Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics.

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

Effect of dipole modifiers on the kinetics of sensitized photoinactivation of gramicidin channels in bilayer lipid membranes

Photodynamic inactivation of gramicidin channels in bilayer lipid membranes induced by single flashes of the visible light in the presence of phthalocyanine has been studied. The kinetic curves of the flash-induced decrease in the gramicidin-mediated electric current are used for determination of the rate constants of formation and termination of gramicidin channels in terms of the channel dimer model. It is revealed that the kinetics of the sensitized photoinactivation of gramicidin in the membrane is altered by agents which modify the dipole potential drop at the membrane-water interface. Addition of phloretin, which is known to decrease the dipole potential drop, slows down the kinetics, whereas the addition of RH421 or 6-ketocholestanol, which increase the dipole potential drop, accelerates the kinetics. It is shown that the photoinactivation kinetics is also slowed down upon the addition of the thyroid hormone L-thyronine, which reduces the dipole potential drop similar to phloretin, as it was found earlier (M. V. Tsybulskaya, Yu. N. Antonenko, A. E. Tropsha, and L. S. Yaguzhinsky, Biofizika 29:801-805 (1984) (in Russian)). It is demonstrated that the changes in the dissociation rate constant of gramicidin dimers under the action of different dipole modifiers correlate with the changes in the dipole potential drop. It is concluded that the process of the gramicidin channel termination corresponding to the dimer dissociation is sensitive to the dipole potential drop. This conclusion is supported by the data on the effect of dipole modifiers on the lifetime of single gramicidin channels.     

Interaction of phloretin with lipid monolayers: relationship between structural changes and dipole potential change.

Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model.     

Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel.

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H(+) concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.     

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.     

Interaction of phloretin with membranes: on the mode of action of phloretin at the water-lipid interface

 The interaction of phloretin with single lipid bilayers on a spherical support and with multilamellar vesicles was studied by differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR). The results indicated that phloretin interacts with the lipid layer and changes its structural parameters. In DSC experiments, phloretin in its neutral form strongly decreased the lipid phase transition temperature and slightly reduced the cooperativity of the phase transition within the lipid layer. In NMR measurements, phloretin led to an increase of the transverse relaxation time constant but had no effect on the spin-lattice relaxation time constant. The overall dipole moment of phloretin was experimentally determined and was found to be roughly 40% lower than has been published previously. This result suggested that the size of the dipole moment of phloretin does not provide such a high contribution to the effect of phloretin on the dipole potential of monolayers and bilayers as has been published previously. To understand the discrepancy between phloretin adsorption and dipole potential change, we performed computational conformational analysis of phloretin in the gas phase. The results showed that a wide distribution of the dipole moments of phloretin conformers exists, which mainly depends on the orientation of the OH moieties. The adsorption of phloretin as determined from its binding to solid supported bilayers differed from the one determined from dipole potential measurements on black lipid membranes. The difference between the phloretin dissociation constants of both types of experiments suggested a change of its dipole moment normal to the membrane surface in a concentration-dependent manner, which was in agreement with the results of the computational conformational analysis. Received: 21 June 1999 / Revised version: 7 January 2000 / Accepted: 31 March 2000     

The dipole-modifying effect of styrylpyridinium dyes and flavonoids on model membranes of different lipid compositions

Changes in dipole potential of lipid bilayers ?d mimicking cell membranes induced by the adsorption of low-molecular-weight amphiphiles, flavonoids (phloretin and quercetin), and styrylpyridinium dyes (RH 421 and RH 237) were measured. A method based on the determination of ionophore-induced transmembrane current was used to evaluate changes in ?d after modifier addition. The characteristic parameters of the Langmuir adsorption isotherm and the greatest changes in ?d at an infinitely large concentration of flavonoid and its desorption constant, which reflects the affinity of the flavonoid to the lipid phase, were determined. The slopes of linear dependences of ?d increasing on the concentration of the styrylpiridinium dyes in membrane-bathing solution were defined. It was found that the dipole-modifying effect of phloretin depends on the charge of the lipids forming the membranes, while the ability of quercetin to reduce ?d is determined by the initial hydration of the bilayer. The results indicate that there are different mechanisms of the decrease in ?d upon the adsorption of the tested flavonoids. It was shown that the changes in ?d at the incorporation of styrylpyridinium dyes into bilayers are determined by the interaction of modifiers with membrane components.     

Probing amphotericin B single channel activity by membrane dipole modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.     

The Interaction of Dipole Modifiers with Polyene-Sterol Complexes

Recently, we showed that the effect of dipole modifiers (flavonoids and styrylpyridinium dyes) on the conductance of single amphotericin B (AmB) channels in sterol-containing lipid bilayers primarily resulted from changes in the membrane dipole potential. The present study examines the effect of dipole modifiers on the AmB multi-channel activity. The addition of phloretin to cholesterol-containing membranes leads to a significant increase in the steady-state AmB-induced transmembrane current. Quercetin significantly decreases and RH 421 increases the current through ergosterol-containing bilayers. Other tested flavonoids and styrylpyridinium dyes do not affect the channel-forming activity of AmB independently on the sterol composition of the bilayers. The effects obtained in these trials may instead be attributed to the direct interaction of dipole modifiers with AmB/sterol complexes and not to the effect of dipole potential changes. The presence of double bonds in the Δ7 and Δ22 positions of sterol molecules, the number of conjugated double bonds and amino sugar residues in polyene molecules, and the conformation and adsorption plane of dipole modifiers are important factors impacting this interaction.     

Transport of large organic ions through syringomycin channels in the membranes containing dipole modifiers

The effect of the membrane dipole potential (Phid) on a conductance and a steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate and chloride was shown. The magnitude of Phid was varied with the introduction to membrane bathing solutions of phloretin, which reduces the Phid, and RH 421, increasing the Phid. It was established that in all studied systems the increase in the membrane dipole potential cause a decrease in the steady-state number of open channels. In the systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are in an order of magnitude smaller than in systems containing sodium chloride. At the same time, the conductance (g) of single SRE-channels on the membranes bathed in NaCI solution increases with the increase in Phid, and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the Phid. The latter is due to the lack of cation/anion selectivity of the SRE-channels in these systems. The different channel-forming activity of SRE in the experimental systems is defined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition

The influence of agents, known to affect the membrane dipole potential, phloretin and RH 421, on the multi channel activity of amphotericin B in lipid bilayers of various compositions, was studied. It was shown that the effects were dependent on the membrane’s phospholipid and sphingolipid type. Phloretin enhanced amphotericin B induced steady-state transmembrane current through bilayers made from binary mixtures of POPC (DOPC) and ergosterol and ternary mixture of DPhPC, ergosterol and stearoylphytosphingosine. RH 421 increased steady-state polyene induced transmembrane current through membranes made from binary mixtures of DPhPC (DPhPS) and ergosterol and ternary mixture of DPhPS, ergosterol and stearoylphytosphingosine. It was proposed that the observed effects reflect the fine balance of the interactions between the various components present: amphotericin B, ergosterol, phospholipid, sphingolipid and dipole modifier. The shape of lipid molecules seems to be an important factor impacting the responses of amphotericin B modified bilayers to dipole modifiers. The influence of different phospholipids and sphingolipids on the physical and structural properties of ordered lipid microdomains, enriched in AmB, was also discussed. It was also shown that RH 421 enhanced the antifungal activity of amphotericin B in vitro.     

Effect of arbutin on the dipole potential and area per lipid of ester and ether phosphatidylcholine and phosphatidyl ethanolamine monolayers

The present results report for the first time a systematic study of the effect of arbutin on the dipole potential of lipid membranes. The dipole potential and the area per lipid were measured in monolayers of dimyristoylphosphatidylcholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoylphosphatidylethanolamine (DMPE) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE), spread on aqueous solutions of different concentrations of arbutin. The decrease of the dipole potential of DMPC, both in condensed and expanded monolayers, is parallel to an increase in the area per lipid. In contrast, for dietherPC, the area per lipid is not affected, in spite of the fact that arbutin is also able to decrease the dipole potential in a less drastic extent. In the case of DMPE, the response is similar to that observed with dietherPC: the dipole potential decreases, while the area per lipid remains unchanged. However, when the carbonyl groups are absent in phosphatidylethanolamine derivatives such as the dietherPE, the dipole potential is not affected by arbutin, with a small decrease in the area. The effect of arbutin on the dipole potential differs from that of sucrose, trehalose and phloretin and is congruent with previous results obtained by FTIR on its interaction with the CO groups. Arbutin binding is interpreted in terms of the exposure to water of the phosphate and carbonyl groups at the membrane interface of the different monolayers.     

Interaction of two phenothiazine derivatives with phospholipid monolayers

This paper addresses the cooperative interaction of two phenothiazine drugs, viz. trifluoperazine (TFP) and chlorpromazine (CPZ), with phospholipid monolayers as the model membrane system. Surface pressure and surface potential isotherms were obtained for mixed Langmuir monolayers of either dipalmitoyl-phosphatidyl-choline (DPPC) or dipalmitoyl-phosphatidyl-glycerol (DPPG) co-spread with TFP or CPZ. The changes in monolayer behavior caused by incorporation of a few molar ratio of drug molecules were practically within the experimental dispersion for the zwitterionic DPPC, and therefore a more refined analysis will be required to probe the interactions in an unequivocal way. For the charged DPPG, on the other hand, the surface pressure and the dipole moment were significantly affected even for TFP or CPZ concentrations as low as 0.002 molar ratio. Overall, the effects from CPZ and TFP are similar, but small differences exist which are probably due to the different protonation properties of the two drugs. For both drugs, changes are more prominent at the liftoff of the surface pressure, i.e. at the gas-condensed phase transition, with the surface pressure and surface potential isotherms becoming more expanded with the drug incorporation. With DPPG/CPZ monolayers, in particular, an additional phase transition appears at higher CPZ concentrations, which resembles the effects from increasing the subphase temperature for a pure DPPG monolayer. The dipole moment for DPPG/CPZ and DPPG/TFP monolayers decreases with the drug concentration, which means that the effects from the charged drugs are not associated with changes in the double-layer potential. Otherwise, the effective dipole moment should increase with the drug concentration. The changes caused in surface pressure and dipole moment by small concentrations of TFP or CPZ can only be explained by some cooperative effect through which the contribution from DPPG molecules changes considerably, i.e. even DPPG molecules that are not neighbor to a CPZ or TFP molecule are also affected. Such changes may occur either through a significant reorientation of the DPPG molecules or to a change in their hydration state. We discuss the cooperativity semi-quantitatively by estimating the number of lipid molecules affected by the drug interaction. CPZ and TFP also affect the morphology of DPPG monolayers, which was confirmed with Brewster angle microscopy. The biological implications from the cooperative, non-specific interaction of CPZ and TFP with membranes are also commented upon.     

Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.

The electrostatic potentials associated with cell membranes include the transmembrane potential (delta psi), the surface potential (psi s), and the dipole potential (psi D). psi D, which originates from oriented dipoles at the surface of the membrane, rises steeply just within the membrane to approximately 300 mV. Here we show that the potential-sensitive fluorescent dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6- naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS) can be used to measure changes in the intramembrane dipole potential. Increasing the content of cholesterol and 6-ketocholestanol (KC), which are known to increase psi D in the bilayer, results in an increase in the ratio, R, of the dye fluorescence excited at 440 nm to that excited at 530 nm in a lipid vesicle suspension; increasing the content of phloretin, which lowers psi D, decreases R. Control experiments show that the ratio is insensitive to changes in the membrane's microviscosity. The lack of an isosbestic point in the fluorescence excitation and emission spectra of the dye at various concentrations of KC and phloretin argues against 1:1 chemical complexation between the dye and KC or phloretin. The macromolecular nonionic surfactant Pluronic F127 catalyzes the insertion of KC and phloretin into lipid vesicle and cell membranes, permitting convenient and controlled modulation of dipole potential. The sensitivity of R to psi D is 10-fold larger than to delta psi, whereas it is insensitive to changes in psi S. This can be understood in terms of the location of the dye chromophore with respect to the electric field profile associated with each of these potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide.

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Two phases of gramicidin photoinactivation in bilayer lipid membranes in the presence of a photosensitizer

The kinetics of the light-induced decrease in the gramicidin-mediated current across a bilayer lipid membrane in the presence of a photosensitizer has been shown to include a slow phase with a characteristic time of the order of 1 s and a fast phase. Based on the dependence of the slow phase relative amplitude and characteristic time on the gramicidin-mediated stationary conductance we concluded that the slow phase reflected the establishment of an equilibrium between gramicidin monomers and dimers in the membrane after the distortion of this equilibrium resulting from modification of a portion of gramicidin molecules by reactive oxygen species generated upon excitation of the photosensitizer. The dependence of the fast phase contribution to the overall kinetics on the stationary conductance allowed us to conclude that the fast phase is associated with transition of gramicidin dimers into a nonconducting state. The characteristic time of the fast phase measured with nanosecond laser excited pulses is 1.5 ms. The slow phase of the decrease in the gramicidin-mediated current was considerably decelerated in the presence of Rose Bengal. The results obtained indicate that adsorption of Rose Bengal on the bilayer interface leads to a reduction of the dipole potential drop at the membrane-solution boundary, similarly to the action of phloretin.     

Thallous ion movements through gramicidin channels incorporated in lipid monolayers supported by mercury.

The potential independent limiting flux of hydrated Tl(+) ions through gramicidin (GR) channels incorporated in phospholipid monolayers self assembled on a hanging mercury-drop electrode is shown to be controlled both by diffusion and by a dehydration step. Conversely, the potential independent limiting flux of dehydrated Tl(+) ions stemming from Tl amalgam electro-oxidation is exclusively controlled by diffusion of thallium atoms within the amalgam. Modulating the charge on the polar heads of dioleoylphosphatidylserine (DOPS) by changing pH affects the limiting flux of hydrated Tl(+) ions to a notable extent, primarily by electrostatic interactions. The dipole potential of DOPS and dioleoylphosphatidylcholine (DOPC), positive toward the hydrocarbon tails, does not hinder the translocation step of Tl(+) ions to such an extent as to make it rate limiting. Consequently, incorporation in the lipid monolayer of phloretin, which decreases such a positive dipole potential, does not affect the kinetics of Tl(+) flux through GR channels. In contrast, the increase in the positive dipole potential produced by the incorporation of ketocholestanol causes the translocation step to contribute to the rate of the overall process. A model providing a quantitative interpretation of the kinetics of diffusion, dehydration-hydration, translocation, and charge transfer of the Tl(+)/Tl(0)(Hg) couple through GC channels incorporated in mercury-supported phospholipid monolayers is provided. A cut-off disk model yielding the profile of the local electrostatic potential created by an array of oriented dipoles located in the lipid monolayer along the axis of a cylindrical ion channel is developed.     

The RH 421 styryl dye induced,pore model-dependent modulation of antimicrobial peptides activity in reconstituted planar membranes

Background

Antimicrobial agents, with different pore-formation mechanisms, may be differently influenced by alteration of the dipolar electric field of a lipid membrane.

Methods

By using electrophysiological measurements on reconstituted lipid membranes, we used alamethicin, melittin and magainin to report on how controlled manipulation of the membrane dipole potential by the styrylpyridinium dye RH 421 affects the kinetic and transport features of peptides within membranes.

Results

Our data demonstrate that the increase of the membrane dipole potential caused by RH 421 decreases the activity and single-channel conductance of alamethicin. Surprisingly, we found that RH 421 increases the activity of melittin and magainin, suggesting that RH 421 may contribute via electrostatic repulsions, among others, to an increase in the monolayer spontaneous curvature of the membrane. We propose that RH 421-induced dipole potential and membrane elasticity changes alter the peptide-induced channel dynamics, and the prevalence of one mechanism over the other for particular classes of peptides is dictated by the electrical and mechanical interactions which rule the pore-formation mechanism of such peptides.

General significance

These results point to a novel paradigm in which electrical and mechanical effects promoted by chemicals which preferentially alter the electrostatics of the membrane, may be employed to help distinguish among various pore-formation mechanisms of membrane-permeabilizing peptides.     

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.