Olga S. Ostroumova

Surfactin activity depends on the membrane dipole potential.

The effect of dipole modifying agents phloretin and RH 421 on the membrane conductance induced by surfactin, a lipopeptide antibiotic from Bacillus subtilis, was studied. Surfactin added on both sides of a bilayer formed from diphytanoylphosphocholine in 1 M KCl (pH 6.5) leads to the formation of voltage-independent channels of different conductance levels. The conductance of different states of SA channels varies from tens of picosiemens for small pores up to tens of nanosiemens for large ones. Small channels demonstrate pronounced cationic selectivity, whereas large ones practically lose their K(+)/Cl(-) selectivity, most probably because of their large effective radii. The addition of phloretin to the bilayer bathing solution, the agent known to decrease the membrane dipole potential, results in a decrease in the surfactin-induced membrane conductance. At the same time, increasing the membrane dipole potential because of the introduction of RH 421 leads to a rise in the steady-state conductance. Increasing dipole potential is accompanied by increases in both the number of open channels and their conductance. The observed changes in the channel-forming activity of surfactin might be caused by varying the partition coefficient of lipopeptide between the lipid and aqueous phases.

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.

Ion channel activity of brain abundant protein BASP1 in planar lipid bilayers

BASP1 (also known as CAP‐23 and NAP‐22) is a brain abundant myristoylated protein localized at the inner surface of the presynaptic plasma membrane. Emerging evidence suggests that BASP1 is critically involved in various cellular processes, in particular, in the accumulation of phosphatidylinositol‐4,5‐diphosphate (PIP2) in lipid raft microdomains. We have recently shown that BASP1 forms heterogeneously‐sized oligomers and higher aggregates with an outward similarity to oligomers and protofibrils of amyloid proteins. However, BASP1 is not known to be related to any amyloid disease. In the present study, we show that BASP1 induces single channel currents across negatively‐charged planar lipid bilayers (containing phosphatidylserine or PIP2) bathed in 0.1–0.2 m KCl (pH 7.5). By their characteristics, BASP1 channels are similar to amyloid protein channels. BASP1 channels exhibit multiple conductance levels, in the range 10–3000 pS, with the most frequently observed conductance state of approximately 50 pS. The channels demonstrate a linear current–voltage relationship and voltage‐independent kinetics of opening and closing. Their K+ to Cl− permeability ratio is approximately 14, indicating that BASP1 channels are cation‐selective. The ion channel activity of BASP1 is in accordance with the pore‐like structure of BASP1 oligomers observed by electron microscopy on a lipid monolayer. Neuronal protein GAP‐43, which is functionally related to BASP1 and also forms oligomers, elicited no ion channel currents under the conditions used in the present study. Elucidation of the physiological or pathological roles of ion channel activity of membrane‐bound BASP1 oligomers will help to define the precise mechanism of amyloid protein toxicity.

Asymmetry of syringomycin E channel studied by polymer partitioning.

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of ∼1 nm. Now, motivated by the asymmetric non‐ohmic current–voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one‐sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis‐side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans‐side. We interpret our results to suggest that the water‐filled pore of the channel is conical with cis‐ and trans‐radii differing by a factor of 2–3 and that the smaller cis‐radius is in the 0.25–0.35 nm range. In symmetric, two‐sided addition, polymers entering the pore from the larger opening dominate blockage.

Effect of dipole modifiers on the magnitude of the dipole potential of sterol-containing bilayers

The effects of various subclasses of flavonoids, Rose Bengal, and different styrylpyridinium dyes on the magnitude of the dipole potential of membranes composed of pure phospholipids and sterol-containing bilayers were investigated. Changes in the steady-state membrane conductance induced by cation-ionophore complexes were measured to examine the changes in the dipole potential of lipid bilayers. The characteristic parameters of the Langmuir adsorption isotherm for different flavonoids and Rose Bengal and the slope of the linear dependence of the dipole potential change on the aqueous concentrations of RH dyes were estimated. Chalcones (phloretin and phloridzin) and flavonols (quercetin and myricetin) strictly decrease the dipole potential of phospholipid- and sterol-containing membranes; the unsaturation of the C-ring and the hydrophobicity of the molecule contribute to the ability of the flavonoid to reduce the bilayer dipole potential. Rose Bengal decreases the magnitude of the bilayer dipole potential to a similar extent, but its affinity for membrane lipids is higher; the effects of RH dyes, chalcones, and phloroglucinol are determined by sterol concentration and type.

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.

Channel-forming activity of cecropins in lipid bilayers: effect of agents modifying the membrane dipole potential.

Cecropin A (CecA) and cecropin B (CecB) added to one side of a bilayer formed from equimolar mixtures of DOPS and DOPE, DPhPS and DPhPE, or DOPS, DOPE, and Chol leads to the formation of well-defined and well-reproducible ion channels of different conductance levels while cecropin P1 (CecP1) does not induce pore formation at micromolar concentrations. We found three populations of CecA channels: pores with weak cationic selectivity, pores with weak anionic selectivity, and pores that were nonselective. The dipole modifiers, flavonoids and styryl dyes, were used to modulate the channel-forming activity of CecA and CecB. The mean conductance of single CecA channels is affected by the influence of dipole modifiers on the lipid packing in the membrane. A decrease in the membrane dipole potential is accompanied by a decrease in the steady-state transmembrane current induced by CecA and CecB in cholesterol-free and cholesterol-containing bilayers. The observed changes in the channel-forming activity might be caused by an increase in the energy barrier for the interfacial accumulation of cecropin monomers. This finding indicates that the negative pole of the cecropin dipole is inserted into the membrane.

Altering the activity of syringomycin E via the membrane dipole potential.

The membrane dipole potential is responsible for the modulation of numerous biological processes. It was previously shown (Ostroumova, O. S.; Kaulin, Y. A.; Gurnev, P. A.; Schagina, L. V. Langmuir 2007, 23, 6889-6892) that variations in the dipole potential lead to changes in the channel properties of the antifungal lipodepsipeptide syringomycin E (SRE). Here, data are presented demonstrating the effect of the membrane dipole potential on the channel-forming activity of SRE. A rise in the dipole potential is accompanied by both an increase in the minimum SRE concentration required for the detection of single channels at fixed voltage and a decrease in the steady-state number of open SRE channels at a given SRE concentration and voltage. These alterations are determined by several factors: gating charge, connected with translocations of lipid and SRE dipoles during channel formation, the bilayer-water solution partitioning of SRE, and the chemical work related to conformational changes during channel formation.

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 flavonoids on the phase separation in giant unilamellar vesicles formed from binary lipid mixtures

Confocal fluorescence microscopy have been employed to investigate phase separation in giant unilamellar vesicles prepared from binary mixtures of unsaturated dioleoylphosphocholine with saturated phosphocholines or brain sphingomyelin in the absence and presence of the flavonoids, biochanin A, phloretin, and myricetin. It has been demonstrated that biochanin A and phloretin make uncolored domains more circular or eliminate visible phase separation in liposomes while myricetin remains the irregular shape of fluorescence probe-excluding domains. Influence of the flavonoids on the endotherms of liposome suspension composed of dioleoylphosphocholine and dimyristoylphosphocholine was investigated by the differential scanning calorimetry. Calorimetry data do not contradict to confocal imaging results.