ion-channel proteins. Here we report on an uncharged peptide, Ac-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, designed to form an aggregate of parallel, amphiphilic, membrane-spanning alpha-helices around a central water-filled pore. This peptide in planar lipid bilayers forms ion channels that show single-channel current rectification in symmetric 1 M KCl. The current at a given holding membrane potential is larger than the current measured through the same channel when the potential is reversed. Based on our hypothesized gating mechanism, the larger currents flow from the peptide carboxyl terminus toward the amino terminus. We present an ionic electrodiffusion model based on the helical-dipole potential and the dielectric interfacial polarization energy, which with reasonable values for dipole magnitude and dielectric constants, accurately replicates the current-voltage data.     

Electrical Conductivity of a Stretching Viscoelastic Filament

Long polymeric chains highly stretched and aligned with the flow confer a strong mechanical anisotropy on a viscoelastic solution. The electrically-driven transport of free ions under such conditions is far from being understood. In this paper, we determine experimentally whether the above-mentioned deviation from isotropy affects the electric charge transport across the liquid. To this end, we measure the electrical conductivity in the flow (stretching) direction of the cylindrical liquid filament formed in the elasto-capillary thinning that arises during the breakup of a viscoelastic liquid bridge. First, we examine the behavior of monodisperse solutions of polyethylene oxide (PEO) in a mixture of glycerine and water. For all the concentrations and molecular weights considered, the filament conductivity remains practically the same as the isotropic conductivity measured under hydrostatic conditions. However, we observe a decrease in the electric current at the end of elasto-capillary regime which may partially be attributed to the reduction of the liquid conductivity. Then, we measure the conductivity of bidisperse solutions of PEO with very different molecular weights. In this case, a significant decrease in conductivity is observed as the filament radius decreases. This constitutes the first experimental evidence of ion mobility reduction in stretching viscoelastic filaments, a relevant effect in applications such as electrospinning.     

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Scanning confocal fluorescence microscopy and multiphoton fluorescence microscopy were used to image the uptake of the protein lysozyme into individual ion exchange chromatography particles in a packed bed in real time. Self-sharpening concentration fronts penetrating into the particles were observed at low salt concentrations in all of the adsorbents studied, but persisted to 100 mM ionic strength only in some materials. In other adsorbents, diffuse profiles were seen at these higher salt concentrations, with the transition region exhibiting a pronounced fluorescence peak at the front at intermediate salt concentrations. These patterns in the uptake profiles are accompanied by significant increases in protein uptake rates that are also seen macroscopically in batch uptake experiments. The fluorescence peak appears to be a concentration overshoot that may develop, in part, from an electrokinetic contribution to transport that also enhances the uptake rate. Further evidence for an electrokinetic origin is that the effect is correlated with high adsorbent surface charge densities. Predictions of a mathematical model incorporating the electrokinetic effect are in qualitative agreement with the observations. These findings indicate that mechanisms other than diffusion contribute to protein transport in oppositely charged porous materials and may be exploited to achieve rapid uptake in process chromatography.     

Surface Charge and the Conductance of Phospholipid Membranes

Bilayer membranes, formed from various phospholipids, were studied to assess the influence of the charge of the polar head groups on the membrane conductance mediated by neutral "carriers" of cations and anions. The surface charge of an amphoteric lipid, phosphatidyl ethanolamine, was altered by varying the pH, and the surface charge of several lipids was screened by increasing the ionic strength of the solution with impermeant monovalent and divalent electrolytes. The surface charge should be a key parameter in defining the membrane conductance for a variety of permeation mechanisms; conductance measurements in the presence of carriers may be used to estimate the potential difference, due to surface charge, between the interior of the bilayer and the bulk aqueous phase. The large changes in conductance observed upon varying the surface charge density and the ionic strength agree with those predicted by the Gouy-Chapman theory for an aqueous diffuse double layer. Explicit expressions for the dependence of the membrane conductance on the concentrations of the carrier, the permeant ion, the surface charge density, and the ionic strength are presented.     

Versatile ultrathin nanoporous silicon nitride membranes

Single- and multiple-nanopore membranes are both highly interesting for biosensing and separation processes, as well as their ability to mimic biological membranes. The density of pores, their shape, and their surface chemistry are the key factors that determine membrane transport and separation capabilities. Here, we report silicon nitride (SiN) membranes with fully controlled porosity, pore geometry, and pore surface chemistry. An ultrathin freestanding SiN platform is described with conical or double-conical nanopores of diameters as small as several nanometers, prepared by the track-etching technique. This technique allows the membrane porosity to be tuned from one to billions of pores per square centimeter. We demonstrate the separation capabilities of these membranes by discrimination of dye and protein molecules based on their charge and size. This separation process is based on an electrostatic mechanism and operates in physiological electrolyte conditions. As we have also shown, the separation capabilities can be tuned by chemically modifying the pore walls. Compared with typical membranes with cylindrical pores, the conical and double-conical pores reported here allow for higher fluxes, a critical advantage in separation applications. In addition, the conical pore shape results in a shorter effective length, which gives advantages for single biomolecule detection applications such as nanopore-based DNA analysis.     

Process of inducing pores in membranes by melittin

Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons, corresponding to its antimicrobial activities. Despite extensive studies, aspects of the molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of the melittin-bound membrane before and after the process. By correlating experiments using giant unilamellar vesicles with those of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide–lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area.     

Role of cyclic AMP in regulation of chloride secretion by canine tracheal mucosa

Cyclic adenosine monophosphate (cyclic AMP) mediates the action of many hormones, and it affects ion transport in several different epithelia. The effect of the analogue dibutyryl cyclic AMP was tested in canine tracheal epithelium in vitro under short-circuit conditions. This agent raised short-circuit current and electrical conductance. Dibutyryl cyclic AMP at 1 mM concentration added to the submucosal bath increased net Cl secretion from 1.74 +/- 0.40 to 2.72 +/- 0.34 mu eq/cm2 . h (mean +/- SE, n = 6). Net Na absorption was slightly reduced. Dibutyryl cyclic AMP and theophylline decreased mannitol flux, suggesting that cyclic AMP acts on Cl transport by increasing its flow through cellular rather than through paracellular pathways. Epinephrine, which also stimulates Cl secretion, causes significant elevation in cyclic AMP in epithelial cells scraped from tracheal mucosa. Data suggest that cellular concentrations of cyclic AMP play an important role in the regulation of Cl transport by tracheal mucosa.