Amphotericin B channels in phospholipid membrane-coated nanoporous silicon surfaces: implications for photovoltaic driving of ions across membranes

The antimycotic agent amphotericin B (AmB) functions by forming complexes with sterols to form ion channels that cause membrane leakage. When AmB and cholesterol mixed at 2:1 ratio were incorporated into phospholipid bilayer membranes formed on the tip of patch pipettes, ion channel current fluctuations with characteristic open and closed states were observed. These channels were also functional in phospholipid membranes formed on nanoporous silicon surfaces. Electrophysiological studies of AmB-cholesterol mixtures that were incorporated into phospholipid membranes formed on the surface of nanoporous (6.5 nm pore diameter) silicon plates revealed large conductance ion channels ( approximately 300 pS) with distinct open and closed states. Currents through the AmB-cholesterol channels on nanoporous silicon surfaces can be driven by voltage applied via conventional electrical circuits or by photovoltaic electrical potential entirely generated when the nanoporous silicon surface is illuminated with a narrow laser beam. Electrical recordings made during laser illumination of AmB-cholesterol containing membrane-coated nanoporous silicon surfaces revealed very large conductance ion channels with distinct open and closed states. Our findings indicate that nanoporous silicon surfaces can serve as mediums for ion-channel-based biosensors. The photovoltaic properties of nanoporous silicon surfaces show great promise for making such biosensors addressable via optical technologies.     

Effect of aggregation of amphotericin B on lysophosphatidylcholine micelles as related to its complex formation with cholesterol or ergosterol

The effect of aggregation of amphotericin B (AmB), as well as the complex formation of AmB with cholesterol or ergosterol, was investigated in micelles and vesicles. AmB in lysophosphatidylcholine (LPC) micelles adopted a more favorable monomeric form than that in other drug formulations. At an LPC/AmB ratio of 200, AmB existed only in monomeric form. Such monomeric behavior is likely dependent upon the fluidity and size of the micelles. In LPC micelles composed of 90% monomeric AmB, AmB-ergosterol complex formation occurred with an increase in the sterol concentration, but the complex formation of AmB-cholesterol was slight. On the other hand, in LPC micelles composed of 40% monomeric AmB, the complex formation of AmB-cholesterol as well as AmB-ergosterol was extensive. These results suggest that the complex formation of AmB with both sterols is highly dependent upon the aggregated state of AmB. In addition, using monolayers, mixtures of AmB/LPC/ergosterol were became more stable with rising temperature, while the stability of mixtures of AmB/LPC/cholesterol remained unchanged, implying that complex formation of AmB with cholesterol is different from that of AmB with ergosterol.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Formation of Two Different Types of Ion Channels by Amphotericin B in Human Erythrocyte Membranes

The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (≤1.0 × 10⁻⁶ M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 × 10⁻⁶ M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K⁺ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft–associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.     

Long Open Amphotericin Channels Revealed in Cholesterol-Containing Phospholipid Membranes Are Blocked by Thiazole Derivative

The action of antifungal drug, amphotericin B (AmB), on solvent-containing planar lipid bilayers made of sterols (cholesterol, ergosterol) and synthetic C14–C18 tail phospholipids (PCs) or egg PC has been investigated in a voltage-clamp mode. Within the range of PCs tested, a similar increase was achieved in the lifetime of one-sided AmB channels in cholesterol- and ergosterol-containing membranes with the C16 tail PC, DPhPC at sterol/DPhPC molar ratio ≤1. The AmB channel lifetimes decreased only at sterol/DPhPC molar ratio >1 that occurred with sterol/PC molar ratio of target cell membranes at a pathological state. These data obtained on bilayer membranes two times thicker than one-sided AmB channel length are consistent with the accepted AmB pore-forming mechanism, which is associated with membrane thinning around AmB–sterol complex in the lipid rafts. Our results show that AmB can create cytotoxic (long open) channels in cholesterol membrane with C14–C16 tail PCs and nontoxic (short open) channels with C17–C18 tail PCs as the lifetime of one-sided AmB channel depends on ~2–5 Å difference in the thickness of sterol-containing C16 and C18 tail PC membranes. The reduction in toxic AmB channels efficacy can be required at the drug administration because C16 tails in native membrane PCs occur almost as often as C18 tails. The comparative analysis of AmB channel blocking by tetraethylammonium chloride, tetramethylammonium chloride and thiazole derivative of vitamin B₁, 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl) thiazole chloride (DMHT), has proved that DMHT is a comparable substitute for both tetraalkylammonia that exhibits a much higher affinity.     

Ion channel behavior of amphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes investigated by electrochemistry and spectroscopy

Amphotericin B (AmB) is a popular drug frequently applied in the treatment of systemic fungal infections. In the presence of ruthenium (II) as the maker ion, the behavior of AmB to form ion channels in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes were studied by cyclic votammetry, AC impedance spectroscopy, and UV/visible absorbance spectroscopy. Different concentrations of AmB ranging from a molecularly dispersed to a highly aggregated state of the drug were investigated. In a fixed cholesterol or ergosterol content (5 mol %) in glassy carbon electrode-supported model membranes, our results showed that no matter what form of AmB, monomeric or aggregated, AmB could form ion channels in supported ergosterol-containing phosphatidylcholine bilayer model membranes. However, AmB could not form ion channels in its monomeric form in sterol-free and cholesterol-containing supported model membranes. On the one hand, when AmB is present as an aggregated state, it can form ion channels in cholesterol-containing supported model membranes; on the other hand, only when AmB is present as a relatively highly aggregated state can it form ion channels in sterol-free supported phosphatidylcholine bilayer model membranes. The results showed that the state of AmB played an important role in forming ion channels in sterol-free and cholesterol-containing supported phosphatidylcholine bilayer model membranes.     

Amphotericin B Membrane Action: Role for Two Types of Ion Channels in Eliciting Cell Survival and Lethal Effects

The formation of aqueous pores by the polyene antibiotic amphotericin B (AmB) is at the basis of its fungicidal and leishmanicidal action. However, other types of nonlethal and dose-dependent biphasic effects that have been associated with the AmB action in different cells, including a variety of survival responses, are difficult to reconcile with the formation of a unique type of ion channel by the antibiotic. In this respect, there is increasing evidence indicating that AmB forms nonaqueous (cation-selective) channels at concentrations below the threshold at which aqueous pores are formed. The main foci of this review will be (1) to provide a summary of the evidence supporting the formation of cation-selective ion channels and aqueous pores by AmB in lipid membrane models and in the membranes of eukaryotic cells; (2) to discuss the influence of membrane parameters such as thickness fluctuations, the type of sterol present and the existence of sterol-rich specialized lipid raft microdomains in the formation process of such channels; and (3) to develop a cell model that serves as a framework for understanding how the intracellular K(+) and Na(+) concentration changes induced by the cation-selective AmB channels enhance multiple survival response pathways before they are overcome by the more sustained ion fluxes, Ca(2+)-dependent apoptotic events and cell lysis effects that are associated with the formation of AmB aqueous pores.     

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drug's binding to phospholipid bilayers. Potassium ion flux assays based on 31P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.     

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drug's binding to phospholipid bilayers. Potassium ion flux assays based on ³¹P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.     

Potassium-selective amphotericin B channels are predominant in vesicles regardless of sidedness.

Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Channels Formed by Amphotericin B Covalent Dimers Exhibit Rectification

Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification.     

Binding of antibiotic amphotericin B to lipid membranes: monomolecular layer technique and linear dichroism-FTIR studies

Amphotericin B (AmB) is one of the main antibiotics applied in treatment of deep-seated mycotic infections. Tensiometric technique has been applied to monitor binding of AmB, from the water subphase, to the lipid monomolecular layers, formed with dipalmitoylphosphatidylcholine at the air-water interface. Time dependencies of surface pressure in the monolayers demonstrate strong enhancement of AmB binding to monolayers brought about by sterols present in the membranes. The monolayers have been deposited to a solid support and examined by means of FTIR spectroscopy. FTIR measurements show that majority of the AmB molecules which bind to the membranes are localized in the polar headgroup region. The results of the linear dichroism-FTIR measurements are consistent with the microscopic picture according to which the molecules of the membrane-bound AmB are distributed among two orientational fractions: one horizontal and one vertical with respect to the plane of the membrane (59% versus 41% respectively, in the case of the membrane formed with the pure lipid without sterols). The presence of cholesterol in the membranes (50 mol% with respect to lipid) slightly affects such a distribution (53% horizontal versus 47% vertical) but the presence of ergosterol has a pronounced effect in the increase in population of the fraction of horizontally bound AmB (85% horizontal vs. 15% vertical). The results of the measurements indicate that mode of action of the AmB consists in disruption of the polar headgroup region of biomembranes, brought about by the AmB molecules bound horizontally with respect to the plane of the membrane.     

Binding of antibiotic amphotericin B to lipid membranes: monomolecular layer technique and linear dichroism-FTIR studies

Amphotericin B (AmB) is one of the main antibiotics applied in treatment of deep-seated mycotic infections. Tensiometric technique has been applied to monitor binding of AmB, from the water subphase, to the lipid monomolecular layers, formed with dipalmitoylphosphatidylcholine at the air-water interface. Time dependencies of surface pressure in the monolayers demonstrate strong enhancement of AmB binding to monolayers brought about by sterols present in the membranes. The monolayers have been deposited to a solid support and examined by means of FTIR spectroscopy. FTIR measurements show that majority of the AmB molecules which bind to the membranes are localized in the polar headgroup region. The results of the linear dichroism-FTIR measurements are consistent with the microscopic picture according to which the molecules of the membrane-bound AmB are distributed among two orientational fractions: one horizontal and one vertical with respect to the plane of the membrane (59% versus 41% respectively, in the case of the membrane formed with the pure lipid without sterols). The presence of cholesterol in the membranes (50 mol% with respect to lipid) slightly affects such a distribution (53% horizontal versus 47% vertical) but the presence of ergosterol has a pronounced effect in the increase in population of the fraction of horizontally bound AmB (85% horizontal vs. 15% vertical). The results of the measurements indicate that mode of action of the AmB consists in disruption of the polar headgroup region of biomembranes, brought about by the AmB molecules bound horizontally with respect to the plane of the membrane.     

Molecular aspects of the interaction between amphotericin B and a phospholipid bilayer: molecular dynamics studies

Amphotericin B (AmB) is a polyene macrolide antibiotic used to treat systemic fungal infections. The molecular mechanism of AmB action is still only partly characterized. AmB interacts with cell-membrane components and forms membrane channels that eventually lead to cell death. The interaction between AmB and the membrane surface can be regarded as the first (presumably crucial) step on the way to channel formation. In this study molecular dynamics simulations were performed for an AmB–lipid bilayer model in order to characterize the molecular aspects of AmB–membrane interactions. The system studied contained a box of 200 dimyristoylphosphatidylcholine (DMPC) molecules, a single AmB molecule placed on the surface of the lipid bilayer and 8,065 water molecules. Two molecular dynamics simulations (NVT ensemble), each lasting 1 ns, were performed for the model studied. Two different programs, CHARMM and NAMD2, were used in order to test simulation conditions. The analysis of MD trajectories brought interesting information concerning interactions between polar groups of AmB and both DMPC and water molecules. Our studies show that AmB preferentially took a vertical position, perpendicular to the membrane surface, with no propensity to enter the membrane. Our finding may suggest that a single AmB molecule entering the membrane is very unlikely.Figure The figure presents the whole structure of the system simulated—starting point. AmB is presented as a space-filling model, DMPC molecules—green sticks, water molecules—red sticks     

Ionic channels and nerve membrane constituents. Tetrodotoxin-like interaction of saxitoxin with cholesterol monolayers

Saxitoxin (STX) and tetrodotoxin (TTX) have the same striking property of blocking the Na⁺ channels in the axolemma. Experiments with nerve plasma membrane components of the squid Dosidicus gigas have shown that TTX interacts with cholesterol monolayers. Similar experiments were carried out with STX. The effect of STX on the surface pressure-area diagrams of lipid monolayers and on the fluorescence emission spectra of sonicated nerve membranes was studied. The results indicate a TTX-like interaction of STX with cholesterol monolayers. The expansion of the monolayers caused by 10^-6 M STX was 2.2 A²/cholesterol molecule at 25°C. From surface pressure measurements at constant cholesterol area (39 A²/molecule) in media with various STX concentrations, it was calculated that the STX/cholesterol surface concentration ratio is 0.54. The apparent dissociation constant of the STX-cholesterol monolayer complex is 4.0 x 10^-7 M. The STX/cholesterol ratio and the apparent dissociation constant are similar to those determined for TTX. The presence of other lipids in the monolayers affects the STX-cholesterol association. The interactions of STX and TTX with cholesterol monolayers suggest (a) that cholesterol molecules may be part of the nerve membrane Na⁺ channels, or (b) that the toxin receptor at the nerve membrane shares similar chemical features with the cholesterol monolayers.     

Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance

Amphotericin B (AmB) is a membrane-active antibiotic that increases the permeability of fungal membranes. Thus, the dynamic process of its interaction with membranes poses intriguing questions, which prompted us to elaborate a quick and reliable method for real-time observation of the drug's binding to phospholipid liposomes. We focused on surface plasmon resonance (SPR) and devised a new modification method of sensor chips, which led to a significant reduction in the level of nonspecific binding of the drug in a control lane. With this method in hand, we examined the affinity of AmB for various membrane preparations. As expected, AmB exhibited much higher affinity for sterol-containing palmitoyloleoylphosphatidylcholine membranes than those without sterol. The sensorgrams recorded under various conditions partly fitted theoretical curves, which were based on three interaction models. Among those, a two-state reaction model reproduced well the sensorgram of AmB binding to an ergosterol-containing membrane; in this model, two states of membrane-bound complexes, AB and AB*, are assumed, which correspond to a simple binding to the surface of the membrane (AB) and formation of another assembly in the membrane (AB*) such as an ion channel complex. Kinetic analysis demonstrated that the association constant in ergosterol-containing POPC liposomes is larger by 1 order of magnitude than that in the cholesterol-containing counterpart. These findings support the previous notion that ergosterol stabilizes the membrane-bound assembly of AmB.     

FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol

Langmuir monolayers of amphotericin B (AmB) were investigated by recording π-A isotherms under different pH conditions. To gain a better insight into antibiotic-membrane interactions they were monitored by use of the ATR-FTIR spectroscopy. It was observed for AmB monolayers that the limiting molecular area was larger at high than at neutral pH. Analysis of FTIR spectra at different pH revealed substantial differences, depending on ionic state, for different orientations of AmB molecules. These results enable better understanding of the participation of functional groups in the interactions between AmB and sterol-containing DPPC membranes. AmB molecules incorporated into two-component lipid monolayers bind strongly to the ergosterol-rich membrane (maximum penetration surface pressures ca 35?mN/m). The FTIR spectra revealed that the ionic state of AmB and the presence of sterols led to changes in membrane fluidity and molecular packing of the AmB molecules in the lipid membranes. These investigations should be further investigated to discover the molecular mechanism responsible for the mode of action AmB in biological systems.     

Molecular organization of antibiotic amphotericin B in dipalmitoylphosphatidylcholine monolayers induced by K(+) and Na(+) ions: the Langmuir technique study

The effect of potassium (K(+)) and sodium (Na(+)) ions on the self-association of antibiotic amphotericin B (AmB) in the lipid membrane was reported. Mixed Langmuir monolayers of AmB and dipalmitoylphosphatidylcholine (DPPC) were investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of K(+) and Na(+) ions. The analyses of the π-A isotherms and compressional modulus curves indicate the interactions in the AmB-DPPC system. The strength of the AmB-DPPC interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The obtained results proved a high affinity of AmB towards lipids induced by the presence of K(+) than Na(+) ions. The most stable monolayers in the presence of K(+) and Na(+) ions were formed by AmB and DPPC with the 1:1 and 2:1 stoichiometry. The understanding of the AmB aggregation processes at the molecular level should contribute to elucidate the mechanisms of action and toxicity of this widely used drug. The presented results are potentially valuable in respect to develop more efficient and less toxic AmB formulations.     

Bis[(benzo-15-crown-5)-15-yl methyl] pimelate forms ion channels in planar lipid bilayer: a novel model ion channel

Some crown ethers translocate cations across the liposomal membrane either by a carrier mechanism or by forming ion channels. We report formation of ion channels in lipid bilayer membranes by bis[(benzo-15-crown-5)-15-yl methyl] pimelate, a crown ether known to form ion inclusion complexes with alkali metal cations. The channels have characteristic long openings lasting several seconds and a low conductance (4 pS in 500 mM KCl and 2.5 pS in 500 mM NaCl). A model of the crown ether channel formed by stacking of four monomers is proposed. A large database of structural information on crown ethers and their ion inclusion complexes as well as large family of crown ethers with a variety of substitutions in the ring are commercially available. Thus the crown ether channel is an attractive model system to study the role of various chemical moieties in ion conduction which may provide deeper insight into understanding the mechanism(s) of selectivity, ion transport, etc. in biological ion channels.     

On the existence of an amphotericin B--sterol complex in lipid vesicles and in propanol-water systems

The amphotericin B (AmB) - ergosterol complex, formed by interaction of the antibiotic with ergosterol-containing phospholipid vesicles, is associated with the lipid bilayer. It has been shown by circular dichroism studies that the AmB-ergosterol complex formed in water-propanol binary mixtures has a similar structure to that observed in phospholipid vesicles. A positive cooperativity is found for the interaction of AmB with ergosterol. The similar AmB-cholesterol complex is much less stable and rearranges rapidly to a different conformation.