Ion channel formation by duramycin.

The formation of ion channels by the nonadecapeptide antibiotic duramycin was examined using black lipid membranes and using the patch-clamp technique. In black lipid membranes made from glyceryl monooleate or a phosphatidylcholine/phosphatidylethanolamine mixture, duramycin induced complex fluctuations in membrane conductance, some step-like and some which were incapable of being resolved into discrete conductance states. Both conductance and largest step size increased with time. A similar time-dependent increase in conductance was seen in patch-clamp experiments with HCA-7 Colony 29 human colonic epithelial cell. The channels displayed weak anion selectivity and the smaller channels formed in patches from epithelial cells showed weak inward-rectification. Channel formation by duramycin was achieved at lower concentrations when the black lipid membrane was made with phospholipid rather than with glyceryl monooleate. Lower concentrations were effective in generating conductances in epithelial cells than in bilayers. It is concluded that duramycin forms ion channels in both artificial and biological membranes. Accumulation of duramycin and coalescence of initially small channels into larger ones is considered to be responsible for the recorded behaviour and to final disruption of membranes.     

Membranolytic selectivity of cystine-stabilized cyclic protegrins.

To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin-1 (PG-1), an 18-residue cationic peptide with a broad-spectrum antimicrobial activity. These cyclic protegrins bear end-to-end peptide bonds together with varying numbers (zero to three) of cross-strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low- and high-salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram-positive and Gram-negative bacteria, fungi and one tested virus, HIV-1. Compared to PG-1, the cyclic tricystine ccPG 3 displayed approximately a 10-fold decrease in hemolytic activity against human cells and 6- to 30-fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [DeltaSS]cPG 8, a cyclic protegrin with no disulfide bond, and [DeltaCys6,15]cPG 5, a cyclic mimic of PG-1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG-1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of beta-strand structures in water/trifluoroethanol or phosphate-buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity.     

Membrane selectivity by W-tagging of antimicrobial peptides

A pronounced membrane selectivity is demonstrated for short, hydrophilic, and highly charged antimicrobial peptides, end-tagged with aromatic amino acid stretches. The mechanisms underlying this were investigated by a method combination of fluorescence and CD spectroscopy, ellipsometry, and Langmuir balance measurements, as well as with functional assays on cell toxicity and antimicrobial effects. End-tagging with oligotryptophan promotes peptide-induced lysis of phospholipid liposomes, as well as membrane rupture and killing of bacteria and fungi. This antimicrobial potency is accompanied by limited toxicity for human epithelial cells and low hemolysis. The functional selectivity displayed correlates to a pronounced selectivity of such peptides for anionic lipid membranes, combined with a markedly reduced membrane activity in the presence of cholesterol. As exemplified for GRR10W4N (GRRPRPRPRPWWWW-NH(2)), potent liposome rupture occurs for anionic lipid systems (dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) and Escherichia coli lipid extract) while that of zwitterionic dioleoylphosphatidylcholine (DOPC)/cholesterol is largely absent under the conditions investigated. This pronounced membrane selectivity is due to both a lower peptide binding to the zwitterionic membranes (z≈-8-10mV) than to the anionic ones (z≈-35-40mV), and a lower degree of membrane incorporation in the zwitterionic membranes, particularly in the presence of cholesterol. Replacing cholesterol with ergosterol, thus mimicking fungal membranes, results in an increased sensitivity for peptide-induced lysis, in analogy to the antifungal properties of such peptides. Finally, the generality of the high membrane selectivity for other peptides of this type is demonstrated.     

Membrane thickening by the antimicrobial peptide PGLa

Using x-ray diffraction, solid-state ²H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.     

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.     

Ion channel formation by zervamicin-IIB

Zervamicin-IIB (Zrv-IIB) is a 16 residue peptaibol which forms voltage-activated, multiple conductance level channels in planar lipid bilayers. A molecular model of Zrv-IIB channels is presented. The structure of monomerc Zrv-II3 is based upon the crystal structure of Zervamicin-Leu. The helical backbone is kinked by a hydroxyproline residue at position 10. Zrv-IIB channels are modelled as helix bundles of from 4 to 8 parallel helices surrounding a central pore. The monomers are packed with their C-terminal helical segments in close contact, and the bundles are stabilized by hydrogen bonds between glutamine 11 and hydroxyproline 10 of adjacent helices. Interaction energy profiles for movement of three different probes species (K⁺, Cl^– and water) through the central pore are analyzed. The conformations of: (a) the sidechain of glutamine 3; (b) the hydroxyl group of hydroxyproline 10; and (c) the C-terminal hydroxyl group are optimized in order to maximize favourable interactions between the channel and the probes, resulting in favourable interaction energy profiles for all three. This suggests that conformational flexibility of polar sidechains enables the channel lining to mimic an aqueous environment.     

Membrane channel formation by the lymphocyte pore-forming protein: comparison between susceptible and resistant target cells

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.     

Membrane insertion and bilayer perturbation by antimicrobial peptide CM15

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2-9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an alpha-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2-3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.     

Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: Membrane integrity disruption and inhibition of biofilm formation

With the increasing emergence of resistant fungi, the discovery and development of novel antifungal therapeutics were urgently needed. Compared with conventional antibiotics, the limited propensity of AMPs to induce resistance in pathogens has attracted great interest. In the present study, the antifungal activity and its mechanism-of-action of polybia-MPI, a cationic peptide from the venom of Social wasp Polybia Paulista was investigated. We demonstrated that polybia-MPI could potently inhibit the growth of Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The 50% inhibitory concentrations (IC₅₀) of Polybia-MPI against cancer cells were much higher than the MICs against the tested C. albicans and C. glabrata cells, indicating that polybia-MPI had high selectivity between the fungal and mammalian cells. Our results also indicated that membrane disturbance mechanism was involved in the antifungal activity. Furthermore, polybia-MPI could inhibit the bio film forming of C. glabrata, which was frequently associated with clinically significant biofilm. These results suggest that polybia-MPI has great advantages in the development of antifungal agents.     

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin’s antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.     

Transients of perforin pore formation observed by fluorescence microscopic single channel recording.

A new type of single channel recording is described. Large pores were generated in the membranes of resealed human erythrocyte ghosts by incubation with perforin (cytolysin). The flux of the polar fluorescent probe Lucifer Yellow was measured in single ghosts by the fluorescence microphotolysis (photobleaching) technique. The distribution of flux rates for ghosts treated with a limiting perforin concentration showed equidistantly spaced peaks suggesting that subpopulations of ghosts with 0, 1 and 2 pores were resolved. Furthermore, distributions obtained for very different perforin concentrations could be well simulated by using one common value for the flux rate of the single pore (k = 4.65 x 10(-3) s) and assuming a Poisson distribution of pores among ghosts. The flux rate of the single pore corresponds to a pore radius of approximately 50 A, a value which is much smaller than that obtained previously by electron microscopic studies but which agrees well with recent electrical single channel recordings. Mature perforin pores were observed to be very stable. No closing events were detected at a time resolution of 0.2 s for a wide range of temperatures and Ca2+ concentrations. However, the formation of new pores was an unexpectedly slow process. Fluorescence microscopic single channel recording as introduced by this study is applicable to a variety of cellular systems and fluorescent probes and thus may complement the information obtainable by electrical single channel recording of anorganic ion fluxes.     

Enhanced membrane pore formation by multimeric/oligomeric antimicrobial peptides

The pore-forming antibacterial peptide magainin 2 was made divalent, tetravalent, and octavalent via a copper(I)-mediated 1-3 dipolar cycloaddition reaction ("click" chemistry). This series of pore-forming compounds was tested in vitro for their ability to form pores in large unilamillar vesicles (LUVs). A large increase in the pore-forming capability was especially observed with the tetravalent and octavalent magainin compounds in the LUVs consisting of DOPC, and the octavalent magainin compound showed a marked increase with the DOPC/DOPG LUVs. Activity was observed in the low nanomolar range for these compounds.     

Pore formation induced by an antimicrobial peptide: electrostatic effects

We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomembranes by performing numerical simulations and electrophysiological measurements on membrane vesicles. Using this natural β-sheet antimicrobial peptide secreted during stress as a model we show that a single peptide is able to form a stable membrane pore of 1 nm diameter of 0.25 nS conductance found both from calculation and electrical measurements. The resulting structure does not resemble the barrel-stave or carpet models earlier predicted, but is very close to that found in the simulation of α-helical peptides. Based on the simulation of a mutated peptide and the effects of small external electric fields, we conclude that electrostatic forces play a crucial role in the process of pore formation.     

Intrinsic lattice formation by the ryanodine receptor calcium-release channel

Recent theoretical analysis of a model lattice of interacting transmembrane receptor proteins has indicated that such clustering in the membrane could provide a novel mechanism for regulating receptor signalling in cells. It has been calculated that cooperative interactions between receptors organized into a cluster, or array, in the membrane would dramatically increase their sensitivity to activation by ligand. Sensitivity to ligand would increase with the extent of spread of activity within the receptor lattice. Hence, formation of extensive receptor lattices in the membrane would allow a large population of receptors to be simultaneously switched on, or off, by a very small change in ligand concentration. We show here that lattice formation is an intrinsic property of an integral membrane protein, the ryanodine-sensitive calcium-release channel (RyR) of endoplasmic reticulum. The purified protein spontaneously assembled into two-dimensional lattices in solution, enabling the construction of a 25 A projection map that identifies the mode of interaction between RyR oligomers. Our observations on the RyR provide a new perspective on various properties of cell signalling via this and other receptors.     

Membrane interactions of antimicrobial peptides from Australian frogs

The membrane interactions of four antimicrobial peptides, aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, isolated from Australian tree frogs, are reviewed. All four peptides are amphipathic α-helices with a net positive charge and range in length from 13 to 25 residues. Despite several similar sequence characteristics, these peptides compromise the integrity of model membrane bilayers via different mechanisms; the shorter peptides exhibit a surface interaction mechanism while the longer peptides may form pores in membranes.     

Cell/cell channel formation involves disulfide exchange.

The oocyte cell/cell-channel assay was used to identify amino acids involved in the process of cell/cell-channel formation. The expression of the rat liver gap-junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol-specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin-32 mutants were generated by site-directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process.