alamethicin and 1-2-dielaidoylphosphatidylethanolamine

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.

Topics: Alamethicin; Anti-Bacterial Agents; Biophysical Phenomena; Biophysics; Electric Conductivity; Hydrogen-Ion Concentration; In Vitro Techniques; Ion Channels; Ionophores; Lipid Bilayers; Models, Chemical; Phosphatidylethanolamines; Phosphatidylserines; Sodium Chloride; Static Electricity

Incorporation of the helical antimicrobial peptide alamethicin from aqueous phase into hydrated phases of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) was investigated within a range of peptide concentrations and temperatures by time-resolved synchrotron X-ray diffraction. It was found that alamethicin influences the organizations of the non-bilayer-forming (DOPE) and the bilayer-forming (DOPC) lipids in different ways. In DOPC, only the bilayer thickness was affected, while in DOPE new phases were induced. At low peptide concentrations (<1.10(-4) M), an inverted hexagonal (H(II)) phase was observed as with DOPE dispersions in pure buffer solution. A coexistence of two cubic structures was found at the critical peptide concentration for induction of new lipid/peptide phases. The first one Q224 (space group Pn3m) was identified within the entire temperature region studied (from 1 to 45 degrees C) and was found in coexistence with H(II)-phase domains. The second lipid/peptide cubic structure was present only at temperatures below 16 degrees C and its X-ray reflections were better fitted by a Q212 (P4(3)32) space group, rather than by the expected Q229 (Im3m) space group. At alamethicin concentrations of 1 mM and higher, a nonlamellar phase transition from a Q224 cubic phase into an H(II) phase was observed. Within the investigated range of peptide concentrations, lamellar structures of two different bilayer periods were established with the bilayer-forming lipid DOPC. They correspond to lipid domains of associated and nonassociated helical peptide. The obtained X-ray results suggest that the amphiphilic alamethicin molecules adsorb from the aqueous phase at the lipid head group/water interface of the DOPE and DOPC membranes. At sufficiently high (>1.10(-4) M) solution concentrations, the peptide is probably accommodated in the head group region of the lipids thus inducing structural features of mixed lipid/peptide phases.

Topics: Alamethicin; Amino Acid Sequence; Anti-Bacterial Agents; Lipid Bilayers; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Thermodynamics; Water; X-Ray Diffraction

Under normal conditions, excess water dispersions of liquid crystalline 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE) are known to convert from a liquid crystalline lamellar (L alpha) to inverse hexagonal (HII) phase at about 60 degrees Celsius. The nonlamellar phase behavior of lipid systems is also known to depend on the monolayer spontaneous curvature. The single-channel activity of alamethicin in black lipid bilayer membranes has been shown to be dependent upon the lipid composition of the membrane. Since the monolayer spontaneous curvature properties (e.g., the monolayer spontaneous curvature, curvature coefficients and bilayer thickness) vary with lipid composition, the single-channel activity of alamethicin presumably also correlates with the monolayer spontaneous curvature properties. Accordingly, we reasoned that if alamethicin couples to the curvature properties of a lipid film, then the curvature properties must, in turn, be perturbed by the presence of alamethicin and that this perturbation should be observable in the lipid phase behavior. Here X-ray diffraction and NMR are used to show that the presence of as little as 1% alamethicin introduces a large region of cubic phase into the thermal phase diagram. This suggests that perturbation of the nonlamellar phase behavior of a lipid system may be a method to survey different channel-forming molecules for possible behavior that indicates that the ion channel is sensitive to the monolayer spontaneous curvature properties.

Topics: Alamethicin; Chemical Phenomena; Chemistry, Physical; Crystallization; Crystallography, X-Ray; Ionophores; Liposomes; Magnetic Resonance Spectroscopy; Phosphatidylethanolamines; Temperature

With few exceptions, membrane lipids are usually regarded as a kind of filler or passive solvent for membrane proteins. Yet, cells exquisitely control membrane composition. Many phospholipids found in plasma membrane bilayers favor packing into inverted hexagonal bulk phases. It was suggested that the strain of forcing such lipids into a bilayer may affect membrane protein function, such as the operation of transmembrane channels. To investigate this, we have inserted the peptide alamethicin into bilayer membranes composed of lipids of empirically determined inverted hexagonal phase "spontaneous radii" Ro, which will have expectably different degrees of strain when forced into bilayer form. We observe a correlation between measured Ro and the relative probabilities of different conductance states. States of higher conductance are more probable in dioleoylphosphatidylethanolamine, the lipid of highest curvature, 1/Ro, than in dioleoylphosphatidylcholine, the lipid of lowest curvature.

Topics: Alamethicin; Biophysical Phenomena; Biophysics; Electric Conductivity; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Phosphatidylethanolamines