Victor Levadny

Kinetic Pathway of Antimicrobial Peptide Magainin 2-Induced Pore Formation in Lipid Membranes

The pore formation in lipid membranes induced by the antimicrobial peptide magainin 2 is considered to be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10,000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage followed by a stage of slow leakage. In contrast, magainin 2 induced a transient, but very small (10-20%), leakage of fluorescent probes of a larger size such as Texas-Red dextran 40,000 and FITC-BSA. These results indicate that magainin 2 molecules initially induce a large, transient pore in lipid membranes following which the radius of the pore decreases to a stable smaller size. We estimated the radius of these pores, which increases with an increase in magainin 2 concentration. On the basis of these data, we propose a hypothesis on the mechanism of magainin 2-induced pore formation.

Stretch-Activated Pore of the Antimicrobial Peptide, Magainin 2

Antimicrobial peptide magainin 2 forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is likely the main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of magainin 2 with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of magainin 2 to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of magainin 2, X. This indicates that the rate constant of the magainin 2-induced two-state transition from the intact state to the pore state greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activated magainin 2-induced pore formation. To reveal the location of magainin 2, the interaction of carboxyfluorescein (CF)-labeled magainin 2 (CF-magainin 2) with single GUVs containing a water-soluble fluorescent probe, AF647, was investigated using confocal microscopy. In the absence of tension due to aspiration, after the interaction of magainin 2 the fluorescence intensity of the GUV rim due to CF-magainin 2 increased rapidly to a steady value, which remained constant for a long time, and at 4-32 s before the start of leakage of AF647 the rim intensity began to increase rapidly to another steady value. In contrast, in the presence of the tension, no increase in rim intensity just before the start of leakage was observed. These results indicate that magainin 2 cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we conclude that a magainin 2-induced pore is a stretch-activated pore and the stretch of the inner monolayer is a main driving force of the pore formation.

Rate constant of tension-induced pore formation in lipid membranes.

We investigated the effects of tension induced by micropipet aspiration on giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). We analyzed the time course of the fraction of intact GUVs among all of the GUVs under constant tension σ and obtained the rate constants of pore formation kp(σ). To determine kp, we developed an approach using the mean first passage time. The fitting of the theoretical curves of kp versus σ to the experimental data determined the line tension of a prepore, Γ. The value of Γ of a DOPG/DOPC bilayer was smaller than that of a DOPC bilayer.

Electrostatics Explains the Shift in VDAC Gating with Salt Activity Gradient

We have analyzed voltage-dependent anion-selective channel (VDAC) gating on the assumption that the states occupied by the channel are determined mainly by their electrostatic energy. The voltage dependence of VDAC gating both in the presence and in the absence of a salt activity gradient was explained just by invoking electrostatic interactions. A model describing this energy in the main VDAC states has been developed. On the basis of the model, we have considered how external factors cause the redistribution of the channels among their conformational states. We propose that there is a difference in the electrostatic interaction between the voltage sensor and fixed charge within the channel when the former is located in the cis side of membrane as opposed to the trans. This could be the main cause of the shift in the probability curve. The theory describes satisfactorily the experimental data (Zizi et al., Biophys. J. 1998. 75:704-713) and explains some peculiarities of VDAC gating. The asymmetry of the probability curve was related to the apparent location of the VDAC voltage sensor in the open state. By analyzing published experimental data, we concluded that this apparent location is influenced by the diffusion potential. Also discussed is the possibility that VDAC gating at high voltage may be better described by assuming that the mobile charge consists of two parts that have to overcome different energetic barriers in the channel-closing process.

Access resistance of a single conducting membrane channel

We have considered the access resistance (AR) of a single conducting channel placed in a membrane bathed by an electrolyte. The classical expression for AR is due to Hall, who modeled the electrolyte as an ohmic conducting homogeneous medium. This approach is discussed in the present paper and it is shown that it is not valid in all cases, but depends on the ion concentration in solution and the ratio between solution and channel resistivities. To get a new expression for AR, we have combined the use of one-dimensional Nernst-Planck and Poisson (NPP) equations for the mouth of the channel and three-dimensional NPP equations for the outside solution. The influence of ion gradients and the channel itself on AR tums out to be considerable in diluted solutions (and also in the case of small channels in any solution). This influence is weaker in concentrated solutions, for which AR is well described by Halls expression.

Passive transport of small ions through human stratum corneum

Some previous streaming potential and EMF measurements on human stratum corneum in vitro (J. Control. Release 32 (1994) 249) are explained in terms of a simple physical model based on the intercellular pathway for passive transport of small inorganic ions. These are assumed to diffuse across the hydrophilic region of the lipid lamellae. The polar region is assumed to have a finite thickness and to be partly penetrable by ions from solution. The hypothesis that the lipid ionized groups give rise to a point charge distribution together with a permanent dipole distribution helps to explain the observed, apparent change in skin selectivity with concentration.

A model of pressure-induced interdigitation of phospholipid membranes

The behaviour of lipid lamellae upon an applied hydrostatic pressure in different phase states is considered. We analyse the phase transitions from the bilayer phase states La or Lb0 to the interdigitated phase LbI. By considering separately the energies of chains and polar heads we obtain the expressions for the chemical potential of each state. The criteria for the existence of La or Lb0 states upon an applied pressure are derived. We conclude that the steeper increase of internal tension in the region of lipid heads in these states in comparison with the LbI one is the main physical cause of phase transitions to interdigitated phase upon an applied pressure. 2002 Published by Elsevier Science B.V.

The effect of peptides and ions interacting with an electrically neutral membrane interface on the structure and stability of lipid membranes in the liquid-crystalline phase and in the liquid-ordered phase

We investigated the effects of a de novo designed peptide, WLFLLKKK (peptide-1) and La3+, which can bind with the electrically neutral lipid membrane interface, on the stability of the phosphatidylcholine (PC) membrane in the Lα phase and that of the liquid-ordered (lo) phase membranes. The results of spacing of the multilamellar vesicle and shape changes of the giant unilamellar vesicle (GUV) indicate that the peptide-1 can be partitioned into the membrane interface in the Lα phase but not into that in the lo phase. La3+ induced shape changes of GUVs of the lo phase membrane, which are the same as those of GUVs in the Lα phase. This indicates that the binding of La3+ induced an increase in the lateral compression pressure of the membrane, which decreased the surface area of the membrane in the lo phase. The difference of the membrane interface between the Lα phase and the lo phase is discussed.

Ion transport through membranes with soft interfaces. The influence of the polar zone thickness

Abstract A membrane with soft permeable interfaces in an aqueous solution has been modelled by taking into account the spatial distribution of structural electric charges and dipoles in the interfaces. The model was developed with special stress on its application to biological membranes. The electric potential distribution and the ion transport across the membrane are studied and the important role of the interface thickness L on ion transport and membrane selectivity is shown. An interesting result is found for certain combinations of charge and dipole densities: a cation selective membrane may become anion selective when L changes. It is discussed how the results obtained can help to explain experimental results for membrane selectivity.

The size of the pore in lipid membranes induced by antimicrobial peptide magainin 2

Antimicrobial peptides found and isolated from a wide variety of organisms have an activity to kill bacteria. The target of these peptides is thought to be the lipid membrane region of the bacterial and fungal biomembranes. Using the single GUV (giant unilamellar vesicle) method, we have succeeded in revealing the elementary processes of the pore formation in lipid membranes induced by antimicrobial peptide, magainin 2. The statistical analysis of the pore formation in a GUV over many “single GUVs” enabled us to estimate the rate constant of the magainin 2-induced pore formation in lipid membranes. In this report, to reveal the size of the magainin 2-induced pores in lipid membranes, we investigated the interactions of magainin 2 with single GUVs containing various sizes of fluorescent probes. Under the conditions with no photobleaching of fluorescent probes, we investigated the interaction of magainin 2 with single GUVs of 50% dioleoylphosphatidylglycerol (DOPG)/ 50% dioleoylphosphatidylcholine(DOPC) membrane containing various sizes of fluorescent probes in 10 mM PIPES (pH 7.0), 150 mM NaCl (buffer A). Magainin 2 induced a transient (less than 10 s), but very small (10–20 %) leakage of Texas-Red dextran 40,000 (TRD-40k), Texas-Red dextran 70,000 (TRD-70k), and FITC-BSA, although the same concentrations of magainin 2 induced a complete leakage of calcein. In contrast, the magainin 2-induced leakage of Texas-Red dextran 10,000 (TRD-10k) and Texas-Red dextran 3,000 (TRD-3k) had two phases; the transient rapid leakage in the initial stage and the following slow leakage. These results indicate that in the initial stage of the magainin 2-induced pore formation, the size of the pore was large and then transformed into a small, steady size. The radius of the transient, large pore in the initial stage is larger than 6.4 nm (i.e., the Stokes-Einstein radius, RSE, of TRD-70k) and also that the radius of the small steady pore in the final stage is smaller than 3.5 nm (i.e., RSE of FITC-BSA). The amount of the leakage of TRD-10k in the initial stage increased with an increase in magainin 2 concentration. This result indicates that the radius of the large pore in the initial stage increased with an increase in magainin 2 concentration. We discuss these results from a point of view of the pore formation.