The thiazole derivative reduces transmembrane currents via ionic channels formed by alpha-latrotoxin and sea anemone toxin in the bilayer lipid membranes

It was shown that the thiazole derivative 3-decyloxycarbonylmethyl-4-methyl- 5-(2-hydroxyethyl)thiazole chloride (DMHT) (0.1 mM) reversibly reduced the transmembrane current in solutions of 10 mM CaCl2 and 100 mM KCl via ionic channels produced by alpha-latrotoxin from black widow spider (alpha-LT) and sea anemone toxin (RTX) in the bilayer lipid membranes (BLM). Introduction of DMHT from the cis-side of BLM inhibited transmembrane current by 31.6 +/- 3% and by 61.8 +/- 3% from the trans-side of BLM for alpha-LT channels. Application of DMHT to the cis-side BLM decreased the inward current through the RTX channels by 50 +/- 5%. Addition of Cd(2+) (0.1 mM) to the cis- or trans-side of a membrane after the DMHT induced depression of transmembrane current across the alpha-LT channels caused its further decrease by 85 +/- 5% that coincides completely with the intensity of Cd(2+)-inhibition in the control experiments without DMHT. These data suggest that DMHT may exert its inhibitory action on alpha-LT channels without considerable influence on the ionogenic groups inside the channel cavity. The comparative analysis of effective radii measured for alpha-LT and RTX channels on the cis- (0.9 nm and 0.55 nm, respectively) and the trans-side of BLM (< 0.467 nm for alpha-LT) allowed to propose the blocking action of DMHT for alpha-LT and RTX channels to result from direct penetration into the channel, achieved due to similar hydrodynamic size of blocking molecules and the size of toxin pores.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(beta-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl2 and KCl solutions via ionic channels produced by latrotoxins (alpha-latrotoxin (alpha-LT) and alpha-latroinsectotoxin (alpha-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl2 inhibited transmembrane current by 31.6+/-3% and by 61.8+/-3% from the trans-side of BLM for alpha-LT channels. Application of DMHT in the solution of 10 mM CaCl2 to the cis-side of BLM decreased the current through the alpha-LIT and RTX channels by 52+/-4% and 50+/-5%, respectively. Addition of Cd2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca2+-current across the alpha-LT channels caused its further decrease by 85+/-5% that coincides favorably with the intensity of Cd2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on alpha-LT channels without considerable influence on the ionogenic groups of Ca2+-selective site inside the channel cavity. The binding kinetics of DMHT with the alpha-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of alpha-LT channel on either side of the BLM. The comparative analysis of effective radii measured for alpha-LT, alpha-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for alpha-LT channel on the trans-side (0.28+/-0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B₁ (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(β-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl₂ and KCl solutions via ionic channels produced by latrotoxins (α-latrotoxin (α-LT) and α-latroinsectotoxin (α-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl₂ inhibited transmembrane current by 31.6 ± 3% and by 61.8 ± 3% from the trans-side of BLM for α-LT channels. Application of DMHT in the solution of 10 mM CaCl₂ to the cis-side of BLM decreased the current through the α-LIT and RTX channels by 52 ± 4% and 50 ± 5%, respectively. Addition of Cd²⁺ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca²⁺-current across the α-LT channels caused its further decrease by 85 ± 5% that coincides favorably with the intensity of Cd²⁺ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on α-LT channels without considerable influence on the ionogenic groups of Ca²⁺-selective site inside the channel cavity. The binding kinetics of DMHT with the α-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of α-LT channel on either side of the BLM. The comparative analysis of effective radii measured for α-LT, α-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for α-LT channel on the trans-side (0.28 ± 0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

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.     

Effects of thiazole analogs of vitamin B1 on neuromuscular transmission and α-latrotoxin-induced transmitter release in skeletal muscles

Thiazole analogs of vitamin B₁ 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)thiazole chloride (DMHT) and 3-decyloxycarbonylmethyl-4-methylthiazole chloride (DMT) suppress quantum transmitter release from nerve terminals in the frog skeletal muscle. Intraperitoneal administration of these compounds to mice suppresses behavioral motor activity, diminishes motor coordination, and suppresses the corazol-induced seizures. Application of DMHT reduces the -latrotoxin-induced massive transmitter release from nerve terminals in the frog skeletal muscle and suppresses latrotoxin-induced seizures in mice. In model experiments, DMHT blocks Ca²⁺ entry through the ion channels formed by -latrotoxin in a bilayer lipid membrane. It has been suggested that the effectiveness of DMHT and DMT is determined by the presence of a thiazole cycle in their molecules that, among all endogenous biologically active compounds, is possessed only by vitamin B₁ and its metabolites.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 368–374, September–December, 1995.     

Inhibition of lecithin-cholesterol acyltransferase by diphytanoyl phosphatidylcholine

Model high density lipoproteins containing human apolipoprotein A-I, cholesterol, and a variety of phosphatidylcholines (PCs) have been prepared and tested. The PCs included 1-palmitoyl-2-oleoyl PC (POPC) and its diether analog 1-O-hexadecyl-2-oleyl PC (POPC ether), 1,2-diphytanoyl PC (DPhPC), 1-palmitoyl-2-phytanoyl PC, and 1-phytanoyl-2-palmitoyl PC. All ester PCs were good acyl donors for the transesterification of cholesterol catalyzed by human lecithin-cholesterol acyltransferase except DPhPC, which showed no reactivity. The PCs containing one phytanoyl chain donated an acyl chain to cholesterol as fast as non-branched fatty acyl chains. However, the competitive inhibition of lecithin-cholesterol acyltransferase by POPC ether and DPhPC was similar, and both lipids formed a macromolecular matrix that supported the reactivity of other ester PC substrates. The bulk of physicochemical properties of model high density lipoproteins composed of DPhPC were indistinguishable from those of POPC ether. These properties included 1) alpha-helical content of the apoprotein as assessed by circular dichroism, 2) microviscosity as determined from the fluorescence polarization and lifetime of the probe 1,6-diphenyl-1,3,5-hexatriene, 3) macromolecular weight based upon analytical gel filtration chromatography, and 4) surface polarity revealed by the fluorescence of 6-propionyl-2(dimethylamino)naphthalene. The only major difference in a physicochemical property was that the molecular surface area of DPhPC (area = 69 A2 at collapse pressure) determined by monolayer methods was 17 A2 greater than that of POPC (area = 53 A2 at collapse pressure) at all surface pressures measured. We suggest that the properties of DPhPC in being enzymatically nonreactive but a competitive inhibitor are due to its much larger size and that the active site of lecithin-cholesterol acyltransferase cannot bind phospholipid substrates in a catalytically productive way if they have surface areas of 70 A2 or more.     

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.     

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.     

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B,miltefosine and nerolidol

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 μM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.     

Lipid metabolism during aging of high-alpha-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of alpha-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-alpha-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-alpha-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-alpha-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-alpha-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-alpha-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-alpha-linolenate tubers. These age- and high-alpha-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-alpha-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-alpha-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-alpha-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Delta 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.     

Polycystin-1 surface localization is stimulated by polycystin-2 and cleavage at the G protein-coupled receptor proteolytic site

Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery.     

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.     

Interaction of all-<Emphasis Type="Italic">trans</Emphasis>-Retinal with bilayer lipid membranes

The interaction of all-trans-retinal (hereinafter referred to as retinal) with planar bilayer lipid membranes has been studied. Addition of retinal into aqueous solutions on both sides of the membrane formed from diphytanoilphosphatidylcholine (DPhPC) or its mixture with diphytanoilphosphatidylethanolamine (DPhPC/DPhPE in w/w proportion of 3: 5) led to a change of conductance induced by ionophores nonactin (increase of conductance) or pentachlorophenol (decrease). Increase of nonactin-induced conductance was dependent on the membrane lipid composition and was two times higher in the case of DPhPC/DPhPE mixture. The change of conductance caused by ionophores of different signs (plus or minus) had different direction suggesting the influence of the retinal on the dipole potential upon its incorporation into BLM. The boundary potentials difference measured by the intramembrane field compensation method (IFC) after the retinal addition on one side of the membrane did not exceed 2.5 mV suggesting that its distribution in the bilayer is almost symmetrical. The illumination of the retinal-containing BLM caused a decrease in its lifetime when the membranes were formed from unsaturated lipids. Retinal incorporated into BLM led also to photoinactivation of the gramicidin channels. The process was completely inhibited by a singlet oxygen quencher (sodium azide). These results indicate that retinal accumulated in the membrane can affect both membrane proteins and the unsaturated lipids by their oxidation by the singlet oxygen.     

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.     

Two-step impact of Amphotericin B (AmB) on lipid membranes: ESR experiment and computer simulations

Abstract

In this study, the electron spin resonance (ESR) method was used to examine the effect of Amphotericin B (AmB) molecules on the fluidity of model membranes made of dipalmitoylphosphatidylcholine (DPPC). The changes occurring under increased AmB concentrations in the spectroscopic parameters of spin probes placed in liposomes were determined. Three probes were used, penetrating the membrane at different depths which allowed the changes in its fluidity to be found in the transverse section. A computer model of the surface layer of membrane, with AmB admixture, was developed and subjected to computer simulation. The effect of changing concentration of the admixture on the binding energy in the system of dipoles representing the surface of the membrane was examined. The ESR studies showed that the process of accumulation of AmB molecules in the membrane has two stages, marked by local maxima in the ESR spectra. The first appears for concentrations of ca. 0.25–0.5% and the second appears for ca. 2.5–3% AmB of its molar ratio to DPPC. The computer simulations permitted reconstructing the two-stage mechanism of interaction between the molecules and the membrane. They demonstrated that, at low concentrations, the AmB molecules position themselves flat on the membrane surface. After the threshold concentration is exceeded, they re-orientate to a vertical position. This process leads to the perforation of the membrane.     

Composition of the protoplast membrane from Saccharomyces cerevisiae

1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0.8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7.0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13-20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C(16:0), C(16:1) and C(18:1) acids. Whole cells contained a slightly greater proportion of C(16:0) and a somewhat smaller proportion of C(18:1) acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas-liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Delta(5,7,22,24(28))-ergostatetraen-3beta-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C(29) di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes.     

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.     

How does Amphotericin B Work?: Studies on Model Membrane Systems

Abstract

The drug Amphotericin B is a very important antifungal agent as well as one of the first model systems for transmembrane pore structures. The most widely accepted model for the anticellular activity of this drug involves the formation of 1:1 Amphotericin/ sterol aggregates which subsequently associate into a transmembrane barrel with a large -OH lined aqueous pore down the middle. The stronger association of Amphotericin with ergosterol versus cholesterol explains the higher toxicity toward fungi. However, conflicting membrane permeability data concerning Amphotericin channel ion selectivity, sterol requirements, and mode of delivery has accumulated over the past fifteen years and suggests there exists a multiplicity of AmB channel structures and modes of action. Some of these mechanisms of action may be even more relevant clinically than the Amphotericin/sterol pore structure. Some of the anticellular membrane damage caused by Amphotericin may be due to formation of membrane defects and non-bilayer phases, channels without sterol or even induction of oxidative damage. In this article we present a survey of recent observations on AmB's activity on model membrane systems. As such, we are mostly concerned with liposome and planar bilayer studies. Some of the newer models explaining AmB s differential effects on cholesterol versus ergosterol containing membranes are presented along with a brief overview of membrane disruption models based on current research on membrane-active amphiphilic peptides. A synthesis and reconciliation of many of these diverse observations is attempted in a model which can accommodate most aspects of the classical sterol/Amphotericin barrel model and more recent observations as well.     

Comparative physical studies of phosphatidylsulfocholine and phosphatidylcholine. Calorimetry,fluorescence polarization and electron paramagnetic resonance spectroscopy

Transition temperatures of phosphatidylsulfocholines (PSCs; di-14 : 0-, di-16 : 0-, di-18 : 0- and di-18 : 1-) were compared with those of the corresponding phosphatidylcholines (PCs) using the techniques of differential scanning calorimetry, fluorescent polarization with diphenylhexatriene (DPH) and cis- and trans- parinaric acids as probes, and electron paramagnetic resonance (EPR) with 5-doxyl stearic acid as probe. Liposomal dispersions of the sulfonium analogs showed the typical multibilayer structure by electron microscopy (EM) and were in general very similar in physical behaviour to those of the corresponding PCs. However, the fully hydrated saturated PSCs consistently showed sharp main transitions 2–4°C above those of the corresponding PCs, by all three techniques used; the unsaturated PSC (di-18 : 1) had a transition 2–3°C below that of di-18 : 1-PC and only the di-14 : 0-PSC and di-18 : 1-PSC showed a well-defined pretransition. Fluorescence polarization studies with cis- and trans-parinaric acids showed that the PSC bilayers were less ordered than the corresponding PC bilayers in both the gel and liquid crystalline states.These results provide a rationale for the observed ability of the sulfonium analogs to substitute for PC in some natural membranes.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.