Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

Study of the conductivity of single supernarrow pores (1–15 nm in diameter) formed in thin membranes (10–12 μm in thickness) from polyethylene terephthalate (PETP) has revealed discrete changes in the currents passing through such pores when applied from an external source of potential difference of 200–1000 mV. Based on several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical to the so-called currents of single ionic channels in cell membranes. The supernarrow pores whose properties are described in the present work were obtained by alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). On the walls of the pores, carboxyl groups, i.e., negative fixed charges, and their compensating counterion (cation) layer are formed. Upon setting the potential difference onto the PETP membrane, this cation layer is able to transfer current, through a process called surface conductance. In the case of nanometer-sized diameters of the pores, such surface conductance can turn out to be dominating. We have shown that these discrete changes of currents passing through the nanometer pores are associated with metastability of their surface conductance. In the highly cation-selective channels in the cell membranes, there should inevitably exist an area with dominating cation surface conductance and, hence, conductance metastability. Therefore, a new explanation is proposed of the characteristic discreteness of the currents of single cation-specific ionic channels in cell membranes. Such an explanation does not rule out the existence of any other traditional explanation of the discreteness of ion channel currents.     

An anion-selective analogue of the channel-forming peptide alamethicin.

The peptide alamethicin self-assembles to form helix bundle ion channels in membranes. Previous macroscopic measurements have shown that these channels are mildly cation-selective. Models indicate that a source of cation selectivity is a zone of partial negative charge toward the C-terminal end of the peptide. We synthesized an alamethicin derivative with a lysine in this zone (replacing the glutamine at position 18 in the sequence). Microscopic (single-channel) measurements demonstrate that dimeric alamethicin-lysine18 (alm-K18) forms mildly anion-selective channels under conditions where channels formed by the parent peptide are cation-selective. Long-range electrostatic interactions can explain the inversion of ion selectivity and the conductance properties of alamethicin channels.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Urinary proteases degrade epithelial sodium channels

Summary The mammalian urinary bladder epithelium accommodates volume changes by the insertion and withdrawal of cytoplasmic vesicles. Both apical membrane (which is entirely composed of fused vesicles) and the cytoplasmic vesicles contain three types of ionic conductances, one amiloride sensitive, an-other a cation-selective conductance and the third a cation conductance which seems to partition between the apical membrane and the mucosal solution. The transport properties of the apical membrane (which has been exposed to urine in vivo) differ from the cytoplasmic vesicles by possessing a lower density of amiloride-sensitive channels and a variable level of leak conductance. It was previously shown that glandular kallikrein was able to hydrolyze epithelial sodium channels into the leak conductance and that this leak conductance was further degraded into a channel which partitioned between the apical membrane and the mucosal solution. This report investigates whether kallikrein is the only urinary constituent capable of altering the apical membrane ionic permeability or whether other proteases or ionic conditions also irreversible modify apical membrane permeability.Alterations of mucosal pH, urea concentrations, calcium concentrations or osmolarity did not irreversible affect the apical membrane ionic conductances. However, urokinase and plasmin (both serine proteases found in mammalian urine) were found to cause an irreversible loss of amiloride-sensitive current, a variable change in the leak current as well as the appearance of a third conductance which was unstable in the apical membrane and appears to partition between the apical membrane and the mucosal solution. Amiloride protects the amiloride-sensitive conductance from hydrolysis but does not protect the leak pathway. Neither channel is protected by sodium. Fluctuation analysis demonstrated that the loss of amiloride-sensitive current was due to a decrease in the sodium-channel density and not a change in the single-channel current. Assuming a simple model of sequential degradation, estimates of single-channel currents and conductances for both the leak channel and unstable leak channel are determined.     

A patch-clamp study of histamine-secreting cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.     

Alzheimer''s disease amyloid beta-protein forms Zn(2+)-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons.

We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1-40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1-40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1-40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1-40] channel pore. These properties of the A beta P[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1-40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity.     

The effect of prymnesin on the electric conductivity of thin lipid membranes

Summary A proteolipidic toxin, prymnesin, when added to the aqueous solutions around thin lipid membranes causes a marked increase in membrane conductance. The toxin-treated membrane is cation-permselective. The extent of cation permselectivity is dependent upon ionic strength of the aqueous solutions in a fashion similar to the dependence of cation permselectivity of a cation exchanger containing about 100mm of fixed negative sites. Dose-response relationship studies reveal a linear relation between log prymnesin concentration and log membrane conductance. The slope of the curve is around 3 if the toxin is applied to one side of the membrane and is around 7 if the toxin is applied to both sides of the membrane. The membrane treated with toxin on one side only is clearly asymmetric in its properties. These characteristics are expressed by an asymmetric current-voltage relationship, and by asymmetric sensitivity of membrane conductance to pH and to salt concentration. The conductance of the toxin-treated membrane is inversely proportional to temperature. It is suggested that aggregates of toxin moieties assemble in the membrane to form negatively charged aqueous pores. There is roughly a good correlation between the increase in membrane conductance and the increase in membrane permeability to urea if both were attributed to the formation of aqueous channels in the membrane.     

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: A comparison

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.     

Amphotericin B Membrane Action: Role for Two Types of Ion Channels in Eliciting Cell Survival and Lethal Effects

The formation of aqueous pores by the polyene antibiotic amphotericin B (AmB) is at the basis of its fungicidal and leishmanicidal action. However, other types of nonlethal and dose-dependent biphasic effects that have been associated with the AmB action in different cells, including a variety of survival responses, are difficult to reconcile with the formation of a unique type of ion channel by the antibiotic. In this respect, there is increasing evidence indicating that AmB forms nonaqueous (cation-selective) channels at concentrations below the threshold at which aqueous pores are formed. The main foci of this review will be (1) to provide a summary of the evidence supporting the formation of cation-selective ion channels and aqueous pores by AmB in lipid membrane models and in the membranes of eukaryotic cells; (2) to discuss the influence of membrane parameters such as thickness fluctuations, the type of sterol present and the existence of sterol-rich specialized lipid raft microdomains in the formation process of such channels; and (3) to develop a cell model that serves as a framework for understanding how the intracellular K(+) and Na(+) concentration changes induced by the cation-selective AmB channels enhance multiple survival response pathways before they are overcome by the more sustained ion fluxes, Ca(2+)-dependent apoptotic events and cell lysis effects that are associated with the formation of AmB aqueous pores.     

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.     

Permeability Increase Induced by Escherichia coli Hemolysin A in Human Macrophages is Due to the Formation of Ionic Pores: A Patch Clamp Characterization

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K⁺ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches. Received: 14 August 1995/Revised: 2 October 1995     

Activation of a nonselective cation channel by swelling in atrial cells

Cell swelling has been shown to increase the permeability of the plasma membrane to ions such as K⁺, Na⁺, Ca²⁺ or Cl^– in many types of cells. In cardiac cells, swelling has been reported to increase Cl^– conductance, but whether cation-selective currents are activated by swelling is not known. Low Cl^– or Cl^–-free solutions were used to study the presence of such currents. Lowering the osmolarity of the extracellular medium from 299 to 219 mOsm resulted in cell swelling and concurrent activation of a cation-selective whole-cell current. When cell-attached patches were formed on swollen cells, opening of bursting single channel currents were observed in 18% of the patches studied. Ion substitution experiments indicated that the channel discriminated poorly among monovalent cations, and was impermeable to Cl^–. The channel was permeable to Ca²⁺. In symmetrical 140 mM K⁺, the current-voltage relation was linear with a single channel conductance of 36 ± 3 pS. Depolarization increased channel open probability. Interestingly, depending on the membrane patch studied, application of negative pressure to the pipette caused either an increase or a decrease in the open probability of the channel already activated by swelling. Thus, the sensitivity to tension of the swelling-activated channel was different from those of previously reported stretch-activated channels. These findings suggest that nonselective cation channels exist in rat atrial cells and may be involved in swelling-induced changes in cell function.Dr. Kim is an Established Investigator of the American Heart Association.     

Discovery of a novel channel-forming protein in the cell wall of the non-pathogenic Nocardia corynebacteroides

Detergent extracts of whole cells of the Gram-positive, non-pathogenic, strictly aerobic bacterium Nocardia corynebacteroides contain channel-forming activity. The protein responsible for channel formation was identified using lipid bilayer experiments. It was purified to homogeneity and had an apparent molecular mass of about 134 kDa on SDS-PAGE when it was solubilized at 40 degrees C. When the 134 kDa protein was heated to 100 degrees C for 10 min in sample buffer, it dissociated into subunits with a molecular mass of about 23 kDa and focused at pI of 4.5 during isoelectric focusing. The pure 134 kDa protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine-phosphatidylserine mixtures by the formation of ion-permeable channels. The channels had an average single-channel conductance of 5.5 nS in 1 M KCl and were found to be cation-selective. Asymmetric addition of the 134 kDa protein to lipid bilayer membranes resulted in an asymmetric voltage-dependence. The analysis of the single-channel conductance as a function of cation radii using the Renkin correction factor and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.0 nm. The channel characteristics of the cell wall channel of N. corynebacteroides were compared with those of other members of the mycolata. They share common features because they are composed of small molecular mass subunits and form large and water-filled channels.     

Odorant-induced currents in intact patches from rat olfactory receptor neurons: theory and experiment.

Odorant-induced currents in mammalian olfactory receptor neurons have proved difficult to obtain reliably using conventional whole-cell recording. By using a mathematical model of the electrical circuit of the patch and rest-of-cell, we demonstrate how cell-attached patch measurements can be used to quantitatively analyze responses to odorants or a high (100 mM) K+ solution. High K+ induced an immediate current flux from cell to pipette, which was modeled as a depolarization of approximately 52 mV, close to that expected from the Nernst equation (56 mV), and no change in the patch conductance. By contrast, a cocktail of cAMP-stimulating odorants induced a current flux from pipette into cell following a significant (4-10 s) delay. This was modeled as an average patch conductance increase of 36 pS and a depolarization of 13 mV. Odorant-induced single channels had a conductance of 16 pS. In cells bathed with no Mg2+ and 0.25 mM Ca2+, odorants induced a current flow from cell to pipette, which was modeled as a patch conductance increase of approximately 115 pS and depolarization of approximately 32 mV. All these results are consistent with cAMP-gated cation channels dominating the odorant response. This approach, which provides useful estimates of odorant-induced voltage and conductance changes, is applicable to similar measurements in any small cells.     

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.     

Investigation of the electrophysiological properties of enniatins

Enniatins are cyclohexadepsipeptides produced by various species of the genus Fusarium, and are known to have ionophoric, antibiotic, and in vitro hypolipidaemic properties. With the patch clamp technique in the inside-out mode it could be shown that enniatin easily incorporates into the cell membrane in which it forms cation-selective pores. This feature is characterized by unitary transitions to conductance levels typical for channels, ion selectivity, rectification, conductivity in the pS range, and block. A model of vertically stacked enniatin molecules that form sandwich complexes is suggested. Like gramicidin enniatin is a passive channel. Single channel properties for the isomers enniatin A1, B, and B1 differed from each other. This implies an influence of the substituted moieties on the complexation of cations. Electrical activity was followed by changes in intracellular ion concentrations, which are consistent with depolarization of the membrane resting potential, shortening of action potential duration, and reduced contractility.     

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Silent TWIK-1 potassium channels conduct monovalent cation currents

TWIK-1 two-pore domain K(+) channels generally produce nonmeasurable or very low levels of K(+) currents in heterologous expression systems under physiologically ionic conditions. Two controversial mechanisms have been proposed to account for this behavior: TWIK-1 K(+) channels are expressed in the cell surface but silenced by sumoylation at a lysine residue (TWIK-1 K274); constitutive and rapid internalization of TWIK-1 causes TWIK-1 channel silencing. Here we report that TWIK-1 K(+) channels heterologously expressed in Chinese hamster ovary cells, which are silent in physiological K(+) gradients, are able to conduct large monovalent cation currents when extracellular ionic conditions change. These results support the hypothesis that TWIK-1 K(+) channels are expressed in the cell surface but silent, and suggest that the TWIK-1 gating behavior rather than the lack of cell surface expression of TWIK-1 results in nondetectable TWIK-1 K(+) currents in heterologous expression systems.