Antimicrobial peptides and peptaibols, substitutes for conventional antibiotics.

In this review, the antimicrobial properties of a number of peptides are described. We first deal with helical linear peptides such as the well-known gramicidin, magainins, melittin, and other less well-known or more recently discovered peptides. Then, beta-sheet peptides (defensins isolated from insects and also from mammalian tissues) and cyclic peptides like amphotericin B are described before the properties of peptaibols (containing the non-coded amino acid Aib) are discussed.

Structure-function studies on the voltage-gated sodium channel.

Recent research on structure-function relationships aspects of voltage-gated sodium channels (VGSCs) are reviewed. Data issued from the literature are summarized and compared, including results from our own studies. The latter deal with the effects of drug binding, deglycosylation and the role of hydrophobic residues in the voltage sensors.

Peptaibiotics and peptaibols: an alternative to classical antibiotics?

The development and antimicrobial properties of peptaibiotics and peptaibols are discussed. Also, the role of emerging peptaibol analogues, of alamethicin, e.g.

Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel.

The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating.

Secondary structure of an isolated P-region from the voltage-gated sodium channel: a molecular modelling/dynamics study.

Conformational studies of synthetic peptides corresponding to the pore-forming regions of voltage-gated sodium channels show a high tendency for beta-sheet conformation when interacting with lipid vesicles, as revealed by circular dichroism and infrared spectroscopy. These observations have guided our choice of possible molecular models for the P-region peptide of domain II of voltage-gated sodium channels: three alternative beta-hairpins, with differing turn assignments, or an alpha-helical hairpin. After generation of models by distance geometry-based methods, molecular dynamics (MD) simulations were run.

Bilayer lipid composition modulates the activity of dermaseptins, polycationic antimicrobial peptides.

The primary targets of defense peptides are plasma membranes, and the induced irreversible depolarization is sufficient to exert antimicrobial activity although secondary modes of action might be at work. Channels or pores underlying membrane permeabilization are usually quite large with single-channel conductances two orders of magnitude higher than those exhibited by physiological channels involved, e.g.

Neuronal sodium channels in ventricular heart cells are localized near T-tubules openings.

Cardiac voltage-dependent sodium channels (VDSC) are known to be tetrodotoxin (TTX)-resistant. However, recent immunochemical studies suggest the presence of TTX-sensitive neuronal-type VDSC in the heart. Scanning ion conductance microscopy (SICM) coupled to electrophysiology was used to obtain more direct functional evidence.

Effects of deglycosylation of sodium channels on their structure and function.

Voltage-gated sodium channels are important membrane proteins underlying electrical signaling in the nervous and muscular systems. They undergo rapid conformational changes between closed resting, activated, and inactivated states. Approximately 30% of the mass of the sodium channel is carbohydrate, present as glycoconjugate chains, mostly composed of N-acetylhexosamines and sialic acid.

Conductance studies on trichotoxin_A50E and implications for channel structure.

Trichotoxin_A50E is an 18-residue peptaibol whose crystal structure has recently been determined. In this study, the conductance properties of trichotoxin_A50E have been investigated in neutral planar lipid bilayers. The macroscopic current-voltage curves disclose a moderate voltage-sensitivity and the concentration-dependence suggests the channels are primarily hexameric.

Helical kink and channel behaviour: a comparative study with the peptaibols alamethicin, trichotoxin and antiamoebin.

Kinks or bends introduced in peptides and proteins by "helical distorter" residues such as proline, other imino acids and glycine, especially when these are in close proximity in the sequence, are increasingly recognized as playing an essential role in the gating of channel-forming peptides as well as of physiological ion channels. Peptaibols are useful simple models for the much more complex biological ion channels, especially voltage-gated ones. In this short review, we compare the monomeric structures of three selected peptaibols (alamethicin, trichotoxin and antiamoebin) that widely differ with regards their near-central kink angles and dipolar moment orientations.

Conformational changes in alamethicin associated with substitution of its alpha-methylalanines with leucines: a FTIR spectroscopic analysis and correlation with channel kinetics.

Alamethicin, a 20 residue-long peptaibol remains a favorite high voltage-dependent channel-forming peptide. However, the structural significance of its abundant noncoded residues (alpha-methylalanine or Aib) for its ion channel activity remains unknown, although a previous study showed that replacement of all Aib residues with leucines preserved the essential channel behavior except for much faster single-channel events. To correlate these functional properties with structural data, here we compare the secondary structures of an alamethicin derivative where all the eight Aibs were replaced by leucines and the native alamethicin.

Channel properties of template assembled alamethicin tetramers.

The multiple conductance levels displayed by the antibiotic alamethicin in planar lipid bilayers is explained by a dynamic 'barrel-stave' model, the conducting pore resulting from the aggregation of up to ten helical amphipathic helical monomers. However, the precise assignment of an oligomerization state to a particular single-channel conductance substate is far from being experimentally clear. In addition, it could be useful to tailor a given channel geometry to selectively allow the permeation of solutes with different molecular sizes, whilst retaining a high voltage-dependence.

Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers.

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers.

Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation.

Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine.

How do channel- and pore-forming helical peptides interact with lipid membranes and how does this account for their antimicrobial activity?

Animals and plants defend themselves against pathogenic micro-organisms by the rapid mobilization of polycationic helical amphipathic peptides. Interactions with membranes induce optimal orientation and mutual structural changes, allowing for example to form transbilayer ion channels or pores whose properties are compared in this review. Physicochemical studies of peptide-lipid interactions provide attractive approaches for drug design.

Artificial membrane excitability revisited and implications for the gating of voltage-dependent ion channels.

Excitability phenomena in planar lipid bilayers doped with alamethicin and protamines have been first described by Mueller and Rudin (Nature 217, 713-719, 1968). These properties are reinvestigated here with virtually solvent-free bilayers made of synthetic phospholipids doped with alamethicin charged component (Glu18) and protamine or other synthetic basic polypeptides. After retrieving the narrow set of experimental requisites allowing negative resistance and action potentials to develop, the potencies of different basic polypeptides were compared.

Basic properties of an inositol 1,4,5-trisphosphate-gated channel in carp olfactory cilia.

In addition to the activation of cAMP-dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5-trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip-dip method).

Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox.

Correlation between anti-bacterial activity and pore sizes of two classes of voltage-dependent channel-forming peptides.

Anti-bacterial activities were compared for two series of voltage-dependent pore-formers: (i) alamethicin (Alm) and its synthetic analogs (Alm-dUL) where alpha-amino-isobutyric acid residues (Aibs) were replaced by leucines and selected key residues substituted and (ii) homologous voltage sensors of the electric eel sodium channel (repeats S4L45 (III) and S4L45 (IV)). Spiroplasma melliferum, a bacterium related to the mycoplasmas, was used as a target cell. The data show that with respect to growth inhibition, cell deformation and plasma membrane depolarization, the highest efficient peptide remained natural Alm although the minimal inhibitory concentrations of its Leu analogs were within the same range as the parent molecule, except for Alm-dUL P14A.

C-terminally shortened alamethicin on templates: influence of the linkers on conductances.

In order to test the influence of chemical modifications designed to allow covalent coupling of channel-forming peptide motifs into variable sized oligomers, a series of alamethicin derivatives was prepared. The building block encompassing the N-terminal 1-17 residues of alamethicin behaved normally in the conductance assay on planar lipid bilayers, albeit at higher concentration and with a slightly reduced voltage-dependence. A linker Ac-K-OCH(2)C(6)H(4)CH(3)p attached via the epsilon amino group of lysine to the C-terminus of alamethicin(1-17) increased membrane affinity.

Sodium channel fragments: contributions to voltage sensitivity and ion selectivity.

The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue.

The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide.

Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine.

Antiamoebin can function as a carrier or as a pore-forming peptaibol.

Antiamoebin is a 16-residue polypeptide whose crystal structure and lytic activity in membrane vesicles have recently been reported. It is a bent helical molecule and a member of the peptaibol family of antibiotics. Under conditions which produce voltage-dependent conductance activity by other members of the family, no single-channel conductance was detected for antiamoebin, and a carrier-like mechanism was put forward to account for its mode of action.