Potential-dependent formation of single conducting ion channels in lipid bilayers induced by the polyene antibiotic levorin A2

The aromatic polyene antibiotic levorin A₂ forms ion channels permeable to monovalent cations, in lipid membranes containing cholesterol or ergosterol. Channel conductivity is in the range 0.3–0.5 pS. The channel has two main states: conducting (open) and nonconducting (closed). The potential-dependent formation of levorin A₂ channels is observed in lipid membranes. The system responsible for the ion-channel selectivity is localized on the hydrophilic side of the lactone ring of the polyene molecule.     

The Properties of Ion Channels in Lipid Membranes Modified by the Aromatic Antibiotic Levorin А2

It has been shown that the main components of levorin A, that is, A₀, A₁, A₂, or A₃, that contain an aromatic group increase the permeability of membranes in the series A₃ > A₂ > A₁ > A₀ when they are on the same side of the membrane. All levorin components have cationic selectivity. The most studied levorin, А₂, promotes the almost ideal permeability of membranes to potassium ions. The membrane potential for a ten-fold change in the KCl concentration gradient is 56 ± 2 mV. It has been shown that the injection of the same concentration of levorin А₂ into one side of the membrane and then, after achieving the typical membrane permeability, into the other side of the membrane generates a two-fold increase in the total membrane permeability. This means that independent levorin-induced conductive semi-pores are formed on each side of the membrane. It has been found that the injection of levorin А₂ only into one side of the membrane enhances the membrane permeability to monosaccharides and other neutral molecules. The presence of levorin А₂ in cholesterol-, ergosterol-, and stigmasterol-containing phospholipid membranes has been shown to lead to the single-channel conductivity of typical ion channels of 0.2–0.5 pS. The properties of these channels have been studied. The levorin channels exist in two states, open and closed. Most of the time, the channel remains in the open state in the KBr solution. In solutions of different salts of the same concentration, the conductivity value of the levorin channels is approximately the same (0.4–0.5 pS). An increase in the dimethyl sulfoxide concentration in aqueous solutions facilitates the transition of polyene antibiotic molecules from dispersed to monomolecular form. The molecules of polyene antibiotics in the associated form exhibit high membrane activity.

     

Potential-dependent formation of single conducting ion channels in lipid bilayers induced by the polyene antibiotic levorin A

The aromatic polyene antibiotic levorin A2 forms ion channels permeable to monovalent cations, in lipid membranes containing cholesterol or ergosterol. Channel conductivity is in the range 0.3-0.5 pS. The channel has two main states: conducting (open) and nonconducting (closed). The potential-dependent formation of levorin A2 channels is observed in lipid membranes. The system responsible for the ion-channel selectively is localized on the hydrophilic side of the lactone ring of the polyene molecule.     

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.     

Radioprotective and antineoplastic activity of polyene antibiotics combined with dimethyl sulfoxide]

Radioprotective and antineoplastic activity of polyene, its derivatives and combinations with dimethyl sulfoxide (DMSO) was studied. The most potent radioprotective effect was demonstrated by methylated levorin, original levorin and by its isomer--isolevorin. Survival rate of the animals on 12th day after X-ray exposure was 100, 60, 60 per cent, at the control group 33.6, 20 and 0 per cent consequently. Levorin and alkyl derivatives of amphotericin B--methamphocin and buthamphocin inhibited growth of ascites and solid tumors to 46.3-79.0 per cent when compared to control group. Polyen antibiotics combined with DMSO also demonstrated antineoplastic activity at the animals treated with carcinogenic agent--diethyl nitrosoamine (DENA). 5-month survival of the animals was 76 per cent at nystatin and levorin group and 35.7 per cent at the control group (animals treated with DENA only).     

Amphotericin B channel conductance inactivation

Effects induced in bilayer lipid membranes by amphotericin B and its alkyl derivatives was analysed. Inactivation of the antibiotic-dependent multichannel membrane conductance was discovered. Kinetics of membrane conductivity was shown to depend on the antibiotic concentration in the membrane. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to the transient. We suggest that the phenomenon of biphasic kinetics of membrane conductance is the result of a consecutive transformation of polyene channels in the membrane: half-pores are assembled on either side of membrane-nonconducting 1; two half-pores combine to build up a conducting channels-conducting 2, and the conducting channels are disassemled to monomers and nonconducting self-associated forms inside the membrane-disassembled state (nonconducting 3). To explain the transient characteristics of the induced conductance, it is proposed that the antibiotic, present in the solution under self-associated form, binds the membrane and forms pores, then dissociates in the bilayer in a non-active monomeric form. The existence of definite monomers and nonconducting self-associated forms of amphotericin B molecules inside the membrane was estimated from the dependence of kinetic conductance of lipid membranes of amphotericin B and its alkyl derivatives, when the antibiotics are washed out from aqueous medium. Equilibrium between different antibiotic assemblies inside the membrane was demonstrated by the kinetics of conductance decrease following washing the antibiotic. Using circular dichroism measurements, we observed that amphotericin B alkyl derivatives were in self-associated form being susceptible to form pores across cholesterol-containing membranes. The phenomenon of biophasic kinetics was observed only in the cholesterol-containing membrane. The substitution of membrane cholesterol for ergosterol provides monotonic kinetics of membrane conductance at any antibiotic concentration.     

Cation conductance and efflux induced by polyene antibiotics in the membrane of skeletal muscle fiber.

Cation conductance and efflux induced by polyene antibiotics amphotericin B (AMB), amphotericin B methyl ester (AME), nystatin, mycoheptin, and levorin on frog isolated skeletal muscle fibers and whole sartorius muscles were investigated. Conductance was measured under current-clamp conditions using a double sucrose-gap technique. Cation efflux was studied using flame emission photometry. Some new data were obtained concerning the effects of levorin and mycoheptin on biological membranes. The power dependence of polyene-induced cation transport on antibiotic concentration in muscle membrane was lower than that in bilayers. The decline in the equilibrium conductance caused by polyene removal (except for levorin) was very fast. There was reverse temperature dependence of AMB- and nystatin-induced conductances. Both induced conductance and efflux values demonstrated a correlation with the order of antifungal activities: levorin > AMB, mycoheptin > AME > nystatin, except for AME, which was more potent on yeastlike cells. These effects were interpreted in terms of possible differences in the kinetics of channel formation in biological and model membranes and in light of the role of nonconducting antibiotic forms in biological membranes.     

Effect of various microorganisms on the activity of levorin

Levorin added to nutrient media with growing cultures of aerobic gram-positive bacilli, Escherichia, enterococci and filamentous fungi was partially inactivated. The antibiotic activity decrease depended on the strain characteristics, incubation period and temperature. Fermentation broth filtrates of the experimental strains also inactivated levorin while to a lesser extent than the growing organisms. In contaminated levorin pastes, the antibiotic activity was lower. The fermentative nature of inactivation was not proved.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

Composition of membranes and mycelia ofStreptomyces levoris at different stages of growth

The composition of cell membranes and mycelia ofStreptomyces levoris, producer of the polyene antibiotic levorin, was studied. The membrane protein/lipid ratio was shown to be constant during growth of the microorganism. The membrane protein was found to be heterogeneous and to have a low molecular weight. The lipid component of the membranes consisted mainly of polar lipids—phosphatidyl ethanolamine, diphosphatidyl glycerol, and phosphatidyl inositol mannoside being predominant. During growth, the phospholipid content of the polar lipid fraction decreased, apparently due to replacement of phospholipids by phosphorus-free analogs.     

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.     

An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.     

Oxidative destruction of the heptaene antibiotic levorin

Levorin destruction in oxygen, argon or air at various temperature levels was studied. It was found that destruction of the antibiotic molecule was mainly due to its interaction with oxygen. As a result the heptaenic chromophore was oxidized and triene and tetraene formed. Oxidation of levorin was accompanied by splitting out of paraaminoacetophenone. Destruction of levorin in the absence of oxygen was retarded.     

The Phytotoxic Lipodepsipeptide Syringopeptin 25A from Pseudomonas syringae pv syringae Forms Ion Channels in Sugar Beet Vacuoles

Syringopeptin 25A (SP(25)A) belongs to a family of cyclic lipodepsipeptides (LDPs) produced by the gram-negative bacterium Pseudomonas syringae, a phytopathogenic organism that affects several plants of agronomic interest. LDPs increase the permeability of plasma and, possibly, intracellular membranes in plant cells. Consistently, SP(25)A forms ion channels in planar lipid bilayers and other model membranes. Here we used sugar beet tonoplasts as a new biological model system to study toxin action. When applied to the vacuoles by a fast perfusion procedure, SP(25)A increases membrane permeability by forming discrete ion channels even at low applied potentials. The SP(25)A channel displays anion selectivity (with a Cl-/K+ permeability ratio of 6.7 +/- 1.3) and has intrinsic rectification properties that derive from a different channel conductance at negative and positive voltages, presumably owing to an asymmetric distribution of fixed charges on the pore. Substitution of chloride with different anions reveals the following selectivity sequence NO3- approximately Cl-> F- > gluconate-, suggesting that the permeation pore is filled with water. The properties of the SP(25)A channels in vacuolar membranes are similar to those observed in planar lipid membranes prepared with asolectin. This work provides a direct demonstration of toxin effects on a native plant membrane, extending to a biological system previous results obtained on artificial planar lipid membranes.     

Dependence of inhibition by levorin of amino acid incorporation into protoplasts and subcellular proteins of Candida albicans on the change of endocellular potassium concentration]

Levorin is found to decrease more efficiently potassium concentration in C. albicans protoplasts under their incubation in the presence of sodium than in the medium containing the equivalent amount of potassium. Minimal inhibitory concentration of levorin for resistant C. albicans cells incubated on potassium-depeleted medium was in 4 times lower than for cells incubated in potassium-enriched medium. The decrease of membrane permeability for 14C-amino acids and their incorporation into membrane, ribosomal and soluble proteins under the effect of levorin was more pronounced when protoplasts were cultivated in sodium-containing medium than in potassium-containing one. In both media the inhibition of 14C-amino acid incorporation by levorin into ribosomal and cytosol proteins was more efficient than into membrane proteins, but these differences were less pronounced in case of potassium-containing medium.     

Action of the alkyl derivatives of amphotericin B on the conductivity of bilayer lipid membranes

Amphotericin B alkyl derivatives increased conductivity of bilayer membranes by formation of channels in them. The properties of such channels were studied. A new method for determination of the polyene antibiotic toxicity is described. The method is based on measurement of the constant of the relaxation time on the antibiotic removal from the membrane solution. It was shown that the amphotericin B alkyl derivatives had very low toxicity for the mammalian cells and were highly toxic for the fungal cells. These antibiotics may be used as new effective antifungal compounds.     

Isolation, purification and characterization of levorin produced by Streptomyces levoris 99/23

Levorin produced by Streptomyces levoris 99/23 was isolated, purified and characterized. It was established that 80% of the levorin was localized in the mycelium and only 20% was in the cell-free supernatant. Amorphous yellow levorin with activity of 24 000 IU/mg and 96% purity was obtained. The preparation exhibited three absorption maxima: at λ 362, 382 and 404 nm. The antibiotic contained seven components: A₀, A₁, A₂, A₃, A₄, and two unidentified ones. According to its composition, the preparation corresponded to the levorin used for medicinal purposes. However, the levorin produced by S. levoris 99/23 contained half as much levorin A₂ and a more than 100 times larger quantity of the more active and less toxic component levorin A₃.     

The mode of action of some antibiotics on red blood cell membranes

Data are presented on the interaction of gramicidin, primycin and valinomycin with red blood cell membranes and compared with those obtained for artificial lipid bilayer membranes. The channel forming antibiotics gramicidin and primycin show specific kinetic behaviour in living cell membranes. It could be shown that the penetration of these antibiotics into the red blood cell membrane is a cooperative process resulting in the occurrence of aggregates in the lipid lattice of the membrane.     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Physical and Chemical Properties of Erythrocyte Membranes in Interaction with Polyene Antibiotics in the Field of Ultrasonic Waves

Biophysics - Red blood cell hemolysis induced by ultrasound and alkyl derivatives of amphotericin B and levorin modified by the amine and carboxyl groups has been studied. A kinetic method has been...