Properties of ionic channels formed by the antibiotic syringomycin E in lipid bilayers: dependence on the electrolyte concentration in the bathing solution.

Using the planar lipid bilayer technique, organization of ionic channels formed by the lipodepsipeptide antibiotic syringomycin E applied to one (cis) side of a lipid bilayer was studied. Low concentrations of NaCl (0.01-0.1 M) induced the opening and closing of two types of channels - "small" and "large". The large channels had single channel conductances approximately six times greater than those of the small channels. An increase in the NaCl concentration (0.6-1.0 M) decreased almost completely the chance to reveal the large channels. Although the syringomycin channels exhibited the anion selectivity within the entire range of NaCl concentrations in the bathing solutions (from 0.001 to 1.0 M) whereas the concentration gradients across the bilayers were 2 and 4, the transfer numbers for Cl-decreased with an increase in the mean NaCl concentration (from 0.83 for 0.005 M to 0.70 for 0.5 M). Moreover, at each mean value of NaCl concentration, all conductance levels had the same ion selectivity (identical reversal potential). These results suggest that at low NaCl concentrations the large channels are clusters of small channels which synchronously open and close, while at high electrolyte concentrations the screening of the charged groups responsible for channel interactions prevents the cluster formation. A new theoretical approach for the estimation of the channel radius and the number of elementary charges located at its inner surface (based on the experimental curve of the dependence of transfer number on the NaCl concentration) was developed. Based on this theoretical approach, the channel radius equal to 1 nm and one elementary charge located at its inner surface were obtained.     

Activity of toxins produced by Pseudomonas syringae pv. syringae in model and cell membranes

We studied effects of toxins produced by a bacterium Pseudomonas syringae pv. syringae on the conductance of bilayer lipid membranes (BLM). The used toxins were as follows: syringopeptin 22A (SP22A), syringomycin E (SPE), syringostatin A (SSA), syringotoxin B (STB), and methylated syringomycin E (CH3-SRE). All toxins demonstrated channel-forming activity. The threshold sequence for toxin activity was SP22A > SRE approximately equal to SSA > STB > CH3-SRE, and this sequence was independent of lipid membrane composition, and NaCl concentration (pH 6) in the membrane bathing solution (in the range of 0.1-1.0 M). This sequence correlated with relative bioactivities of toxins. In addition, SRE demonstrated a more potent antifungal activity than CH3-SRE. These findings suggest that ion channel formation may underlie the bioactivities of the above toxins. The properties of single ion channels formed by the toxins in BLMs were found to be similar, which points to the similarity in the channel structures. In negatively charged membranes, bathed with diluted electrolyte solutions (0.1 M NaCl), the channels were seen to open with positive transmembrane potentials (V) (from the side of toxin addition), and close with negative potentials. In uncharged membranes the opposite response to a voltage sign was observed. Increasing the NaCl concentration up to 1 M unified the voltage sensitivity of channels in charged and uncharged membranes: channels opened with negative V, and closed with positive V. With all systems, the voltage current curves of single channels were similarly superlinear in the applied voltage and asymmetric in its sign. It was found that the single channel conductance of STB and SSA was higher than that of other toxin channels. All the toxins formed at least two types of ion channels that were multiple by a factor of either 6 or 4 in their conductance. The results are discussed in terms of the structural features of toxin molecules.     

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation. Received: 3 April 1995/Revised: 24 August 1995     

Voltage-dependent synchronization of gating of syringomycin E ion channels

Antifungal lipodepsipeptide syringomycin E (SRE) forms two major conductive states in lipid bilayers: "small" and "large". Large SRE channels are cluster of several small ones, demonstrating synchronous opening and closure. To get insight into the mechanism of such synchronization we investigated how transmembrane potential, membrane surface charge, and ionic strength affect the number of small SRE channels synchronously functioning in the cluster. Here, we report that the large SRE channels can be presented as 3-8 simultaneously gating small channels. The increase in the absolute value of the transmembrane potential (from 50 to 200 mV) decreases the number of synchronously gated channels in the clusters. Voltage-dependence of channel synchronization was influenced by the ionic strength of the bathing solution, but not by membrane surface charge. We propose a mechanism for the voltage-dependent cluster behavior that involves a voltage-induced reorientation of lipid dipoles associated with the channel pores.     

Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.     

Transport of large organic ions through syringomycin channels in the membranes containing dipole modifiers

The effect of the membrane dipole potential (Phid) on a conductance and a steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate and chloride was shown. The magnitude of Phid was varied with the introduction to membrane bathing solutions of phloretin, which reduces the Phid, and RH 421, increasing the Phid. It was established that in all studied systems the increase in the membrane dipole potential cause a decrease in the steady-state number of open channels. In the systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are in an order of magnitude smaller than in systems containing sodium chloride. At the same time, the conductance (g) of single SRE-channels on the membranes bathed in NaCI solution increases with the increase in Phid, and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the Phid. The latter is due to the lack of cation/anion selectivity of the SRE-channels in these systems. The different channel-forming activity of SRE in the experimental systems is defined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

Cluster organization of ion channels formed by the antibiotic syringomycin E in bilayer lipid membranes.

The cyclic lipodepsipeptide, syringomycin E, when incorporated into planar lipid bilayer membranes, forms two types of channels (small and large) that are different in conductance by a factor of sixfold. To discriminate between a cluster organization-type channel structure and other possible different structures for the two channel types, their ionic selectivity and pore size were determined. Pore size was assessed using water-soluble polymers. Ion selectivity was found to be essentially the same for both the small and large channels. Their reversal (zero current) potentials with the sign corresponding to anionic selectivity did not differ by more than 3 mV at a twofold electrolyte gradient across the bilayer. Reduction in the single-channel conductance induced by poly(ethylene glycol)s of different molecular weights demonstrated that the aqueous pore sizes of the small and large channels did not differ by more than 2% and were close to 1 nm. Based on their virtually identical selectivity and size, we conclude that large syringomycin E channels are clusters of small ones exhibiting synchronous opening and closing.     

Mechanism of action of Pseudomonas syringae phytotoxin, syringomycin. Interaction with the plasma membrane of wild-type and respiratory-deficient strains of Saccharomyces cerevisiae

The effects of the phytotoxin, syringomycin, produced by Pseudomonas syringae pv. syringae, were examined on cells of a wild-type and a respiratory-deficient (rho0) mutant of Saccharomyces cerevisiae. The growth of both strains in liquid culture was inhibited by 0.5 micrograms syringomycin per ml and higher. Uptake rates of tetraphenylphosphonium and dimethyloxazolidine ions in cell suspensions of both strains increased when 1.5 micrograms per ml syringomycin was added. These responses were kinetically and quantitatively similar in the two strains and indicated increases in electrical potential (cell interior negative) and pH differences (cell interior alkaline) across the plasma membrane. Glucose (0.1 M) enhanced the effect on the electrical potential, was required for the pH changes, and increased the cellular ATP levels. These results show that the effects of syringomycin are energy-dependent and are due to alterations of plasma membrane and not to mitochondrial function.     

Transport of large organic ions through syringomycin channels in membranes containing dipole modifiers

The effect of the membrane dipole potential (φ _d ) on conductance and the steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate, and chloride was shown. The φ _d value varied with the introduction of phloretin to membrane bathing solutions, which reduces φ _d and RH 421, which increases φ _d . It was established that, in all studied systems, an increase in the membrane dipole potential caused a decrease in the steady-state number of open channels. In systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are one order lower than those of systems that contain sodium chloride. At the same time, the conductance (g) of single SRE channels in the membranes bathed in NaCl solution increases with increase in φ _d and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the φ _d . The latter is due to the lack of cation/anion selectivity of the SRE channels in these systems. The different channel-forming activity of SRE in the experimental systems is determined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

Lipopolysaccharide promoted opening of the porin channel

We show here that the imipenem (a carbapenem, β-lactam antibiotic)-permeable porin channels (protein D2 or OprD2) of Pseudomonas aeruginosa were closed mostly in the lipopolysaccharide (LPS)-free membrane and were openable by adding LPS to the membrane as assayed by ion conductivity measurements using planar lipid bilayers. Open and closed states of the OprD2 channels exhibited conductivities of about 400 and 30 pS, respectively, in 1 M NaCl. The OprD2 channel in the LPS-containing membrane showed very rapid opening and closing events in a second order and the duration of closure became longer at low membrane potentials.     

Syringomycin E channel: a lipidic pore stabilized by lipopeptide?

Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E demonstrate unprecedented involvement of the host bilayer lipids. We find that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the syringomycin E channels to the transmembrane voltage, is modified by the lipid charge and lipid dipolar moment. We show that the type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. We also demonstrate that the replacement of lamellar lipid by nonlamellar with the negative spontaneous curvature inhibits channel formation. These observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlies channel gating. We conclude that the syringomycin E channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.     

Kinetics of opening and closure of syringomycin E channels formed in lipid bilayers

A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels.     

Study of F-actin interaction with planar and liposomal bilayer phospholipid membranes

Interaction of the cytoskeletal protein F-actin with planar bilayer lipid membrane (BLM) induced formation of single ionic channels in both NaCl and KCl bathing solutions. We also recorded noiselike high-currentjumps with a mean conductivity of approximately 160 pS, which might represent the simultaneous opening and closing of several channels of lower conductivity. The ratio of cation to anion permeabilities (Pc/Pa) of the BLM with many channels in KCl was 26 +/- 2. Freeze-fracture electron microscopy revealed fibrillar-like structures on the hydrophobic surfaces of liposomal membranes. We also observed some structural features giving evidence for the penetration of F-actin fibers through an artificial phospholipid membrane. We suggest that the F-actin/lipids complexes can transmit electric signals in synaptic and other intercellular contacts.     

Ion-conducting channels produced by botulinum toxin in planar lipid membranes

The interaction of botulinum neurotoxin (Botx) with planar lipid membranes was studied by measuring the ability of the toxin to form ion-conducting channels. Channel formation was pH dependent. At physiological pH, Botx formed no channels, whereas at pH 6.6, the toxin formed channels with a unit conductance of 12 pS in 0.1 M NaCl. The rate of channel formation increased with decreasing pH, reaching a maximum at pH 6.1, and then decreased at lower values of pH. The channels, once formed, were permanent entities in the membrane throughout the course of an experiment and fluctuated between an open and a closed state. The rate of channel formation depended upon the square of the toxin concentration, suggesting an aggregation step is involved in channel formation. The data were consistent with the hypothesis that Botx enters cells through endocytosis, followed by its release into the cytoplasm at low pH.     

Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.     

Kinetics of light-sensitive channels in vertebrate photoreceptors

We have studied the ion channels mediating the light response of vertebrate rod photoreceptors by analysing fluctuations in the current across the rod membrane, using the whole cell patch-clamp technique on rods isolated from the axolotl retina. Light decreases the membrane current fluctuations. Noise analysis reveals two components to this decrease: a low frequency component due to biochemical noise in the transduction mechanism, and a high frequency component we attribute to the random opening and closing of the ion channels in the dark. The probability of any one channel being open in the dark is low. The spectrum of the high frequency component of the current fluctuations indicates that the current through an open channel is 4 X 10(-15)A, that the mean channel open time is 2 ms, and that about 10000 channels are open in each rod in the dark. The effect of light is to reduce the opening rate constant of these channels, with no effect on the closing rate constant.     

Isoproterenol opens K+(ATP) channels via a beta2-adrenoceptor-linked mechanism in Sertoli cells from immature rats.

In the present study, we investigated the mechanism by which isoproterenol hyperpolarises membrane potential (MP) in Sertoli cells from seminiferous tubules of 15-day-old rat testes. Modification of MP and resistance (R0) was analysed using conventional intracellular glass microelectrodes. Isoproterenol (2 x 10(-6) M) induced an immediate and significant hyperpolarisation in the Sertoli-cell membrane. The beta2-AR antagonist, butoxamine (1 x 10(-6) M), nullified isoproterenol action. The effect of the beta1 antagonist, metoprolol (1 x 10(-6) M), was light and non-significant. Sulphonylurea glibenclamide inhibition of the K+(ATP) channels suppressed isoproterenol action, and testosterone, while depolarising Sertoli-cell MP closing the K+(ATP) channels through the PLC/PIP2 pathway, reduced beta-AR agonist-induced hyperpolarisation. Also, polycations LaCl3 and spermine reversed isoproterenol's hyperpolarisation effect, probably depolarising the membrane potential through ionic interaction neutralising the action of isoproterenol on K+(ATP) channels. Adenylate cyclase agonist forskolin (0.1 microM) rapidly hyperpolarised Sertoli-cell MP, mimicking the isoproterenol effect. These effects indicate that isoproterenol's action on K+(ATP) channel probably involves the known signalling cascade beta-AR/Gs/AC/cAMP/PKA. These results suggest that the isoproterenol-induced hyperpolarisation is mediated by the opening of K+(ATP) channels in Sertoli cells. This beta-adrenergic hyperpolarisation might play a physiological role in the modulation of MP.     

Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.     

Ionic Blockage of Sodium Channels in Nerve

Increasing the hydrogen ion concentration of the bathing medium reversibly depresses the sodium permeability of voltage-clamped frog nerves. The depression depends on membrane voltage: changing from pH 7 to pH 5 causes a 60% reduction in sodium permeability at +20 mV, but only a 20% reduction at +180 mV. This voltage-dependent block of sodium channels by hydrogen ions is explained by assuming that hydrogen ions enter the open sodium channel and bind there, preventing sodium ion passage. The voltage dependence arises because the binding site is assumed to lie far enough across the membrane for bound ions to be affected by part of the potential difference across the membrane. Equations are derived for the general case where the blocking ion enters the channel from either side of the membrane. For H⁺ ion blockage, a simpler model, in which H⁺ enters the channel only from the bathing medium, is found to be sufficient. The dissociation constant of H⁺ ions from the channel site, 3.9 x 10^-6 M (pK_a 5.4), is like that of a carboxylic acid. From the voltage dependence of the block, this acid site is about one-quarter of the way across the membrane potential from the outside. In addition to blocking as described by the model, hydrogen ions also shift the responses of sodium channel "gates" to voltage, probably by altering the surface potential of the nerve. Evidence for voltage-dependent blockage by calcium ions is also presented.     

Voltage dependence of acetylcholine receptor channel gating in rat myoballs

Whole-cell currents from nicotinic acetylcholine receptor (AChR) channels were studied in rat myoballs using a light-activated agonist to determine the voltage dependence of the macroscopic opening and closing rate constants. Myoballs were bathed in a solution containing a low concentration of the inactive isomer of the photoisomerizable azobenzene derivative, cis-Bis-Q. A light flash was then presented to produce a known concentration jump of agonist, trans-Bis-Q, across a wide range of membrane potentials in symmetrical solutions (NaCl or CsCl on both sides) or asymmetrical solutions (NaCl in the bath and CsCl in the pipette). At the low agonist concentration used in this study, the reciprocal of the macroscopic time constants gives an unambiguous measure of the effective closing rate. It showed an exponential decrease with membrane hyperpolarization between +20 and -100 mV, but tended to level off at more depolarized and at more hyperpolarized membrane potentials. The relative effective opening rate was derived from the steady-state conductance, the single-channel conductance, and the apparent closing rate; it decreased sharply in the depolarizing region and tended to level off and then turn up in the hyperpolarizing region. The two effective rate constants were shown to depend on the first, second, and third power of membrane potential.