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We show that voltage alone can inactivate alamethicin channels, which has been previously observed for monazomycin and suzukacillin channels. The voltage required to trigger inactivation is above the potential to form channels, and, like with channel activation, this threshold reduces with increasing peptide concentration and membrane fluidity. Since similar monazomycin channels inactivate via channel break up and translocation, we hypothesized that inactivation of alamethicin channels occurs via the same mechanism. Our data prove this hypothesis to be true through two experiments. First, we show that inactivation of channels at positive voltages when peptides are supplied to only the cis side correlates to new channel activity on the trans side at negative potentials. This result indicates translocation of alamethicin peptides occurs only during voltage-induced inactivation. Second, we measured the ratio of steady-state (with inactivation) to ideal (without inactivation) conductance versus voltage for membranes with equal amounts of alamethicin on both sides and used these values to quantify alamethicin flux. Plotting flux versus steady-state conductance across multiple alamethicin concentrations shows a single linear dependence, signifying that translocated peptides originate from active channels that break up under prolonged voltage. Given the frequent use of alamethicin as model ion channels, these results add important understanding of their kinetic responses when subjected to prolonged, high voltages., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stephen A Sarles reports financial support was provided by The University of Tennessee Knoxville. Stephen A. Sarles reports financial support was provided by Air Force Office of Scientific Research. Joshua J. Maraj reports financial support was provided by Air Force Office of Scientific Research. Jessie D. Ringley reports financial support was provided by Air Force Office of Scientific Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Trichoderma spp. is primarily applied to manage biotic stresses in plants. Still, they also can mitigate abiotic stresses by the stimulation of antioxidative protective mechanisms and enhanced synthesis of secondary metabolites. The study optimized the conditions to enhance peptaibol production by novel Trichoderma spp, characterized and quantified peptaibol- alamethicin using HPLC and LC MS-MS. The present study investigated these isolates efficacy in enhancing growth and the associated physio-biochemical changes in black pepper plants under moisture stress. Under in vitro conditions, out of 51 isolates studied, six isolates viz., T. asperellum (IISR NAIMCC 0049), T. erinaceum (IISR APT1), T. harzianum (IISR APT2), T. harzianum (IISR KL3), T. lixii (IISR KA15) and T. asperellum (IISR TN3) showed tolerance to low moisture levels (5, 10 and 20%) and higher temperatures (35 and 40 °C). In vivo evaluation on black pepper plants maintained under four different moisture levels (Field capacity [FC]; 75%, 50%, and 25%) showed that the plants inoculated with Trichoderma accumulated greater quantities of secondary metabolites viz., proline, phenols, MDA and soluble proteins at low moisture levels (50% and 25% FC). In the present study, plants inoculated with T. asperellum and T. harzianum showed significantly increased growth compared to uninoculated plants. The shortlisted Trichoderma isolates exhibited differences in peptaibol production and indicated that the peptide might be the key factor for their efficiency as biocontrol agents. The present study also demonstrated that Trichoderma isolates T. harzianum and T. asperellum (IISR APT2 & NAIMCC 0049) enhanced the drought-tolerant capabilities of black pepper by improving plant growth and secondary metabolite production., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Membrane active peptides are known to porate lipid bilayers, but their exact permeabilization mechanism and the structure of the nanoaggregates they form in membranes have often been difficult to determine experimentally. For many sequences at lower peptide concentrations, transient leakage is observed in experiments, suggesting the existence of transient pores. For two well-know peptides, alamethicin and melittin, we show here that molecular mechanics simulations i) can directly distinguish equilibrium poration and non-equilibrium transient leakage processes, and ii) can be used to observe the detailed pore structures and mechanism of permeabilization in both cases. Our results are in very high agreement with numerous experimental evidence for these two peptides. This suggests that molecular simulations can capture key membrane poration phenomena directly and in the future may develop to be a useful tool that can assist experimental peptide design., (© 2024. The Author(s).)

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, C α -tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol-membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure-activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes., (© 2023 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

The linear 15-mer peptide gramicidin A (gA) produced by Bacillus brevis is known to form the simplest natural ion channel in lipid membranes representing a head-to-head transmembrane dimer. Its incorporation into a planar lipid bilayer manifests itself in regular electrical current transitions. If two gA subunits are tightly connected by a water-soluble, flexible linker of a certain length, the current transitions become heterogeneous: in a part of them, the amplitude is almost twofold higher than that of a single channel, thereby demonstrating the synchronous opening of two single channels. The lifetime, i.e. the open-state duration, of this dual channel is by several orders of magnitude longer than that of the single channel. Here, we used the ideas of the theory of excitons to hypothesize about the mechanism of synchronous opening and closing of two adjacent channels. Two independent (uncoupled) single channels can be described by two independent conformational coordinates q 1 and q 2, while two closely located channels can exhibit collective behavior, if the coupling between them produces mixing of the individual states ( q 1,0) and (0, q 2). We suppose that a similar phenomenon can occur not only with synthetic derivatives of gA, but also with such natural channel-forming peptide antibiotics and toxins as alamethicin and syringomycin. In particular, channel clustering observed with these peptides may be also associated with formation of collective conductance states, resulting from mixing of their monomeric states, which allows us to explain the fact that clusters of these channels transmit ions and nonelectrolytes of the same size as the original single channels.

We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD + -isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Glutathione reductase (GR; EC 1.6.4.2) is a key NADPH-dependent flavo-protein oxidoreductase which can catalyze the oxidized glutathione (GSSG) to reduced glutathione (GSH) to protect plant cells from oxidative damage induced by Reactive oxygen species (ROS) burst. To investigate the biochemical characteristics and functional divergence of Populus GR family, three GR genes (PtGR1.1/1.2/2) were cloned from Populus trichocarpa and their biochemical characteristics were analyzed in this study. All the three genes were expressed in root, stem, leaf and bud, and the expression of PtGR genes were general upregulated under salicylic acid and alamethicin treatment. PtGR1.1 and PtGR1.2 were localized in cytoplasm, while PtGR2 was in chloroplast. The three PtGR proteins showed different enzymatic activities, apparent kinetic characteristic and thermal stability profiles. However, they have similar bivalent metal ions (Cu 2+ , Cd 2+ , Zn 2+ and Pb 2+ ) sensitivity and optimum pH profiles. Our study sheds light on a comprehensive information of glutathione reductase family in P. trichocarpa, and proved PtGR genes play critical roles when suffering different stresses., Competing Interests: Declaration of Competing Interest No conflict of interest exits in the submission of the revised manuscript, and it is approved by all authors for publication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Filamentous fungi have evolved to succeed in nature by efficient growth and degradation of substrates, but also due to the production of secondary metabolites including mycotoxins. For Trichoderma reesei, as a biotechnological workhorse for homologous and heterologous protein production, secondary metabolite secretion is of particular importance for industrial application. Recent studies revealed an interconnected regulation of enzyme gene expression and carbon metabolism with secondary metabolism., Results: Here, we investigated gene regulation by YPR2, one out of two transcription factors located within the SOR cluster of T. reesei, which is involved in biosynthesis of sorbicillinoids. Transcriptome analysis showed that YPR2 exerts its major function in constant darkness upon growth on cellulose. Targets (direct and indirect) of YPR2 overlap with induction specific genes as well as with targets of the carbon catabolite repressor CRE1 and a considerable proportion is regulated by photoreceptors as well. Functional category analysis revealed both effects on carbon metabolism and secondary metabolism. Further, we found indications for an involvement of YPR2 in regulation of siderophores. In agreement with transcriptome data, mass spectrometric analyses revealed a broad alteration in metabolite patterns in ∆ypr2. Additionally, YPR2 positively influenced alamethicin levels along with transcript levels of the alamethicin synthase tex1 and is essential for production of orsellinic acid in darkness., Conclusions: YPR2 is an important regulator balancing secondary metabolism with carbon metabolism in darkness and depending on the carbon source. The function of YPR2 reaches beyond the SOR cluster in which ypr2 is located and happens downstream of carbon catabolite repression mediated by CRE1.

The insertion and ion-conducting channel properties of alamethicin reconstituted into a 1,2-di- O-phytanyl- sn-glycero-3-phosphocholine bilayer floating on the surface of a gold (111) electrode modified with a 1-thio-β-d-glucose (β-Tg) self-assembled monolayer were investigated using a combination of electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The hydrophilic β-Tg monolayer separated the bilayer from the gold substrate and created a water-rich spacer region, which better represents natural cell membranes. The EIS measurements acquired information about the membrane resistivity (a measure of membrane porosity), and the PM-IRRAS experiments provided insight into the conformation and orientation of the membrane constituents as a function of the transmembrane potential. The results showed that the presence of alamethicin had a small effect on the conformation and orientation of phospholipid molecules within the bilayer for all studied potentials. In contrast, the alamethicin peptides assumed a surface state, where the helical axes adopted a large tilt angle with respect to the surface normal, at small transmembrane potentials, and inserted into the bilayer at sufficiently negative transmembrane potentials forming pores, which behaved as barrel-stave ion channels for ionic transport across the membrane. The results indicated that insertion of alamethincin peptides into the bilayer was driven by the dipole-field interactions and that the transitions between the inserted and surface states were electrochemically reversible. Additionally, the EIS measurements performed on phospholipid bilayers without alamethicin also showed that the application of negative transmembrane potentials introduces defects into the bilayer. The membrane resistances measured in both the absence and presence of alamethicin show similar dependencies on the electrode potential, suggesting that the insertion of the peptide may also be assisted by the electroporation of the membrane. The findings in this study provide new insights into the mechanism of alamethicin insertion into phospholipid bilayers.

The antimicrobial action of the peptide antibiotic alamethicin (Alm) is commonly related to peptide self-assembling resulting in the formation of voltage-dependent channels in bacterial membranes, which induces ion permeation. To obtain a deeper insight into the mechanism of channel formation, it is useful to know the dependence of self-assembling on peptide concentration. With this aim, we studied Alm F50/5 spin-labeled analogs in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, for peptide-to-lipid (P/L) ratios varying between 1/1500 and 1/100. Pulsed electron-electron double resonance (PELDOR) spectroscopy reveals that even at the lowest concentration investigated, the Alm molecules assemble into dimers. Moreover, under these conditions, electron spin echo envelope modulation (ESEEM) spectroscopy of D 2 O-hydrated membranes shows an abrupt change from the in-plane to the trans-membrane orientation of the peptide. Therefore, we hypothesize that dimer formation and peptide reorientation are concurrent processes and represent the initial step of peptide self-assembling. By increasing peptide concentration, higher oligomers are formed. A simple kinetic model of equilibrium among monomers, dimers, and pentamers allows for satisfactorily describing the experimental PELDOR data. The inter-label distances in the oligomers obtained from PELDOR experiments become better resolved with increasing P/L ratio, thus suggesting that the supramolecular organization of the higher-order oligomers becomes more defined.

A simple and efficient strategy is proposed to significantly improve the antibacterial activity of peptaibols and other antimicrobial peptides by N-terminal capping with 1,2,3-triazole bearing various hydrophobic substituents on C-4. Such N-terminal insertions on alamethicin F50/5 could enhance its antimicrobial activity on Gram-positive bacteria without modification of its overall three-dimensional structure. Although the native peptide and its analogues shared comparable helical contents, the crystal structure of one of the most active derivative showed a local slight distortion of the N-terminal extremity, which was also observed in solution using NMR spectroscopy. Importantly, fluorescence studies showed that the N-capped derivatives had increased affinity for liposomes, which may indicate they interacted more strongly with the bacterial membrane than alamethicin F50/5., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The bacterial membrane represents an attractive target for the design of new antibiotics to combat widespread bacterial resistance. Understanding how antimicrobial peptides (AMPs) and other membrane-active agents attack membranes could facilitate the design of new, effective antimicrobials. Despite intense study of AMPs on model membranes, we do not know how well the mechanism of attack translates to real biological membranes. To that end, we have characterized the attack of AMPs on Escherichia coli cytoplasmic membranes and directly compared this action to model membranes. AMPs induce membrane permeability in E. coli spheroplasts or giant unilamellar vesicles (GUVs) under well-defined concentrations of AMPs and fluorescent molecules. The action of AMPs on spheroplasts is unique in producing an intracellular fluorescence intensity time curve that increases in a sigmoidal fashion to a steady state. This regular pattern is reproducible by melittin, LL37, and alamethicin but not by CCCP or daptomycin, agents known to cause ion leakage. Remarkably, a similar pattern was also reproduced in GUVs. Indeed the steady-state membrane permeability induced by AMPs is quantitatively the same in spheroplasts and GUVs. There are, however, interesting dissimilarities in details that reveal differences between bacterial and lipid membranes. Spheroplast membranes are permeabilized by a wide range of AMP concentrations to the same steady-state membrane permeability. In contrast, only a narrow range of AMP concentrations permeabilized GUVs to a steady state. Tension in GUVs also influences the action of AMPs, whereas the spheroplast membranes are tensionless. Despite these differences, our results provide a strong support for using model membranes to study the molecular interactions of AMPs with bacterial membranes. As far as we know, this is the first time the actions of AMPs, on bacterial membranes and on model membranes, have been directly and quantitatively compared., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To understand the mechanism of development of cross-resistance in food pathogen Bacillus cereus against an antimicrobial peptide pediocin and antibiotic alamethicin, the present study was designed. Pediococcus pentosaceus was taken as a source of pediocin, and it was purified by ammonium sulphate precipitation followed by cation exchange chromatography with 14.01-fold purity and 14.4 % recovery. B. cereus strains alamethicin-resistant strains (IC50 3.23 µg/ml) were selected from sensitive population with IC50 2.37 µg/ml. The development of resistance in B. cereus against alamethicin was associated with decrease in alamethicin-membrane interaction observed by in vitro assay. Resistant strain of B. cereus was found to harbour one additional general lipid as compared to sensitive strain, one amino group lacking phospholipid and one amino group containing phospholipid (ACP). In addition, ACP content was increased in resistant mutant (29.7 %) as compared to sensitive strain (14.56 %). The alamethicin-resistant mutant B. cereus also showed increased IC50 (58.8 AU/ml) for pediocin as compared to sensitive strain (IC50 47.8 AU/ml). Cross-resistance to pediocin and alamethicin in resistant mutant of B. cereus suggested a common mechanism of resistance. Therefore, this understanding could result in the development of peptide which will be effective against the resistant strains that share same mechanism of resistance.

An all-atom molecular dynamics simulation of the archetype barrel-stave alamethicin (alm) pore in a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer at 313 K indicates that ∼7 μs is required for equilibration of a preformed 6-peptide pore; the pore remains stable for the duration of the remaining 7 μs of the trajectory, and the structure factors agree well with experiment. A 5 μs simulation of 10 surface-bound alm peptides shows significant peptide unfolding and some unbinding, but no insertion. Simulations at 363 and 413 K with a -0.2 V electric field yield peptide insertion in 1 μs. Insertion is initiated by the folding of residues 3-11 into an α-helix, and mediated by membrane water or by previously inserted peptides. The stability of five alm pore peptides at 413 K with a -0.2 V electric field demonstrates a significant preference for a transmembrane orientation. Hence, and in contrast to the cationic antimicrobial peptide described in the following article, alm shows a strong preference for the inserted over the surface-bound state., (Published by Elsevier Inc.)

The structure, topology and orientation of membrane-bound antibiotic alamethicin were studied using solid state nuclear magnetic resonance (NMR) spectroscopy. (13)C chemical shift interaction was observed in [1-(13)C]-labeled alamethicin. The isotropic chemical shift values indicated that alamethicin forms a helical structure in the entire region. The chemical shift anisotropy of the carbonyl carbon of isotopically labeled alamethicin was also analyzed with the assumption that alamethicin molecules rotate rapidly about the bilayer normal of the phospholipid bilayers. It is considered that the adjacent peptide planes form an angle of 100° or 120° when it forms α-helix or 310-helix, respectively. These properties lead to an oscillation of the chemical shift anisotropy with respect to the phase angle of the peptide plane. Anisotropic data were acquired for the 4 and 7 sites of the N- and C-termini, respectively. The results indicated that the helical axes for the N- and C-termini were tilted 17° and 32° to the bilayer normal, respectively. The chemical shift oscillation curves indicate that the N- and C-termini form the α-helix and 310-helix, respectively. The C-terminal 310-helix of alamethicin in the bilayer was experimentally observed and the unique bending structure of alamethicin was further confirmed by measuring the internuclear distances of [1-(13)C] and [(15)N] doubly-labeled alamethicin. Molecular dynamics simulation of alamethicin embedded into dimyristoyl phophatidylcholine (DMPC) bilayers indicates that the helical axes for α-helical N- and 310-helical C-termini are tilted 12° and 32° to the bilayer normal, respectively, which is in good agreement with the solid state NMR results., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Microbial electrosynthesis (MES) systems with mixed cultures often generate a variety of gaseous and soluble chemicals. Methane is the primary end product in mixed-culture MES because it is the thermodynamically most favorable reduction product of CO2. Here, we show that the peptaibol alamethicin selectively suppressed the growth of methanogens in mixed-culture MES systems, resulting in a shift of the solution and cathode communities to an acetate-producing system dominated by Sporomusa, a known acetogenic genus in MES systems. Archaea in the methane-producing control were dominated by Methanobrevibacter species, but no Archaea were detected in the alamethicin-treated reactors. No methane was detected in the mixed-culture reactors treated with alamethicin over 10 cycles (∼ 3 days each). Instead, acetate was produced at an average rate of 115 nmol ml(-1) day(-1), similar to the rate reported previously for pure cultures of Sporomusa ovata on biocathodes. Mixed-culture control reactors without alamethicin generated methane at nearly 100% coulombic recovery, and no acetate was detected. These results show that alamethicin is effective for the suppression of methanogen growth in MES systems and that its use enables the production of industrially relevant organic compounds by the inhibition of methanogenesis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Many antimicrobial peptides (AMPs) kill bacteria by disrupting the lipid bilayer structure of their inner membrane. However, there is only limited quantitative information in the literature to differentiate between AMPs of differing molecular properties, in terms of how they interact with the membrane. In this study, we have used quartz crystal microbalance with dissipation monitoring (QCM-D) to probe the interactions between a supported bilayer membrane of egg phosphatidylcholine (egg PC) and four structurally different AMPs: alamethicin, chrysophsin-3, indolicidin, and sheep myeloid antimicrobial peptide (SMAP-29). Multiple signatures from the QCM-D measurements were extracted, differentiating the AMPs, that provide information on peptide addition to and lipid removal from the membrane, the dynamics of peptide-membrane interactions and the rates at which the peptide actions are initiated. The mechanistic variations in peptide action were related to the fundamental structural properties of the peptides including the hydrophobicity, hydrophobic moment, and the probability of α-helical secondary structures., (Published by Elsevier B.V.)

In this work, we investigate the structure, dynamic and thermodynamic properties of noncanonical disubstituted amino acids (α,α-dialkyl glycines), also known as non-natural amino acids, in the peptaibol Alamethicin. The amino acids under study are Aib (α-amino isobutyric acid or α-methyl alanine), Deg (α,α-diethyl glycine), Dpg (α,α-dipropyl glycine), Dibg (α,α-di-isobutyl glycine), Dhg (α,α-dihexyl glycine), DΦg (α,α-diphenyl glycine), Dbzg (α,α-dibenzyl glycine), Ac6c (α,α-cyclohexyl glycine), and Dmg (α,α-dihydroxymethyl glycine). It is hypothesized that these amino acids are able to induce well-defined secondary structure in peptidomimetics. To test this hypothesis, new peptidomimetics of Alamethicin were constructed by replacing the native Aib positions of Alamethicin by one or more new α,α-dialkyl glycines. Dhg and Ac6c demonstrated the capacity to induce well-defined α-helical structures. Dhg and Ac6c also promote the thermodynamic stabilization of these peptides in a POPC model membrane and are better alternatives to the Aib in Alamethicin. These noncanonical amino acids also improved secondary structure properties, revealing preorganization in water and maintenance of α helical structure in POPC. We show that it is possible to optimize the helicity and thermodynamic properties of native Alamethicin, and we suggest that these amino acids could be incorporated in other peptides with similar structural effect.

Peptaibols, mainly produced by Trichoderma, play a pivotal role in controlling plant disease caused by fungi, virus, and Gram-positive bacteria. In the current study, we evaluated the control effect of Trichokonins, antimicrobial peptaibols from Trichoderma pseudokoningii SMF2, on soft rot disease of Chinese cabbage caused by a Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum and analyzed the mechanism involved. Trichokonins treatment (0.3 mg L(-1) ) enhanced the resistance of Chinese cabbage against Pcc infection. However, Trichokonins could hardly inhibit the growth of Pcc in vitro, even at high concentration (500 mg L(-1) ). Therefore, the direct effect of Trichokonins on Pcc may not the main reason why Trichokonins could control soft rot of Chinese cabbage. Trichokonin treatment led to an obvious increase in the production of reactive oxygen species hydrogen peroxide and superoxide radical, a significant enhance of the activities of pathogenesis-related enzymes catalase, polyphenoloxidase and peroxidase, and upregulation of the expression of salicylic acid - responsive pathogenesis-related protein gene acidic PR-1a in Chinese cabbage. These results indicate that Trichokonins induce resistance in Chinese cabbage against Pcc infection through the activation of salicylic acid signaling pathway, which imply the potential of Trichoderma and peptaibols in controlling plant disease caused by Gram-negative bacteria., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Ion channels are specialized proteins that enable the movement of charges through otherwise impermeable lipidic membranes. Their action is essential in living organisms facilitating electric signaling, muscle contraction or osmotic stress response among other effects. The protein and the lipid charges configure a polarized interface that yields local ionic concentrations and electric potentials that are very different from those of the bulk electrolyte. The combined effect of gradients of ionic concentration and electric potential causes the transport of ions through channels. Here we analyze charge regulation effects in different protein-lipid conformations, stressing how important is the role of electrostatic interactions in the ion channel function that traditionally has been rationalized paying attention mainly to changes in pore size. Tuning lipid charge combined with conductance and selectivity measurements is shown to be a complementary method to evidence lipid involvement in the structure of a biological ion channel.

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP-mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL-37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL-37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion-induced LL-37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol-rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion-induced resistance. Our data highlight the importance of Rho GTPase-dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL-37., (© 2013 John Wiley & Sons Ltd.)

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

We induced stochastic resonance in planar lipid bilayer systems with alamethicin ion channels, and varied alamethicin concentration, membrane area, and applied voltage. We found that membrane-induced microphonic noise significantly affects the signature of stochastic resonance, and that this noise can be used to optimize ion channel-based biosensors.

Antibiosis is one of the widespread strategies used by Trichoderma spp. against plant fungal pathogens, the mechanism of which, however, remains poorly understood. Peptaibols are a large family of antimicrobial peptides produced by Trichoderma spp. Our previous study showed that trichokonins, a type of peptaibol from Trichoderma pseudokoningii SMF2, exhibited antibiotic activities against plant fungal pathogens. In this study, we first demonstrated that trichokonin VI (TK VI) induced extensive apoptotic programmed cell death in plant fungal pathogens. For a deeper insight into the apoptotic mechanism involved in the action of TK VI, Fusarium oxysporum was used as a model. Cells of F. oxysporum treated with TK VI showed apoptotic hallmarks, such as exposure of phosphatidylserine, the appearance of reactive oxygen species and fragmentation of nuclear DNA. Moreover, TK VI-treated cells exhibited an accumulation of cytoplasmic vacuoles with loss of the mitochondrial transmembrane potential, and this process was independent of metacaspases. Therefore, TK VI induces metacaspase-independent apoptotic cell death in F. oxysporum. This represents what is believed to be the first report to reveal the antibiotic mechanism of peptaibols against plant fungal pathogens.

Alamethicin is the archetypal antimicrobial pore-forming peptide. Although the peptide has long been known to form pores of characteristic conductances in lipid membranes, the precise nature of these pores is not known. Simultaneous calcium-flux imaging and single-channel recording in a droplet interface bilayer allowed us to directly attribute multiple conductance states to a single point diffusing in the bilayer.

Mechanosensitive (MS) channels can prevent bacterial bursting during hypo-osmotic shocks by responding to increases in lateral tension at the membrane level through an integrated and coordinated opening mechanism. Mechanical regulation in protocells could have been one of the first mechanisms to evolve in order to preserve their integrity against changing environmental conditions. How has the rich functional diversity found in present cells been created throughout evolution, and what did the primordial MS channels look like? This review has been written with the aim of identifying which factors may have been important for the appearance of the first osmotic valve in a prebiotic context, and what this valve may have been like. It highlights the mechanical properties of lipid bilayers, the association of peptides as aggregates in membranes, and the conservation of sequence motifs as central aspects to understand the evolution of proteins that gate below the tension required for spontaneous pore formation and membrane rupture. The arguments developed here apply to both MscL and MscS homologs, but could be valid to mechano-susceptible proteins in general.

Biological molecules perform a sophisticated array of transport and signaling functions that rival anything that the modern electronics industry can create. Incorporating such building blocks into nanoelectronic devices could enable new generations of electronic circuits that use biomimetics to perform complicated tasks. Such types of circuits could ultimately blur the interface between living biological organisms and synthetic structures. Our laboratory has recently developed a versatile and flexible platform for integrating ion channels and pumps into single-walled carbon nanotube (SWNT) and silicon nanowire (SiNW) transistor devices, in which membrane proteins are embedded in a lipid bilayer shell covering the nanotube or nanowire component. In this chapter, we provide details for the fabrication of these devices and outline procedures for incorporating biological molecules into them. In addition, we also provide several examples of the use of these devices to couple biological transport to electronic signaling.

A method for quantitatively predicting the hepatic clearance of drugs by UDP-glucuronosyltransferases (UGTs) from in vitro data has not yet been established. We examined the relationship between in vitro and in vivo intrinsic clearance by rat hepatic UGTs using 10 drugs. For these 10 drugs, the in vitro intrinsic clearance by UGTs (CL(int, in vitro)) measured using alamethicin-activated rat liver microsomes was in the range 0.10-4500 ml/min/kg. Microsomal binding (f(u, mic)) was determined to be in the range 0.29-0.95 and the unbound intrinsic clearance (CL(uint, in vitro)) to be in the range 0.11-9600 ml/min/kg. The contribution of rat hepatic glucuronidation to drug elimination was 12.0%-76.6% and in vivo intrinsic clearance by UGTs was 5.7-9000 ml/min/kg. To evaluate the discrepancy between the in vitro and in vivo values, a scaling factor was calculated (CL(int, in vivo)/CL(int, in vitro)); the values were found to be in the range 0.89-110. The average fold error of the scaling factor values incorporating f(u, mic) was closer to unity than that without f(u, mic). The scaling factor values incorporating f(u, mic) were <10 in 8/10 drugs and <2 in 6/10 drugs, indicating a small discrepancy between in vitro and in vivo values. Thus, using alamethicin-activated liver microsomes, incorporating f(u, mic) into CL(int, in vitro), and considering the contribution of glucuronidation may enable us to quantitatively predict in vivo hepatic glucuronidation from in vitro data.

In Arabidopsis thaliana cell cultures, the peptaibol alamethicin induced a form of active cell death that was associated with cell shrinkage and DNA fragmentation. The transfer of mature A. thaliana plants from a peptide-free medium to a medium containing a moderate concentration of alamethicin caused the development of lesions in leaves after a few days. These lesions were characterized by cell death, deposition of callose, production of autofluorescent phenolic compounds, and transcription of defense genes, just like in the hypersensitive response to a pathogen attack. The induction of defense-like responses in Arabidopsis by other membrane-disrupting peptides was also evaluated. The peptides selected for comparison included the natural antimicrobial melittin and the peptaibol ampullosporin A, as well as synthetic analogues of the peptaibols cervinin and trichogin. The response amplitude in A. thaliana increased with the peptaibol's ability to permeabilize biological membranes through a pore-forming mechanism and was strongly associated with their content in the helicogenic α-aminoisobutyric acid residue.

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH approximately 7, the net charge of alamethicin is -1 whereas that of melittin is +5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions., (Copyright 2010 Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Suspended planar lipid membranes (or black lipid membranes (BLMs)) are widely used for studying reconstituted ion channels, although they lack the chemical and mechanical stability needed for incorporation into high-throughput biosensors and biochips. Lipid polymerization enhances BLM stability but is incompatible with ion channel function when membrane fluidity is required. Here, we demonstrate the preparation of a highly stable BLM that retains significant fluidity by using a mixture of polymerizable and nonpolymerizable phospholipids. Alamethicin, a voltage-gated peptide channel for which membrane fluidity is required for activity, was reconstituted into mixed BLMs prepared using bis-dienoyl phosphatidylcholine (bis-DenPC) and diphytanoyl phosphatidylcholine (DPhPC). Polymerization yielded BLMs that retain the fluidity required for alamethicin activity yet are stable for several days as compared to a few hours prior to polymerization. Thus, these polymerized, binary composition BLMs feature both fluidity and long-term stability.

Alamethicin, a member of the peptaibol family of antibiotics, is a typical channel-forming peptide with a helical structure. The self-assembly of the peptide in the membranes yields voltage-dependent channels. In this study, three alamethicin analogs possessing a charged residue (His, Lys, or Glu) on their N-termini were designed with the expectation of stabilizing the transmembrane structure. A slight elongation of channel lifetime was observed for the Lys and Glu analogs. On the other hand, extensive stabilization of certain channel open states was observed for the His analog. This stabilization was predominantly observed in the presence of metal ions such as Zn(2+), suggesting that metal coordination with His facilitates the formation of a supramolecular assembly in the membranes. Channel stability was greatly diminished by acetylation of the N-terminal amino group, indicating that the N-terminal amino group also plays an important role in metal coordination., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Probing the interaction of biological systems with radio frequencies holds great promise for research and drug screening applications. While a common assumption is that biological systems do not operate at radio frequencies, we find that currents due to ion transport through channels and pores in cell membranes are in the pA to nA range. These values translate via the average current I = ne/tau(d) = nef to frequencies in the range of 1 MHz-1 GHz, where n is the average number of ions transported and tau(d) is the dwell time of the ions in the channel. It is thus desirable to have circuitry available which facilitates radio frequency spectroscopy of ion transport. This will yield real-time in vitro information on ion channel operation. Here we present measurements on the local interaction of a radio frequency signal with single ion channels and pores. We find radio frequency rectification and pumping on the channels and pores embedded in suspended bilipid membranes, recorded in direct current measurements. This electromagnetic modulation can be used to probe the dynamics of ion channel conformational changes.

(31)P and (15)N solid-state NMR with the magic angle-oriented sample spinning (MAOSS) strategy was used to investigate the effect of two model peptides on phospholipid bilayers mimicking biological membrane. One of the peptides, alamethicin, used as a reference of transmembrane alignment, has been shown to disrupt the lipid bilayer organisation, affecting the DMPC packaging. On the other hand, a alpha-helix alanine-rich peptide, K(3)A(18)K(3), with a (15)N labelled alanine, did not present any effect in the DMPC bilayer organisation. The mean orientation of this peptide in the bilayer gave a transmembrane alignment of about 80%.

Detailed insight into the interplay between antimicrobial peptides and biological membranes is fundamental to our understanding of the mechanism of bacterial ion channels and the action of these in biological host-defense systems. To explore this interplay, we have studied the incorporation, membrane-bound structure, and conformation of the antimicrobial peptide alamethicin in lipid bilayers using a combination of 1H liquid-state NMR spectroscopy and molecular dynamics (MD) simulations. On the basis of experimental NMR data, we evaluate simple in-plane and transmembrane incorporation models as well as pore formation for alamethicin in DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine) bicelles. Peptide-lipid nuclear Overhauser effect (NOE) and paramagnetic relaxation enhancement (PRE) data support a transmembrane configuration of the peptide in the bilayers, but they also reveal that the system cannot be described by a single simple conformational model because there is a very high degree of dynamics and heterogeneity in the three-component system. To explore the origin of this heterogeneity and dynamics, we have compared the NOE and PRE data with MD simulations of an ensemble of alamethicin peptides in a DMPC bilayer. From all-atom MD simulations, the contacts between peptide, lipid, and water protons are quantified over a time interval up to 95 ns. The MD simulations provide a statistical base that reflects our NMR data and even can explain some initially surprising NMR results concerning specific interactions between alamethicin and the lipids.

The lipid matrix, or the lipid bilayer, of cell membranes is a natural binding site for amphipathic molecules, including antimicrobial peptides, pore-forming proteins, and many drugs. The unique property of pore-forming antimicrobial peptides is that they exhibit a threshold concentration (called the lethal concentration or the minimum inhibitory concentration) for activity, below which no effect is seen. Without this property, antimicrobial peptides would not be effective self-defense weapons, because they would have harmed all cells at any concentration. The question is what gives rise to this unique property? This study provides a free energy description for the origin of a threshold concentration. The same free energy applied differently also explains the binding of drugs that shows no threshold concentrations. The idea is compared with theories of micellar solutions that require a large oligomer size (n 15) to achieve a threshold concentration. The elasticity of lipid bilayers makes the phenomena in membranes different. The majority of antimicrobial peptides have a large negative binding energy to the bilayer interface, but the binding causes an expansion in the membrane area, or equivalently a thinning in the membrane thickness. This elastic energy of membrane thinning elevates the energy level of interfacial binding with the peptide concentration, hence gives rise to a threshold concentration for forming pores containing as few as four peptides.

Alamethicin is a 20-residue peptaibiotic that induces voltage-dependent ion channels in lipid membranes. The mode by which alamethicin inserts into membranes was investigated using measurements of peptide-lipid interactions by spin-label electron paramagnetic resonance (EPR) and of peptide orientation by polarized infrared (IR) spectroscopy. In fluid membranes, spin-labeled stearic acid shows no evidence of a specific motionally restricted population of lipid chains, such as that found at the intramembranous surface of integral membrane proteins or oligomeric assemblies of transmembrane alpha-helices. In agreement with recent results from TOAC-substituted alamethicin analogues, native alamethicin is predominantly monomeric in fluid lipid membranes and presents an intramembrane surface that integrates well with the lipid chains but is insufficiently extensive to induce specific motional restriction. Channel formation takes place by transient association of transmembrane monomers. In aligned fluid membranes, alamethicin exhibits a large tilt in short chain-length lipids that decreases first rapidly with increasing chain-length and then more gradually for the lipids with longer chains. This macroscopically low order contrasts with the high local order, relative to the local membrane normal, that is found by EPR for alamethicins spin-labeled with TOAC. The macroscopic behavior is consistent with predictions for the chain-length dependence of elastic bending fluctuations of the membrane surface, which was invoked recently to explain the spontaneous insertion of beta-barrel proteins in short-chain lipid membranes.

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D2O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D2O-ESEEM intensities decrease from TOAC(1) to TOAC(8) and TOAC(16) but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC(16) is located in the bilayer leaflet opposite to that of TOAC(1) and TOAC(8). Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D2O-ESEEM intensity is greatest for TOAC(8), indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.

We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by alamethicin peptides in a hydrated lipid bilayer. In the 1-micros CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 micros is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the alpha-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of alamethicin oligomers formed by perfectly aligned helical alamethicin peptides in a lipid bilayer.

Electric features of biological membranes are major determinants of the function and physiological manifestation of membrane-penetrating peptides, and such features are prone to be modulated by the properties of the surrounding aqueous medium. In this work, we demonstrate that pH plays crucial roles in modulating electric characteristics of zwitterionic-based artificial lipid membranes. The effect of pH on electrical properties of such membranes was probed by evaluating the transport properties of embedded alamethicin oligomers over a wide range of pH values (i.e., 0.65, 2.08, 2.94, 7 and 10.1). Our data strongly support the paradigm of a pH-dependent variation of the surface and membrane dipole potential which, in conjunction with possible lateral pressure effects within the lipid membrane, lead to a non-monotonic modulation of the electrical conductance of alamethicin oligomers. As expected, pH modulation of transport properties through the alamethicin oligomer is more visible for narrower pores (that is, the 1st conductive state) with slightly better cation selectivity as compared to larger oligomers.

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.

Catalytic reactions performed with enzymes localized in lipid vesicles or in whole cells represent a new, promising approach in biocatalysis. The delivery of different substrates into these micro- or nano-'reactors' requires a sufficient permeability of lipid membranes. To increase the permeability of lipid bilayers, one may use different membrane-active peptides, including peptaibols. In the present study, the trypsin-catalyzed hydrolysis of N(alpha)-benzoyl-L-arginine-para-nitroanilide (BAPA; 1) was studied in a phospholipid vesicular system made of phosphatidylcholine (POC), in the presence of the peptaibols alamethicin (ALM) or zervamicin IIB (ZER). Two different manners of compartmentalization of substrate and enzyme (enzyme- vs. substrate-containing vesicles) were used. The kinetics parameters of the reaction in homogeneous solution and in the vesicular systems were determined. The rate of the extra- or intravesicular enzymatic reaction was found to be controlled by substrate diffusion through the lipid bilayer. In comparison with untreated vesicular systems, an up to seven-fold increase in reaction rate was observed in the presence of either ALM or ZER.

We have investigated the x-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide alamethicin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius corresponding to the geometric radius of the pore, we find a repulsive lipid-mediated interaction with a range of approximately 30 A and a contact value of 2.4 k(B)T. This result is in qualitative agreement with recent theoretical models.

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-beta-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (maximal rate of inactivation) of 0.21 min(-1). Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

Release of cytochrome c from mitochondria is considered a critical, early event in the induction of an apoptosis cascade that ultimately leads to programmed cell death. Mitochondrial Ca(2+) loading is a trigger for the release of cytochrome c, although the molecular mechanism underlying this effect is not fully clarified. This study tested the hypothesis that distinct Ca(2+) thresholds may induce cytochrome c release from rat liver mitochondria by membrane permeability transition (MPT)-dependent and independent mechanisms. The involvement of reactive oxygen species (ROS) and cardiolipin in the Ca(2+)-induced cytochrome c release was also investigated. Cytochrome c was quantitated by a new, very sensitive, and rapid reverse-phase high performance liquid chromatography method with a detection limit of 0.1 pmol/sample. We found that a low extramitochondrial Ca(2+) level (2 microM) promoted the release of approximately 13% of the total alamethicin releasable pool of cytochrome c from mitochondria. This release was not depending of MPT; it was mediated by Ca(2+)-induced ROS production and cardiolipin peroxidation and appears to involve the voltage-dependent anion channel. High extramitochondrial Ca(2+) level (20 microM) promoted approximately 45% of the total releasable pool of cytochrome c. This process was MPT-dependent and was also mediated by ROS and cardiolipin. It is suggested that distinct Ca(2+) levels may determine the mode and the amount of cytochrome c release from rat liver mitochondria. The data may help to clarify the molecular mechanism underlying the Ca(2+)-induced release of cytochrome c from rat liver mitochondria and the role played by ROS and cardiolipin in this process.

Most ion channel proteins exhibit some degree of charge selectivity, that is, an ability to conduct ions of one charge more efficiently than ions of the opposite charge. The structural origins of charge selectivity remain incompletely understood despite recent advances in the determination of cation-selective and anion-selective channel protein structures. Helix bundle channels formed via self-assembly of the peptide alamethicin provide a tractable model system for exploring the structural basis of charge selectivity. We synthesized covalently-linked alamethicin dimers, with amino acid substitutions at position 18 [lysine (Lys), arginine (Arg), glutamine (Gln), 2,3-diaminopropionic acid (Dpr)] in each helix, to assess the role of this position as a charge-selectivity determinant in alamethicin channels. Of the position 18 substitutions investigated, the Lys derivative exhibited the greatest degree of anion selectivity. Arg-containing channels were slightly less anion-selective than Lys. Interestingly, Dpr channels showed cation selectivity nearly equivalent to that exhibited by the neutral Gln derivative. We suggest that this result is due to a wider pore diameter that permits a greater number of counter-ions leading to enhanced charge screening and a lower effective side-chain positive charge.

Ceratotoxin A (CtxA), a 36-residue alpha-helical cationic peptide isolated from the medfly Ceratitis capitata, exhibits strong antibacterial activity. To determine its mode of action against bacteria, we investigated the behavior of ceratotoxin A by incorporating it into planar lipid bilayers. Macroscopic and single channel conductance experiments showed that ceratotoxin A forms voltage-dependent ion channels in bilayers according to the barrel-stave model. The characteristics of the channel suggest that the C-terminal regions form bundles of five or six helices embedded in the membrane, such that the N-terminal moieties lie on the polar side of the lipid bilayer.

We present a straightforward, accessible method for the fabrication of micropores with diameters from 2 to 800 micro m in films of amorphous Teflon (Teflon AF). Pores with diameters

The binding state of the antibiotic peptide alamethicin with phospholipid bilayers was investigated in terms of the changes induced in lipid mobility. Fluorescence anisotropy was used for the study. It was found that an increase in peptide concentration induced different changes in lipid mobility above and below a critical peptide concentration. This concentration was also critical for an increase in the cooperative binding of the peptide, as detected by circular dichroism. Above the critical peptide concentration, the mobility of both lipid regions, around the polar head and hydrocarbon chain, became restricted with an increased peptide concentration. Below the critical level, however, an increased peptide concentration induced a "wobbling" of the lipid hydrocarbon chain. These results show that an increase in the cooperative binding of the peptide is accompanied by a change in the dominant configuration of the binding peptide. When the binding peptide increases, the dominant configuration appears to shift from surface association to deep incorporation within the membrane. This shift in configuration means that in the formation of ion-conductive pores, voltage-driven insertion of the peptide is a prominent step below a critical peptide concentration.