Diffusion of nystatin in plasma membrane is inhibited by a glass-membrane seal.

In perforated patch recording, the pore former nystatin is incorporated into a cell-attached patch, to increase its conductance. The possibility of lateral diffusion of nystatin through the membrane and under the glass-membrane seal was examined by reversing the nystatin gradient. Namely, a cell-attached patch on a cell was examined while placing nystatin into the bath. The reversal potential and current-voltage relationship of single Ca2+ activated K+ channels in the patch were readily changed by varying the K+ concentration in the bath, showing that nystatin was active in the cell membrane outside of the patch. However, the patch itself did not become leaky. The absence of a conductance induced in the patch by the nystatin in the rest of the plasma membrane of the cell suggests that the lateral diffusion of nystatin is inhibited by the glass-membrane seal.     

Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules.

We have systematically investigated the probe size and shape dependence of lateral diffusion in model dimyristoyl phosphatidylcholine membranes. Linear hydrophobic polymers, which differ in length by an order of magnitude, were used to explore the effect on the lateral diffusion coefficient of hydrodynamic restrictions in the bilayer interior. The polymers employed are isoprenoid alcohols--citronellol, solanesol, and dolichol. Tracer lateral diffusion coefficients were measured by fluorescence photobleaching recovery. Despite the large difference in lengths, the nitrobenzoxadiazole labelled alcohols all diffuse at the rate of lipid self-diffusion (5.0 x 10(-12) m2 s-1, 29 degrees C) in the liquid crystal phase. Companion measurements in isotropic polymer solution, in gel phase lipid membranes and with nonpolar fluorescent polyaromatic hydrocarbons, show a marked dependence of the lateral diffusion coefficient on the probe molecule size. Our results in the liquid crystal phase are in accord with free area theory which asserts that lateral diffusion in the membrane is restricted by the surface-free area. Probe molecules which are significantly longer than the host phospholipid, seven times longer in the case of dolichol, are still restricted in their lateral motion by the surface properties of the bilayer in the liquid crystal phase. Fluorescence quenching experiments indicate that the nitrobenzoxadiazole label does not reside at the aqueous interface, although it must reside in close proximity according to the diffusion measurements.     

Permeability and Electrical Properties of Thin Lipid Membranes

We present and discuss the permeability and electrical properties of thin lipid membranes, and the changes induced in these properties by several agents added to the aqueous phases after the membranes have formed. The unmodified membrane is virtually impermeable to ions and small "hydrophilic" solutes, but relatively permeable to water and "lipophilic" molecules. These properties are consistent with those predicted for a thin film of hydrocarbon through which matter is transported by dissolving in the membrane phase and then diffusing through it. The effect of cholesterol in reducing the water and "lipophilic" solute permeability is attributed to an increase of the "viscosity" of the hydrocarbon region, thus reducing the diffusion coefficient of molecules within this phase. The selective permeability of the membrane to iodide (I^-) in the presence of iodine (I₂) is attributed to the formation of polyiodides (perhaps I₅ ^-), which are presumed to be relatively soluble in the membrane because of their large size, and hence lower surface charge density. Thus, I₂ acts as a carrier for I^-. The effects of "excitability-inducing material" and the depsipeptides (particularly valinomycin) on ion permeability are reviewed. The effects of the polyene antibiotics (nystatin and amphotericin B) on ion permeability, discussed in greater detail, are the following: (a) membrane conductance increases with the 10th power of nystatin concentration; (b) the membrane is anion-selective but does not discriminate completely between anions and cations; (c) the membrane discriminates among anions on the basis of size; (d) membrane conductance decreases extraordinarily with increasing temperatures. Valinomycin and nystatin form independent conductance pathways in the same membrane, and, in the presence of both, the membrane can be reversibly shifted between a cation and anion permeable state by changes in temperature. It is suggested that nystatin produces pores in the membrane and valinomycin acts as a carrier.     

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

Energy metabolism in wheat root cells under modification of plasma membrane permeability by antibiotic nystatin

This paper reports changes in ion transport and energy metabolism of plant cells during short- and long-term expositions, resp., to antibiotic nystatin, which is known to specifically bind with plasma membrane sterols to form channels. The excised roots of 5 days old wheat seedlings were used as a model system in this research. It has been shown that treatment of excised roots with nystatin leads to activation of energy metabolism expressed as an increase of respiration and heat production by root cells. Furthermore, in the presence of nystatin increased pH of incubation medium, plasma membrane depolarization and a significant loss of potassium ions were observed. Nystatin-induced stimulation of respiration was prevented by malonate, an inhibitor of succinate dehydrogenase, electron acceptor dichlorophenolindophenol, and AgNO3, an inhibitor of H(+)-ATPase. Based on the data obtained it can be suggested that nystatin-induced stimulation of respiration is related to electron transport activation via mitochondrial respiratory chain, and is connected with activation of plasmalemma proton pump. Moreover, nystatin-induced increase of oxygen consumption was prevented by cerulenin, an inhibitor of fatty acid and sterol synthesis. This indicates that additional sterols and phospholipids may be synthesized in root cells to "heal" nystatin-caused damage of plasma membrane. A supposed chain of events of cell response to nystatin action may by as following: formation of nystatin channels-influx of protons--depolarization of plasmalemma-efflux of potassium ions-disturbance of ion homeostasis--activation of H(+)-ATPase work-increase in energy "requests" for H(+)-ATPase function--increase in the rate of oxygen consumption and heat production. The increased energy production under the action of nystatin, may provide the work of proton pump and synthesis of sterols and phospholipids, which are necessary for membrane regeneration.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

Study of the association of nystatin and amphotericin B in nonaqueous solvent systems]

Association of nystatin and amphotericin B in non-aqueous systems was studied with the method of equilibria dialysis. A specially treated celophane membrane arresting colloid associats and macromolecules with a molecular weight of more than 30000 in the systems of dimethylformamide-ethylacetate was used for the dialysis. Relation between the dialysis rate and the difference of the concentrations at every side of the membrane was used for estimation of the antibiotic colloid association level. It was found that nystatin formed stable associates of the colloid type in the system of dimethylformamide-ethylacetate, close by the composition to the critical one or that providing precipitation of the antibiotic. Unlike nystatin, amphotericin B formed not colloid but larger conglomerates which precipitated. Neither of the antibiotics formed colloid associates in dimethylformamide. The level of the nystatin colloid association increased with a rise in the solution concentration and reached 80%. On the basis of the results obtained the following supposition concerning the mechanism of formation of the antibiotic complexes with polyvinylpyrrolidone (PVP) in non-aqueous systems is possible: sorption of PVP on the colloids formed or larger associates of the polyenic antibiotics must take place during coprecipitation which is accompanied by formation of a precipitate of the sorption complex.     

Cholesterol and ergosterol influence nystatin surface aggregation: relation to pore formation

Nystatin interaction with liposomes mimicking fungal and mammalian membranes (ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles, respectively) was studied by fluorescence spectroscopy. The activity of this antibiotic was also measured using a pyranine fluorescence detected K+/H+ exchange assay. Nystatin mean fluorescence lifetime varied with the antibiotic concentration and ergosterol content (0-30 mol%) of the lipid vesicles. It sharply increased from 5 to 37 ns upon reaching 100 molecules per liposome, reporting nystatin oligomerization in the membrane. Concomitantly, spectral alterations typical of excitonic coupling were detected and there was a pronounced increase in the initial rate of pore formation by nystatin. These findings suggest that nystatin exerts its antibiotic activity via a two-stage mechanism: at low antibiotic concentrations, surface-adsorbed monomeric antibiotic molecules perturb the lipid packing, changing the permeability properties of the ergosterol-rich liposomes. Upon reaching a critical threshold, nystatin mode of action switches to the classical model of transmembrane aqueous channel formation. In the presence of cholesterol-containing POPC liposomes, neither nystatin spectroscopic properties, nor the kinetics of K+ efflux varied with the antibiotic concentration suggesting that in this case the first stage of antibiotic mode of action always prevails or the assemblies formed by nystatin and cholesterol are very loose.     

Effects of sterol-binding agent nystatin on wheat roots: the changes in membrane permeability, sterols and glycoceramides

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion.     

An ionometric study of Na+ and K+ fluxes across the nystatin-modified human erythrocyte membrane]

The application of ion-selective electrodes is discussed for the kinetic determination of K+ and Na+ concentrations in the system, containing human red blood cells modified by nystatin. A series of mixed solutions was worked out, according to which the Na(+)-glass and the K(+)-thick membrane valinomycin electrodes were calibrated. The human erythrocytes were washed for 3 times with the basic solution (in mol per liter: 0.141 NaCl, 0.004 KCl, 0.002 CaCl2, 0.003 MgCl2, 0.01 glucose), and then were resuspended in it. The suspension was kept in a shaking bath at 37 degrees C. The modification of the cell membranes was performed by the introduction of different amounts of the antibiotic nystatin into the probe. Under these conditions the concentration of Na+ decreased, while K+ concentration increased. The values of concentration were registered ionometrically. In an hour and a half the stationary lines were obtained. Being based on the values of the stationary cation concentrations and the final concentrations, registered after the complete lysis of erythrocytes promoted by saponin, the ratio of cation fluxes across the modified membrane to the flux across the nonmodified membrane was calculated in accordance with the Hodgkin-Katz equation.     

Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranes

Nystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the present study, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free and ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). The mean tetraene to NBD separating distance determined from fluorescence energy transfer measurements increased from 18 to 25.6 A upon antibiotic binding to the lipid vesicles, indicating that the monomeric labelled antibiotic adopts a more extended conformation in its lipid-bound state than in aqueous solution. The oligomeric state of membrane-bound NBD-Nys was also studied by resonance energy homotransfer between the NBD fluorophores. The decrease measured in its steady state fluorescence anisotropy upon increasing the surface concentration of the NBD-Nys is shown to be consistent with a random distribution of molecules on the surface of the liposomes. This data contradicts the sharp increase measured for nystatin mean fluorescence lifetime in the presence of 10 mol% ergosterol-containing POPC LUV within the same antibiotic concentration range and which is known to report nystatin oligomerization in the lipid vesicles. Therefore, we conclude that the amine group of nystatin is an essential requisite for the supramolecular organization/pore formation of this antibiotic.     

Evidence that nystatin channels form at the boundaries, not the interiors of lipid domains

Nystatin (nys) is an antifungal agent that preferentially forms ion channels in membranes containing the sterol, ergosterol (erg). The structure of the nystatin channel is not clear, but it is known that multiple nystatin monomers must aggregate to form channels in a sterol-rich membrane. When nys/erg containing vesicles are fused to a sterol-free bilayer, characteristic spikelike changes in membrane conductance are observed. An abrupt increase in conductance is followed by a decay that is generally stepwise linear and the decay time depends strongly on [erg]. These data are inconsistent with the hypothesis that nys channels form uniformly throughout the membrane and decay independently (which would produce exponential decay). We propose that channels are located at the boundaries of lipid superlattices such that diffusion of erg out of the lattice results in correlated channel decay. This was tested using a statistical mechanical analysis and Monte Carlo simulations, which reveal details of the diffusion process and provide insight into conditions at superlattice boundaries during decay. This analysis predicts the linear decay schemes and the dramatic drop in channel decay time observed at erg mol % = 50. This interpretation also explains puzzling data relating conductance spike height to vesicle diameter.     

Nystatin as a probe for investigating the electrical properties of a tight epithelium

We show how the antibiotic nystatin may be used in conjunction with microelectrodes to resolve transepithelial conductance Gt into its components: Ga, apical membrane conductance; Gbl, basolateral membrane conductance; and Gj, junctional conductance. Mucosal addition of nystatin to rabbit urinary bladder in Na+-containing solutions caused Gt to increase severalfold to ca. 460 micrometerho/muF, and caused the transepithelial voltage Vt to approach +50 mV regardless of its initial value. From measurements of Gt and the voltage-divider ratio as a function of time after addition or removal of nystatin, values for Ga, Gbl, and Gj of untreated bladder could be obtained. Nystatin proved to have no direct effect on Gbl or Gj but to increase Ga by about two orders of magnitude, so that the basolateral membrane then provided almost all of the electrical resistance in the transcellular pathway. The nystatin channel in the apical membrane was more permeable to cations than to anions. The dose-response curve for nystatin had a slope of 4.6. Use of nystatin permitted assessment of whether microelectrode impalement introduced a significant shunt conductance into the untreated apical membrane, with the conclusion that such a shunt was negligible in the present experiments. Nystatin caused a hyperpolarization of the basolateral membrane potential in Na+- containing solutions. This may indicate that the Na+ pump in this membrane is electrogenic.     

The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin

The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membrane’s molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membrane's expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin.     

Clustering of the B cell receptor is not required for the apoptotic response

We examined the role of BCR cell membrane redistribution in anti-IgM-induced apoptosis in three human B cell lines, RA#1, 2G6, and MC116, that differ in their relative levels of sIgM expression. The apoptotic response was found to be dependent on the nature of the anti-IgM and the cell line. In the cell lines, RA#1 and MC116, sIgM aggregated into patches that were insensitive to the disruption of cholesterol-rich membrane microdomains by nystatin or beta-MCD. The B cell line 2G6 was able to reorganize sIgM into a tight coalescent cap upon anti-IgM treatment. However, in this case, the lipid raft inhibitors nystatin and beta-MCD disrupted the patching. In 2G6 cells, BCR-mediated apoptosis was not affected by nystatin treatment, whereas it increased in beta-MCD pretreated cells. Thus, no evident correlation was found between apoptosis and BCR cell membrane redistribution or lipid raft formation in either of the three cell lines. The data indicate that the apoptotic signal transduction pathway is independent of BCR translocation into lipid rafts and/or aggregation.     

In vivo staining of cytoskeletal actin by autointernalization of nontoxic concentrations of nitrobenzoxadiazole-phallacidin

The blue light-excited fluorescent phallotoxin derivative nitrobenzoxadiazole phallacidin (NBD-Ph) was used to stain entire tissue culture monolayers of live L6 mouse cells and other mammalian cell lines without the aid of permeabilization treatment. Although cells tend to exclude the fluorescent toxin, reducing the internal concentration by approximately 1,000 times, some of it enters the cells, probably by pinocytosis, and stains actin structures at low intracellular NBD-Ph concentrations (approximately 5-15 nM), where cell toxicity was negligible or at least not detectable by phase-contrast microscopy. Protracted treatments with NBD-Ph did induce pharmacological responses similar to those of phalloidin. The dissociation constant for NBD-Ph with F-actin in fixed and extracted L6 cells was determined, from staining intensity measurements at various NBD-Ph concentrations, to be 1.5-2.5 x 10(-8) M.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Cooperative partition model of nystatin interaction with phospholipid vesicles

Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, . The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in nystatin mean fluorescence lifetime from approximately 7-10 to 35 ns, begins to occur at a critical concentration of 10 nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric nystatin is anchored at the phospholipid/water interface and suggest that nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6 +/- 2 antibiotic molecules.     

Effects of the pore-forming agent nystatin on giant phospholipid vesicles

The effects of the polyene pore-forming agent nystatin were investigated on individual giant unilamellar phospholipid vesicles (GUVs), made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in different methanol-water solutions using phase-contrast optical microscopy. Three characteristic effects were detected in three different nystatin concentration ranges: vesicle shape changes (between 150 and 250μM); transient, nonspecific, tension pores (between 250 and 400μM); and vesicle ruptures (above 400μM). Both the appearance of the transient tension pores and the vesicle ruptures were explained as being a consequence of the formation of size-selective nystatin channels, whose membrane area density increases with the increasing nystatin concentrations. Our results also show that nystatin is able to form pores in the absence of sterols. In addition, study of the cross-interactions between nystatin and methanol revealed mutually antagonizing effects on the vesicle behavior for methanol volume fractions higher than 10%.