Excess electrical noise during current flow through porous membranes separating ionic solutions

Summary Spectral analysis of electrical noise from various artificial membrane systems suggests that excess noise of anf ^–n spectral form, wheren is approximately unity, is not primarily a bulk phenomenon simply dependent on the number of charge carriers. Measurements from aqueous and nonaqueous electrolytic resistors, comprised of several different ionic species, show only flat power density spectra under applied currents, even at extreme dilutions. Excess noise off ^–n form is observed under applied d-c current in single pore membranes, as previously reported, but is also seen in multipore and polymer mesh membranes. Calculations based on single pore membrane noise data are in significant variance with the bulk charge carrier model proposed by Hooge. These observations suggest that such excess noise occurs in conjunction with anisotropic constraints to ion flow.1 Fishman, H. M., Moore, L. E., Poussart, D. J. M. 1975. Potassium ion conduction noise in squid axon membrane.J. Membrane Biol. (Submitted for publication).     

Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes

The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.     

ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis.

The "molecular Coulter counter" concept has been used to study transport of ATP molecules through the nanometer-scale aqueous pore of the voltage-dependent mitochondrial ion channel, VDAC. We examine the ATP-induced current fluctuations and the change in average current through a single fully open channel reconstituted into a planar lipid bilayer. At high salt concentration (1 M NaCl), the addition of ATP reduces both solution conductivity and channel conductance, but the effect on the channel is several times stronger and shows saturation behavior even at 50 mM ATP concentration. These results and simple steric considerations indicate pronounced attraction of ATP molecules to VDAC's aqueous pore and permit us to evaluate the effect of a single ATP molecule on channel conductance. ATP addition also generates an excess noise in the ionic current through the channel. Analysis of this excess noise shows that its spectrum is flat in the accessible frequency interval up to several kilohertz. ATP exchange between the pore and the bulk is fast enough not to display any dispersion at these frequencies. By relating the low-frequency spectral density of the noise to the equilibrium diffusion of ATP molecules in the aqueous pore, we calculate a diffusion coefficient D = (1.6-3.3)10(-11) m2/s. This is one order of magnitude smaller than the ATP diffusion coefficient in the bulk, but it agrees with recent results on ATP flux measurements in multichannel membranes using the luciferin/luciferase method.     

Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes

A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes. By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore. We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS). Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution. In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration. The channel is cation-selective at high pH but nearly unselective at low pH. We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances. In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane. Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane. We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores. This was effective only when the enzyme was applied on the cis side.     

GCEMC Simulations of “Swiss Cheese” Ion Exchange Membranes in Dilute Solutions of Primitive Model 1:1 Electrolytes with Different Sizes of Ions or 2:1 Electrolytes with Different Bjerrum Parameters

Abstract

Monte Carlo simulations using a Markov process corresponding to a (generalized) Grand Canonical Ensemble have been performed for a number of spherical micropores in equilibrium with dilute external bulk solutions of primitive model electrolytes. Dilute solutions of 1:1 electrolytes with a Bjerrum parameter B = 1.546 with cations three times larger than the anions have been simulated. Also, dilute solutions of 2:1 electrolytes with ions of equal size and reduced Bjerrum parameters B_r = 1.546 and 3 have been simulated. The pores are primitive pores with hard walls and the same dielectric permittivity in the wall and in the pore solution. They range from a pore radius = 5 times the mean ionic diameter to 35 times this diameter, and they carry a fixed charge equal to + 5,0 and ?5 elementary charges. The fixed charge is modelled as smoothly distributed on the pore-wall interface. In addition to the electric potential of the interfacial charge and the electric potential of the spherical double layer, a potential Δ between the pore solution and the bulk solution may be deliberately added. For single pores we may take Δ = 0, but then the pore is generally not electroneutral. In a “Swiss cheese” membrane with a lot of (equally sized) pores, the membrane phase has to approach electroneutrality for growing size of the phase. This is approximated by means of a membrane generated potential Δ in each pore (from the electrostatic interactions with the other pores). The potential A so chosen to obtain electroneutrality is the GCEMC Donnan potential. These non-ideal Donnan potentials are compared to the ideal values (with activity coefficients equal to zero). From the mean occupation numbers of cations and anions in the pores, the average pore values of the mean ionic and the single ionic activity coefficients of the ions are calculated. These are very dependent on pore sizes and on the potential in the pore. The excess energy and the electrostatic Helmholtz free energy of the ions in the pores are also simulated directly. The electrostatic entropy is found as the difference.     

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation

Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein- containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae.     

Non-equilibrium voltage noise generated by ion transport through pores

In this paper, we describe a systematic approach to the theoretical analysis of non-equilibrium voltage noise that arises from ions moving through pores in membranes. We assume that an ion must cross one or two barriers in the pore in order to move from one side of the membrane to the other. In our analysis, we consider the following factors: a) surface charge as a variable in the kinetic equations, b) linearization of the kinetic equations, c) master equation approach to fluctuations. To analyze the voltage noise arising from ion movement through a two barrier (i.e., one binding site) pore, we included the effects of ions in the channel's interior on the voltage noise. The current clamp is considered as a white noise generating additional noise in the system. In contrast to what is found for current noise, at low frequencies the voltage noise intensity is reduced by increasing voltage across the membrane. With this approach, we demonstrate explicity for the examples treated that, apart from additional noise generated by the current clamp, the non-equilibrium voltage fluctuations can be related to the current fluctuations by the complex admittance.     

Use of protein charge ladders to study electrostatic interactions during protein ultrafiltration

Recent studies have demonstrated the importance of electrostatic interactions in membrane systems, but there is still controversy about the underlying phenomena. Protein charge ladders, consisting of a set of chemical derivatives of a given protein that differ by single charge groups, were used to quantify the electrostatic interactions during protein ultrafiltration. Myoglobin charge ladders were generated by acylation, with the different derivatives analyzed simultaneously by capillary electrophoresis. Filtration experiments were performed using polyethersulfone and composite regenerated cellulose membranes, with the membrane charge determined from the streaming potential. As expected, the rejection increased as the protein became more heavily charged due to the increase in electrostatic repulsion. However, the transmission of the weakly charged myoglobin species increased dramatically at very low ionic strength. This increase in transmission was attributed to a shift in pH within the pore caused by hydrogen ion partitioning into the charged membrane. The sieving data were in good agreement with theoretical calculations accounting for the effects of this pH shift on the electrostatic interactions.     

Structure and activity of the N-terminal region of the eukaryotic cytolysin equinatoxin II

The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.     

The pho-controlled outer membrane porin PhoE does not contain specific binding sites for phosphate or polyphosphates

Purified PhoE-porins were reconstituted into black lipid bilayer membranes, and the selectivity and size of the reconstituted pores were determined. Addition of polyphosphates influenced the internal charge situation of the pore resulting in a shift from anion to cation selectivity. However, the pore size as judged from single channel conductances was not influenced by the addition of polyphosphates. A strong inhibition of the pore conductance only occurred when Mg2+ was also present in the aqueous phase. The inhibition of the pore function is presumably caused by the formation of a chelate between the divalent cation and the polyphosphate. Nevertheless, neither this inhibition nor the selectivity shift are specific to phosphate, because both effects can be mimicked by other polyvalent anions such as citrate. Inhibition of the PhoE pore function by polyphosphate in in vivo experiments confirmed the results of in vitro experiments that polyphosphate is only able to affect the permeability of the outer membrane toward beta-lactam antibiotics if Mg2+ is present. The outcome of the in vivo and the in vitro experiments are consistent with the assumption that the PhoE-porins do not contain a specific binding site for phosphate or polyphosphates but are anion selective because of an excess of positively charged amino acids inside or at the surface of the pore.     

Fluctuation of motor charge in the lateral membrane of the cochlear outer hair cell

Functioning of the membrane motor of the outer hair cell is tightly associated with transfer of charge across the membrane. To obtain further insights into the motor mechanism, we examined kinetics of charge transfer across the membrane in two different modes. One is to monitor charge transfer induced by changes in the membrane potential as an excess membrane capacitance. The other is to measure spontaneous flip-flops of charges across the membrane under voltage-clamp conditions as current noise. The noise spectrum of current was inverse Lorentzian, and the capacitance was Lorentzian, as theoretically expected. The characteristic frequency of the capacitance was approximately 10 kHz, and that for current noise was approximately 30 kHz. The difference in the characteristic frequencies seems to reflect the difference in the modes of mechanical movement associated with the two physical quantities.     

Partitioning of Differently Sized Poly(ethylene glycol)s into OmpF Porin

To understand the physics of polymer equilibrium and dynamics in the confines of ion channel pores, we study partitioning of poly(ethylene glycol)s (PEGs) of different molecular weights into the bacterial porin, OmpF. Thermodynamic and kinetic parameters of partitioning are deduced from the effects of polymer addition on ion currents through single OmpF channels reconstituted into planar lipid bilayer membranes. The equilibrium partition coefficient is inferred from the average reduction of channel conductance in the presence of PEG; rates of polymer exchange between the pore and the bulk are estimated from PEG-induced conductance noise. Partition coefficient as a function of polymer weight is best fitted by a “compressed exponential” with the compression factor of 1.65. This finding demonstrates that PEG partitioning into the OmpF channel pore has sharper dependence on polymer molecular weight than predictions of hard-sphere, random-flight, or scaling models. A 1360-Da polymer separates regimes of partitioning and exclusion. Comparison of its characteristic size with the size of a 2200-Da polymer previously found to separate these regimes for the α-toxin shows good agreement with the x-ray structural data for these channels. The PEG-induced conductance noise is compatible with the polymer mobility reduced inside the OmpF pore by an order of magnitude relatively to its value in bulk solution.     

Are fusion peptides a good model to study viral cell fusion?

Fusion peptides are hydrophobic and conserved sequences located within glycoprotein ectodomains that protrude from the virion surface. Direct participation of fusion peptides in the viral membrane fusion phenomenon has been inferred from genetic analyses showing that even a single residue substitution or a deletion within these sequences may completely block the process. However, the specific fusion peptide activities associated to the multi-step fusion mechanism are not well defined. Based on the assumption that fusion peptides are transferred into target membranes, biophysical methodologies have been applied to study integration into model membranes of synthetic fragments representing functional and non-functional sequences. From these studies, it is inferred that, following insertion, functional sequences generate target membrane perturbations and adopt specific structural arrangements within. Further characterization of these artificial systems may help in understanding the molecular processes that bring initial bilayer destabilizations to the eventual opening of a fusion pore.     

Are fusion peptides a good model to study viral cell fusion?

Fusion peptides are hydrophobic and conserved sequences located within glycoprotein ectodomains that protrude from the virion surface. Direct participation of fusion peptides in the viral membrane fusion phenomenon has been inferred from genetic analyses showing that even a single residue substitution or a deletion within these sequences may completely block the process. However, the specific fusion peptide activities associated to the multi-step fusion mechanism are not well defined. Based on the assumption that fusion peptides are transferred into target membranes, biophysical methodologies have been applied to study integration into model membranes of synthetic fragments representing functional and non-functional sequences. From these studies, it is inferred that, following insertion, functional sequences generate target membrane perturbations and adopt specific structural arrangements within. Further characterization of these artificial systems may help in understanding the molecular processes that bring initial bilayer destabilizations to the eventual opening of a fusion pore.     

Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization

Streptolysin O belongs to the class of thiol-activated toxins, which are single chain, four-domain proteins that bind to membranes containing cholesterol and then assemble to form large oligomeric pores. Membrane binding involves a conserved tryptophan-rich sequence motif located within the C-terminally located domain 4. In contrast, sites involved in oligomerization and pore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly expressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to membranes, but it did not form oligomers nor lyse cell membranes. Moreover, the labeled fragment did not detectably become incorporated into hybrid oligomers when combined with lytically active full-length toxin. However, when present in large excess over the active toxin, the domain 4 fragment effected reduction of hemolytic activity and of functional pore size, which indicates interference with oligomerization of the lytically active species. Our findings support the notion that domain 4 of the streptolysin O molecule may fold autonomously, is essential for membrane binding and is capable not of irreversible but of reversible association with the entire toxin molecule.     

The mode of action of Vibrio cholerae cytolysin. The influences on both erythrocytes and planar lipid bilayers.

The interaction with erythrocytes of cholera cytolysin (CC) obtained from a non-01 Vibrio cholerae strain results in the osmotic rupture of target cells upon formation by CC of the waterfilled pores in their membranes. The aggregation of several toxin monomers is required for the formation of one CC channel with a radius of 0.9-1.0 nm. The investigations using planar bilayer lipid membranes suggest that the CC-induced pore is an interprotein anion selective channel carrying a fixed positive charge. The role of the charge was supported by the influence of pH on the selectivity, single conductance and voltage gating of the CC channels. The ability of the CC to modify both model and natural membranes has a maximum at pH 6.0-7.0. It was found that CC channels insert into the membrane asymmetrically. The effect of proteolytic treatment of the channel by papain also indicates that the two entrances of the channel protrude from the plane of the membrane into the solution for different distances. It is proposed that the biological effects of the non-01 V. cholera cytolysin are based on its channel-forming activity.     

Mechanism of pore formation in stearoyl-oleoyl-phosphatidylcholine membranes subjected to lateral tension

Theoretical model of a through pore formation in lipid bilayer membrane under applied lateral tension was developed. In the framework of elastic theory of liquid crystals adapted to lipid membranes, we calculated a continuous trajectory from intact bilayer through a hydrophobic defect to a through pore. It was shown that the major energetic characteristic of membrane stability with respect to the pore formation, i. e., line tension, depends both on the pore radius and on the value of the applied lateral tension. This leads to a non-monotonous dependence of the average waiting time of the pore formation on the lateral tension: at low tensions the waiting time was large, then there was a local minimum, after which the average waiting time was increasing again. For membranes formed from stearoyl oleoyl phosphatidylcholine, the local minimum corresponded to the lateral tension of 7 mN/m; the calculated value of the edge line tension of a large pore was 16.5 pN. These results are consistent with available experimental data.     

Highly aligned lipid membrane systems in the physiologically relevant "excess water" condition

The "excess water" condition in biologically relevant systems is met when a membrane mesophase coexists with excess bulk water. Further addition of water to such a system results in no change to any of the system's physical properties (e.g., transition temperature, repeat spacing, and structural mesophases). Moreover, because biological membranes are anisotropic systems, many of their properties are best studied using aligned samples. Although model membrane systems are routinely aligned, they have traditionally been hydrated with water vapor. It is well known that membranes exposed to water vapor at 100% humidity do not imbibe the same quantity of water as a sample in contact with liquid water. As such, membranes that have been hydrated with water vapor have physical properties different from those of membranes dispersed in water. Because of this shortcoming, aligned membranes have not been utilized to their full potential. Here we present a novel and simple method of aligning model membrane systems under conditions of excess water, which will make possible, for the first time, a variety of techniques (e.g., neutron and x-ray diffraction, nuclear magnetic resonance, electron spin resonance, attenuated total reflection infrared spectroscopy, etc.) for studying such systems under physiologically relevant conditions. In addition, when dealing with samples of limited availability, the system allows for the conditions (buffer pH and ionic strength) to be altered without any effect on the sample's alignment.     

Cellular membrane potentials induced by alternating fields

Membrane potentials induced by external alternating fields are usually derived assuming that the membrane is insulating, that the cell has no surface conductance, and that the potentials are everywhere solutions of the Laplace equation. This traditional approach is reexamined taking into account membrane conductance, surface admittance, and space charge effects. We find that whenever the conductivity of the medium outside the cell is low, large corrections are needed. Thus, in most of the cases where cells are manipulated by external fields (pore formation, cell fusion, cell rotation, dielectrophoresis) the field applied to the cell membrane is significantly reduced, sometimes practically abolished. This could have a strong bearing on present theories of pore formation, and of the influence of weak electric fields on membranes.