The ion-channel activity of longibrachins LGA I and LGB II: effects of pro-2/Ala and gln-18/Glu substitutions on the alamethicin voltage-gated membrane channels.

Longibrachins LGA I (Ac Aib Ala Aib Ala Aib(5) Ala Gln Aib Val Aib(10) Gly Leu Aib Pro Val(15) Aib Aib Gln Gln Pheol(20), with Aib: alpha-aminoisobutyric acid, pheol: phenylalaninol) and LGB II are two homologous 20-residue long-sequence peptaibols isolated from the fungus Trichoderma longibrachiatum that differ between them by a Gln-18/Glu substitution. They distinguish from alamethicin by a Pro-2 for Ala replacement, which allowed to examine for the first time with natural Aib-containing analogues, the effect of Pro-2 on the ion-channel properties exhibited by alamethicin. The influence of these structural modifications on the voltage-gated ion-channel forming activity of the peptides in planar lipid bilayers were analysed. The general 'barrel-stave' model of ion-channel activity, already described for alamethicin, was preserved with both longibrachins. The negatively charged LGB II promoted higher oligomerisation levels, which could presumably dilute the repulsive effect of the negative Glu ring near the entrance of the channel and resulted in lower lifetimes of the substates, confirming the strong anchor of the peptide C-terminus at the cis-interface. Reduction of the channel lifetimes was observed for the longibrachins, compared to alamethicin. This argues for a better stabilisation of the channels formed by peptaibols having a proline at position 2, which results in better anchoring of the peptide monomer N-terminus at the trans-bilayer interface. Qualitative assays of the temperature dependence on the neutral longibrachin channel properties demonstrated a high increase of channel lifetimes and a markedly reduced voltage-sensitivity when the temperature was decreased, showing that such conditions may allow to study the channel-forming properties of peptides leading to fast current fluctuations.     

Template-free self-assembling fullerene and lipopeptide conjugates of alamethicin form voltage-dependent ion channels of remarkable stability and activity.

N- and C-terminally modified with fullerene or lipopeptide alamethicin molecules were designed for the formation of template-free, self-assembling, voltage-dependent ion conducting channels. The automated solid phase synthesis of the alamethicin-F30 sequence was performed by in situ fluoride activation on 2-chlorotritylchloride-polystyrene resin and the conjugation with fullerenes-C60 and -C70 was carried out in solution. Voltage-dependent bilayer experiments revealed preferred channel sizes for C-terminal alamethicin F30-fullerene-C60 and -C70 conjugates and higher activity compared with native alamethicin, whereas N-terminally linked fullerene balls destabilize pore formation. C-terminal alamethicin F30-fullerene-C70 conjugates show pore states with remarkably long lifetimes of seconds. C-terminal lipopeptide conjugates of alamethicin were prepared by coupling via short peptide spacers with synthetic tripalmitoyl-S-glyceryl-cysteine. which represents the strong membrane anchoring N-terminus of bacterial lipoprotein. Alamethicin-lipopeptide conjugates exhibit high channel forming activities, whereby they self-assemble and adopt preferred pore states with extremely long lifetimes. The novel membrane modifying peptaibol constructs are valuable lead compounds for developments in sensorics related to transmembrane ion conductance.     

Design and conformation of non-Aib synthetic peptides enjoying alamethicin-like ionophore activity

Analogues of alamethicin, a 20-mer amphipathic helical peptide with ionophore activity, in the sequence of which all Aib residues were substituted by Ala (A1) or Leu (L1), were synthesized by the solid phase method, purified by high performance liquid chromatography and characterized by fast atomic bombardment mass spectrometry. Infrared and CD studies showed that A1 easily underwent a transconformation to beta-structure whereas L1 displayed a predominant alpha-helical character, thus being a potential ionophore model. Its voltage-dependent multistate activity in model membranes showed that Aib is not a requisite residue to observe an alamethicin-like behavior. However, as the lifetime of the single channels was much shorter than for alamethicin, the peptide chain was lengthened by a Leu (LL1) or a Ser (SL1) residue. The last peptide gave an increased channel lifetime, but the design of other non-Aib peptides, taking into account the hydroxyl C-terminus and side-chain interactions between helices in a barrel-stave bundle, is desirable to approach more closely the alamethicin activity.     

Alamethicin channel conductance modified by lipid charge

The membrane surface charge modifies the conductance of ion channels by changing the electric potential and redistributing the ionic composition in their vicinity. We have studied the effects of lipid charge on the conductance of a multi-state channel formed in planar lipid bilayers by the peptide antibiotic alamethicin. The channel conductance was measured in two lipids: in a neutral dioleoylphosphatidylethanolamine (DOPE) and a negatively charged dioleoylphosphatidylserine (DOPS). The charge state of DOPS was manipulated by the pH of the membrane-bathing solution. We find that at high salt concentrations (e.g., 2 M NaCl) the effect of the lipid charge is below the accuracy of our measurements. However, when the salt concentration in the membrane-bathing solution is decreased, the surface charge manifests itself as an increase in the conductance of the first two channel levels that correspond to the smallest conductive alamethicin aggregates. Our analysis shows that both the salt and pH dependence of the surface charge effect can be rationalized within the nonlinear Poisson-Boltzmann approach. Given channel conductance in neutral lipids, we use different procedures to account for the surface charge (e.g., introduce averaging over the channel aperture and take into account Na+ adsorption to DOPS heads), but only one adjustable parameter: an effective distance from the nearest lipid charge to the channel mouth center. We show that this distance varies by 0.3-0.4 nm upon channel transition from the minimal conducting aggregate (level L0) to the next larger one (level L1). This conclusion is in accord with a simple geometrical model of alamethicin aggregation.     

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.     

Detection of protein-ligand interaction on the membranes using C-terminus biotin-tagged alamethicin

C-terminal biotin-tagged alamethicin, which has several alpha-aminoisobutyric acid (Aib) residues in its sequence, was synthesized by the preparation of the protected peptide segment using the 2-chlorotrityl resin, followed by conjugation with biotin hydrazide. Suppression of the channel current of the biotin-tagged alamethicin by the addition of streptavidin to the electrolyte was monitorable in real time using the planar lipid-bilayer method. The system was also applicable to the detection of interaction of the biotin-tagged alamethicin with the anti-biotin antibody.     

Pressure effects on alamethicin conductance in bilayer membranes.

We report here the first observations of the effects of elevated hydrostatic pressure on the kinetics of bilayer membrane conductance induced by the pore-forming antibiotic, alamethicin. Bacterial phosphatidylethanolamine-squalene bilayer membranes were formed by the apposition of lipid monolayers in a vessel capable of sustaining hydrostatic pressures in the range, 0.1-100 MPa (1-1,000 atm). Principal observations were (a) the lifetimes of discrete conductance states were lengthened with increasing pressure, (b) both the onset and decay of alamethicin conductance accompanying application and removal of supra-threshold voltage pulses were slowed with increasing pressure, (c) the onset of alamethicin conductance at elevated pressure became distinctly sigmoidal, suggesting an electrically silent intermediate state of channel assembly, (d) the magnitudes of the discrete conductance levels observed did not change with pressure, and, (e) the voltage threshold for the onset of alamethicin conductance was not altered by pressure. Apparent activation volumes for both the formation and decay of conducting states were positive and of comparable magnitude, namely, approximately 100 A3/event. Observation d indicates that channel geometry and the kinetics of ion transport through open channels were not affected by pressure in the range employed. The remaining observations indicate that, while the relative positions of free-energy minima characterizing individual conducting states at a given voltage were not modified by pressure, the heights of intervening potential maxima were increased by its application.     

The properties of ion channels formed by zervamicins

The zervamicins (Zrv) are a family of 16 residue peptaibol channel formers, related to the 20 residue peptaibol alamethicin (Alm), but containing a higher proportion of polar sidechains. Zrv-1113 forms multi-level channels in planar lipid (diphytanoyl phosphatidylcholine) bilayers in response to cis positive voltages. Analysis of the voltage and concentration dependence of macroscopic conductances induced by Zrv-IIB suggests that, on average, channels contain ca. 13 peptide monomers. Analysis of single channel conductance levels suggests a similar value. The pattern of successive conductance levels is consistent with a modified helix bundle model in which the higher order bundle are distorted within the plane of the bilayer towards a torpedo shaped cross-section. The kinetics of intro-burst switching between adjacent conductance levels are shown to be approximately an order of magnitude faster for Zrv-IIB than for Alm. The channel forming properties of the related naturally occurring peptaibols, Zrv-Leu and Zrv-IC, have also been demonstrated, as have those of the synthetic apolar analogue Zrv-Al-16. The experimental studies on channel formation are combined with the known crystallographic structures of Zrv-Al-16 and Zrv-Leu to develop a molecular model of Zrv-II3 channels.Abbreviations Alm Alamethicin - Zrv Zervamicin - CFP Channel forming peptide - Aib -aminoisobutyric acid Correspondence to: M. S. P. Sansom     

Influence of proline-14 substitution on the secondary structure in a synthetic analogue of alamethicin

Due to the bend introduced by proline 14 in the conformation of alamethicin (AcUPUAUQUVUGLU P VUUEQFol), the role of this residue was assumed essential in the barrel-stave model for voltage-gated ion channels. Taking advantage of a previous synthetic alamethicin analogue (L2) in which all eight α-aminoisobutyric(U) Were replaced by leucines (Ac L P L A L AQ L V L GL L PV LL EQFol), another analogue (L5) was synthesized in order to test the effects ofproline-14 substitution by an alanine (Ac L P L A L AQ L V L G LL PV LL EQFol). Previous conductance experiments showed that both high voltage dependence and multistate behavior were conserved. In order to complement these functional results, a conformational study of L5 has been undertaken and compared to L2 using CD. high field nmr, and molecular dynamics. Results show that L5 presents a better ordered structure than L2 particularly in the region of the substitution and in the C-terminal part. These results are discussed as regards the previous hypothesis of the nonessential character of helix bending for the gating of voltage-dependent ion channels. © 1995 John Wiley & Sons, Inc.     

Influence of the secondary structure on the pore forming properties of synthetic alamethicin analogs: NMR and molecular modelling studies

Synthetic alamethicin analogs, in which all Aib residues had been replaced by Leu (L2) then proline 14 replaced by an alanine (L5), were studied in SDS micelles using circular dichroism and NMR spectroscopy. Nuclear Overhauser effects were used as constraints for molecular modelling. The structures determined for both peptides in SDS micelles were compared with those previously obtained in methanol in order to establish a secondary structure/ionophore activity relationship. Our results indicated that a shortening of peptide helices could be responsible for the observed decrease in ion channel lifetimes. However, the length of helices may not by itself explain the drastic destabilization of channels when Pro14 of alamethicin is replaced by Ala in L5. Indeed analysis of the helical wheel of L5 reveals heterogeneity in the amphipathicity depending on the medium. Thus, loss of amphipathicity seems to underly the observed destabilization of channels. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational Changes in Alamethicin Associated with Substitution of Its α-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 (α-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. Fourier transform infrared (FTIR) spectra of these peptides were recorded in methanol and in aqueous phospholipid membranes. Results obtained show a significant conformational change in alamethicin upon substitution of its Aib residues with Leu. The amide I band occurs at a lower frequency for the Leu-derivative indicating that its α-helices are involved in stronger hydrogen-bonding. In addition, the structure of the Leu-derivative is quite sensitive to membrane fluidity changes. The amide I band shifts to higher frequencies when the lipids are in the fluid phase. This indicates either a decreased solvation due to a more complete peptide insertion or a peptide stretching to match the full thickness of the bilayer. These results contribute to explain the fast single-channel kinetics displayed by the Leu-derivative.     

Effects of polycations on ion channels formed by neutral and negatively charged alamethicins

The effects of the peptide polycations salmon protamine (M _r = 4332,z = + 21) and poly-l-lysine (M _r 100,00,z + 775) on ion channels formed by synthetic alamethicin Alm-F30 (one negative charge), natural Alm-F50 (neutral) and phosphorylated Alm-F50 (two negative charges) reconstituted in planar lipid bilayers have been studied at the single channel level. It was observed that both polycations in micromolar concentrations transiently block ion permeation through the channels formed by each alamethicin analogue, although in case of the neutral Alm-F50 to a significantly lesser extent. Poly-l-lysine showed to be more effective than protamine in blocking these channels. If either polycation is present in the cis-compartment, blockade occurs only at cis positive membrane voltages. At constant polycation concentration, dwell times in the blocked state increase when salt concentration is lowered, and decrease at acidic pH with an apparent pK of 4.8. Mean lifetime of blockade events shortens when membrane voltage is increased, which suggests that both polycations may permeate through the oligomeric alamethicin channels if conductance levels are > 2. We suggest that blockade is caused by electrostatic binding of a single polycation molecule to the C-terminal channel mouth; in case of Alm-F30, Glu18 has to be considered as the putative binding site. Our results provide further evidence for the barrel-stave model and a parallel orientation of dipole monomers in the channel aggregate, the C-termini facing the membrane side with the more positive membrane potential.     

Voltage-dependent conductance induced by alamethicin-phospholipid conjugates in lipid bilayers.

Alamethicin, a linear 20-amino acid antibiotic, forms voltage-dependent channels in lipid bilayer membranes. We show here that alamethicin-phospholipid conjugates can be prepared by photolysis of unilamellar vesicles containing alamethicin and a phosphatidylcholine analogue with a carbene precursor at the end of the C-2 fatty acyl chain. This result indicates that at least a portion of the alamethicin molecule is in contact with the hydrocarbon moiety of the membrane in the absence of an applied voltage. Furthermore, the alamethicin-phospholipid photoproduct is able to induce a voltage-gated conductance similar to that of natural alamethicin. The importance of these results in terms of mechanisms for channel gating is discussed.     

Conformational analysis of TOAC-labelled alamethicin F50/5 analogues

In the preceding paper in this issue, we reported the total syntheses in solution of a set of four TOAC-containing analogues of the [L-Glu(OMe)(7,18,19)] F50/5 component of alamethicin, the prototype of peptaibol antibiotics forming channels in the biological membranes. In this article, we have expanded this work to the examination of their preferred conformation in solution by use of a combination of CD, FT-IR absorption, and NMR spectroscopies. The results are strongly in favor of the view that replacement of the Aib residues at positions 1, 8, and 16 with TOAC (both are members of the helicogenic sub-class of C(alpha)-tetrasubstituted alpha-amino acids) does not significantly affect the overwhelmingly populated alpha-helical 3D structure of alamethicin. The X-ray diffraction crystal structure of the N(alpha)-protected, C-terminal, hexapeptide amide segment Z-L-Pro-L-Val-(Aib)(2)-[L-Glu(OMe)](2)-Fol lends further support to our conformational conclusions.     

Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.     

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. The latter was further enhanced in a dimer and a tetramer in which alamethicin(1-17) chains were tethered to di- or tetra-lysine linkers, respectively, but macroscopic current-voltage curves displayed much reduced voltage-dependencies and reversed hysteresis. An usual behaviour with high voltage-dependence was restored with the modified dimer of alamethicin(1-17) in which alanine separated the two consecutive lysine residues in the linker. Of special interest was the development of a 'negative resistance' branch in macroscopic current-voltage curves for low concentrations of this dimer with the more flexible linker. Single channel events displayed only one single open state with fast kinetics and whose conductance matches that of the alamethicin heptamer or octamer.     

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

Dynamics and aggregation of the peptide ion channel alamethicin. Measurements using spin-labeled peptides.

Two spin-labeled derivatives of the ion conductive peptide alamethicin were synthesized and used to examine its binding and state of aggregation. One derivative was spin labeled at the C-terminus and the other, a leucine analogue, was spin labeled at the N-terminus. In methanol, both the C and N terminal labeled peptides were monomeric. In aqueous solution, the C-terminal derivative was monomeric at low concentrations, but aggregated at higher concentrations with a critical concentration of 23 microM. In the membrane, the C-terminal label was localized to the membrane-aqueous interface using 13C-NMR, and could assume more than one orientation. The membrane binding of the C-terminal derivative was examined using EPR, and it exhibited a cooperativity seen previously for native alamethicin. However, this cooperativity was not the result of an aggregation of the peptide in the membrane. When the spectra of either the C or N-terminal labeled peptide were examined over a wide range of membrane lipid to peptide ratios, no evidence for aggregation could be found and the peptides remained monomeric under all conditions examined. Because electrical measurements on this peptide provide strong evidence for an ion-conductive aggregate, the ion-conductive form of alamethicin likely represents a minor fraction of the total membrane bound peptide.     

A three state model for alamethicin conductance in bilayer membranes

Alamethicin is an antibiotic which produces voltage gated channels in lipid bilayer membranes. Recently completed studies of the pressure dependence of alamethicin conductance have shown that its onset following application of a suprathreshold voltage step at a pressure of 100 MPa (1000 atm) is markedly slowed relative to that observed at ambient pressure. Furthermore, the time course of the onset of conductance becomes distinctly sigmoidal at elevated pressure, a condition which is not evident at atmospheric pressure. The decay of alamethicin conductance upon removal of suprathreshold applied voltage is also slowed by application of hydrostatic pressure, but it follows a single exponential time course at all pressures. In addition, kinetic parameters characterizing the onset and decay of conductance show distinctly different pressure dependences. These observations cannot be explained by a two state model in which alamethicin moves reversibly between nonconducting and conducting states. Therefore we re-examine critically a hypothesis made by previous workers, namely that alamethicin, in monomeric or aggregate form, moves upon application of suprathreshold voltage first from a nonconducting surface state to a nonconducting preassembly or precursor state, and then finally into a conducting state. Parameters of this three state model are related to a geometric factor which measures the degree of sigmoidal conductance response and which can be evaluated directly from experimental data. An alternative aggregation-type analysis, equivalent to that applied by Hodgkin & Huxley to the potassium conductance in squid axon, is also considered in the context of this same geometric factor. The possibility of distinguishing between these analyses on the basis of experimental data is discussed.     

Voltage-dependent insertion of alamethicin at phospholipid/water and octane/water interfaces

Understanding the binding and insertion of peptides in lipid bilayers is a prerequisite for understanding phenomena such as antimicrobial activity and membrane-protein folding. We describe molecular dynamics simulations of the antimicrobial peptide alamethicin in lipid/water and octane/water environments, taking into account an external electric field to mimic the membrane potential. At cis-positive potentials, alamethicin does not insert into a phospholipid bilayer in 10 ns of simulation, due to the slow dynamics of the peptide and lipids. However, in octane N-terminal insertion occurs at field strengths from 0.33 V/nm and higher, in simulations of up to 100 ns duration. Insertion of alamethicin occurs in two steps, corresponding to desolvation of the Gln7 side chain, and the backbone of Aib10 and Gly11. The proline induced helix kink angle does not change significantly during insertion. Polyalanine and alamethicin form stable helices both when inserted in octane and at the water/octane interface, where they partition in the same location. In water, both polyalanine and alamethicin partially unfold in multiple simulations. We present a detailed analysis of the insertion of alamethicin into the octane slab and the influence of the external field on the peptide structure. Our findings give new insight into the mechanism of channel formation by alamethicin and the structure and dynamics of membrane-associated helices.