Lipid Phase Coexistence Favors Membrane Insertion of Equinatoxin-II, a Pore-forming Toxin from Actinia equina
2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês
10.1074/jbc.m313817200
ISSN1083-351X
AutoresAriana Barlič, Ion Gutiérrez‐Aguirre, José M. M. Caaveiro, Antonio Cruz, M. Begoña Ruiz-Argüello, Jesús Pérez‐Gil, Juan Manuel González‐Mañas,
Tópico(s)Marine Sponges and Natural Products
ResumoEquinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed. Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed. Equinatoxin II (Eqt-II) 1The abbreviations used are: Eqt-II, equinatoxin-II; ANS, 1-anilinonaphtalene-8-sulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; Chol, cholesterol; DAG, diacylglycerol; DPX, p-xylene-bis-pyridinium bromide; Ld, liquid-disordered phase; Lo, liquid-ordered phase; LUV, large unilamellar vesicle(s); NBD, 2-nitrobenzo-2-oxa-1,3-diazole; NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadizole-1-yl)amino]dodecanoyl] sn-glycero-3-phosphocholine; PC, egg phosphatidylcholine; PLC, phospholipase C; SM, bovine brain sphingomyelin; TR, Texas Red™; mN, millinewton. 1The abbreviations used are: Eqt-II, equinatoxin-II; ANS, 1-anilinonaphtalene-8-sulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; Chol, cholesterol; DAG, diacylglycerol; DPX, p-xylene-bis-pyridinium bromide; Ld, liquid-disordered phase; Lo, liquid-ordered phase; LUV, large unilamellar vesicle(s); NBD, 2-nitrobenzo-2-oxa-1,3-diazole; NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadizole-1-yl)amino]dodecanoyl] sn-glycero-3-phosphocholine; PC, egg phosphatidylcholine; PLC, phospholipase C; SM, bovine brain sphingomyelin; TR, Texas Red™; mN, millinewton. is a member of the actinoporins, a group of sea anemone cytolysins (1Anderluh G. Maček P. Toxicon. 2002; 40: 111-124Crossref PubMed Scopus (342) Google Scholar). It is a 179-amino acid residue protein with a molecular mass of 19.8 kDa and an isoelectric point of 10.5 (2Maček P. Lebez D. Toxicon. 1988; 26: 441-451Crossref PubMed Scopus (135) Google Scholar). Its three-dimensional structure has been solved by x-ray crystallography and NMR (3Athanasiadis A. Anderluh G. Maček P. Turk D. Structure. 2001; 9: 341-346Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 4Hinds M.G. Zhang W. Anderluh G. Hansen P.E. Norton R.S. J. Mol. Biol. 2002; 315: 1219-1229Crossref PubMed Scopus (126) Google Scholar). Eqt-II forms cation-selective pores with a diameter of ∼2 nm in cell and model membranes (5Zorec R. Tester M. Maček P. Mason W.T. J. Membr. Biol. 1990; 118: 243-249Crossref PubMed Scopus (76) Google Scholar, 6Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (189) Google Scholar, 7Maček P. Belmonte G. 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Structure. 2003; 11: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Membrane insertion of Eqt-II and sticholysins is favored by the presence of sphingomyelin within the target membrane (6Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (189) Google Scholar, 8Maček P. Zechinni M.G. Pederzolli C. Dalla Serra M. Menestrina G. Eur. J. Biochem. 1995; 234: 329-335Crossref PubMed Scopus (55) Google Scholar, 14Caaveiro J.M.M. Echabe I. Gutiérrez-Aguirre I. Nieva J.L. Rodríguez-Arrondo J.L. González-Mañas J.M. Biophys. J. 2001; 80: 1343-1353Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Tejuca M. Dalla Serra M. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (153) Google Scholar, 16De los Ríos V. Mancheño J.M. Lanio M.E. Oñaderra M. Gavilanes J.G. Eur. J. Biochem. 1998; 252: 284-289Crossref PubMed Scopus (101) Google Scholar). The recent finding of a phosphocholine binding site in the three-dimensional structure of sticholysin-II (13Mancheño J.M. Martín-Benito J. Martínez-Ripoll M. Gavilanes J.G. Hermoso J. Structure. 2003; 11: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) supports the role of sphingomyelin as a specific receptor for actinoporins, as other authors have suggested (17Bernheimer A.W. Avigad L.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 467-471Crossref PubMed Scopus (165) Google Scholar, 18Turk T. Maček P. Period. Biol. 1986; 88: 216-217Google Scholar). However, the presence of sphingomyelin is not strictly necessary for the lytic activity of these toxins, which are also active in phosphatidylcholine/cholesterol mixtures (14Caaveiro J.M.M. Echabe I. Gutiérrez-Aguirre I. Nieva J.L. Rodríguez-Arrondo J.L. González-Mañas J.M. Biophys. J. 2001; 80: 1343-1353Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16De los Ríos V. Mancheño J.M. Lanio M.E. Oñaderra M. Gavilanes J.G. Eur. J. Biochem. 1998; 252: 284-289Crossref PubMed Scopus (101) Google Scholar). Therefore, other factors are likely to govern their mechanism of action. Mixtures of sphingomyelin, phosphatidylcholine, and cholesterol are characteristic of the so-called rafts, microdomains in which the concentration of membrane components (lipids or proteins) and their physicochemical properties are different from the surrounding environment. The increasing amount of information pointing to the existence of lipid domains in cell and model membranes and their implication in many crucial biological processes has been extensively reviewed (19Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8052) Google Scholar, 20Rietveld A. Simons K. Biochim. Biophys. Acta. 1998; 1376: 467-479Crossref PubMed Scopus (452) Google Scholar, 21Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2546) Google Scholar, 22Brown D.A. London E. J. 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Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (611) Google Scholar, 30Patra S.K. Alonso A. Arrondo J.L.R. Goñi F.M. J. Liposome Res. 1999; 9: 247-260Crossref Scopus (55) Google Scholar). This property is associated with the fact that lipids in rafts exist in the liquid-ordered (Lo) phase, where their acyl chains are extended and ordered as in the gel phase but possess lateral and rotational mobilities characteristic of the liquid-disordered (Ld) phase (31Ipsen J.H. Karlström G. Mouritsen O.G. Wennerström H.W. Zuckermann M.J. Biochim. Biophys. Acta. 1987; 905: 162-172Crossref PubMed Scopus (898) Google Scholar, 32Ipsen J.H. Mouritsen O.G. Zuckermann M.J. Biophys. J. 1987; 56: 661-667Abstract Full Text PDF Scopus (121) Google Scholar). In monolayers, bilayers, and animal cell membranes, Lo and Ld fluid phases are immiscible (33McConnell H.M. Vrljic M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 469-492Crossref PubMed Scopus (224) Google Scholar). Other examples of lipid phase coexistence are known, the most common one being probably the coexistence of gel and fluid phases in certain lipid mixtures or in pure lipid bilayers near the gel-fluid transition temperature (34Papahadjopoulos D. Nir S. Oki S. Biochim. Biophys. Acta. 1972; 266: 561-583Crossref PubMed Scopus (286) Google Scholar). In the present work, we have analyzed a variety of parameters that determine the formation of distinct lipid phases (lipid composition, temperature, presence of different sterols, and enzymatic activity of phospholipase C). A strong correlation was found between the coexistence of lipid phases and the pore-forming activity of Eqt-II. Epifluorescence microscopy imaging of supported lipid monolayers revealed the preferential localization of this eukaryotic toxin at the interface between lipid phases. Materials—Egg phosphatidylcholine (PC), bovine brain sphingomyelin (SM), and cholesterol (Chol) were from Avanti Polar Lipids (Alabaster, AL). Ergosterol, cholestenone (4-cholesten-3-one), and Triton X-100 were from Sigma. 8-Aminonaphthalene-1,3,6-trisulfonic acid (ANTS), 1-anilinonaphtalene-8-sulfonic acid (ANS), p-xylene-bispyridinium bromide (DPX), 1-palmitoyl-2-[12-[(7-nitro-2–1,3-benzoxadizole-1-yl)amino]dodecanoyl]sn-glycero-3-phosphocholine (NBD-PC), and the FluoReporter® Texas Red™-X (TR) protein labeling kit were obtained from Molecular Probes, Inc. (Eugene, OR). Phospholipase C from Bacillus cereus (PLC) (EC 3.1.4.3) was supplied by Roche Applied Science, and o-phenantroline was from Merck. Eqt-II Purification—Eqt-II was purified from the liquid exuded by Actinia equina specimens freshly collected in the Bay of Biscay. We followed the purification protocol described in Ref. 2Maček P. Lebez D. Toxicon. 1988; 26: 441-451Crossref PubMed Scopus (135) Google Scholar. The purified protein was concentrated to ∼10 mg/ml with an Amicon 8050 concentrator (Danvers, MA) ultrafiltration unit equipped with a regenerated nitrocellulose filter (Millipore Corp., Bedford, MA) with a molecular mass cut-off of 10 kDa. Aliquots were stored at –20 °C, and once thawed they were not refrozen. Protein concentration was estimated spectrophotometrically using a molar extinction coefficient at 280 nm of 3.61 × 104m–1 cm–1 (35Norton R.S. Maček P. Reid G.E. Simpson R.J. Toxicon. 1992; 30: 13-23Crossref PubMed Scopus (25) Google Scholar). Labeling of Equinatoxin II with Texas Red™—To a 190 μm solution of Eqt-II in distilled water, 130 μl of 1 m NaHCO3 were added to raise the pH to 8.3. The labeling reaction was started by the addition of 200 μl of TR stock solution (5 mg/ml) (final Eqt-II/TR molar ratio of 5:1). The mixture was incubated for 60 min at room temperature with constant stirring and protected from light. To inactivate any remaining free dye, 47 μl of hydroxylamine were added to the mixture, and the solution was stirred for an additional 30 min at room temperature. To purify the labeled protein, the mixture was loaded on a Sephadex G-15 column and eluted with 10 mm Hepes, 200 mm NaCl, pH 7.5. 500-μl fractions were collected, and absorption spectra from 250 to 650 nm were measured. Protein concentration and the degree of labeling were determined as indicated by the manufacturer of the protein labeling kit. Leakage of Liposomal Contents—The appropriate lipids were mixed in organic solvent, evaporated thoroughly, and resuspended in 10 mm Hepes 200 mm NaCl, pH 7.5, containing 25 mm ANTS and 90 mm DPX. Large unilamellar vesicles (LUV) were prepared by the extrusion method (36Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1566) Google Scholar), using polycarbonate filters with a pore size of 0.1 μm (Nucleopore, Pleasanton, CA). Nonencapsulated fluorescent probes were separated from the vesicle suspension through a Sephadex G-75 gel filtration column (Amersham Biosciences). Solution osmolarities were checked with an Osmomat 030 instrument (Gonotec, Berlin, Germany). Phospholipid concentration was measured according to Bartlett (37Bartlett G.R. J. Biol. Chem. 1959; 334: 466-468Abstract Full Text PDF Google Scholar). The leakage of encapsulated solutes was assayed as described by Ellens et al. (38Ellens H. Bentz J. Szoka F.C. Biochemistry. 1985; 26: 3099-3106Crossref Scopus (445) Google Scholar). The probe-loaded liposomes (final lipid concentration = 0.1 mm) were treated with the appropriate amounts of Eqt-II in a fluorometer cuvette at 25 °C with constant stirring. Changes in fluorescence intensity were recorded in a PerkinElmer Life Sciences LS-50 spectrofluorometer (Beaconsfield, UK) with excitation and emission wavelengths set at 350 and 510 nm, respectively. An interference filter with a nominal cut-off value of 470 nm was placed in the emission light path to minimize the contribution of the light scattered by the vesicles to the fluorescence signal. The percentage of leakage was calculated after the complete release of the fluorescent probe by the addition of the nonionic detergent Triton X-100 (final concentration = 0.1% w/v). When PLC was used, the assay was carried out under optimal conditions for its activity; buffer was 10 mm Hepes, 200 mm NaCl, 10 mm CaCl2, pH 7.5, and the experiment was carried out at 37.6 °C with constant stirring. Concentrations were 0.1 mm, 0.3 μm, and 1.5 units/ml for lipid, Eqt-II, and PLC, respectively. To stop the enzyme reaction, o-phenantroline was added at a final concentration of 6 mm. Surface Pressure Measurements—Surface pressure measurements were carried out with a MicroTrough-S system from Kibron (Helsinki, Finland) at 25 °C with constant stirring. The aqueous phase consisted of 1.1 ml of 10 mm Hepes, 200 mm NaCl, pH 7.5. The lipid, dissolved in chloroform/methanol (2:1), was gently spread over the surface. The desired initial surface pressure was attained by changing the amount of lipid applied to the air-water interface. After 10 min to allow for solvent evaporation, the protein was injected through a hole connected to the subphase. The final protein concentration in the Langmuir trough was 1 μm. The increment in surface pressure versus time was recorded until a stable signal was obtained. Supported Phospholipid Monolayers—Monolayers were formed by spreading chloroform/methanol (3:1, v/v) solutions (1 mm) of the phospholipid mixture on top of a buffered subphase (10 mm Hepes, 200 mm NaCl, pH 7.5) in a thermostated Langmuir-Blodgett ribbon trough (NIMA Technologies, Coventry, UK). To allow the observation by epifluorescence microscopy, 1% (mol/mol) of NBD-PC was included. After 10 min to allow for solvent evaporation, monolayers were compressed at 25 cm2/min to an initial surface pressure of 20 mN/m. After 10 min for equilibration, TR-labeled Eqt-II from a 45 μm buffered stock solution was injected into the subphase. Insertion was followed by monitoring the increase in surface pressure. The surface pressure stabilized at 25 mN/m, and at this point, the monolayer was transferred onto glass coverslips at a velocity of 5 mm/min. The ribbon trough was provided with a feedback mechanism that kept the surface pressure constant by compressing the monolayer, thereby compensating the loss of material that took place during the transfer. Epifluorescence microscopy observation of the planar supported monolayers was carried out with a Zeiss Axioplan II fluorescence microscope (Carl Zeiss, Jena, Germany). Images from NBD-labeled phospholipid and TR-labeled protein were recorded separately from the same sample by switching fluorescence filters to select the proper emission wavelength range. The experiment was carried out at 25 °C. Interaction of Eqt-II with SM-PC Mixtures—In most cases, the interaction of Eqt-II and other actinoporins with model membranes requires the presence of SM in the target membrane (6Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (189) Google Scholar, 15Tejuca M. Dalla Serra M. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (153) Google Scholar). To gain more information on the interaction of Eqt-II with model membranes, we prepared LUV composed of SM and PC in different proportions. Protein-vesicle interaction was monitored through the release of fluorescent dyes that had been entrapped in the vesicles. In Fig. 1A, we observe a strong dependence of the release of encapsulated ANTS/DPX on the SM content of the vesicles. For SM molar fractions between 0.3 and 0.7, the percentages of leakage ranged from 44 to 54%, and maximum leakage was obtained when the mixture was approximately equimolar. When one of the two phospholipids predominated, the release was reduced to ∼25%, and for LUV made of 100% PC or 100% SM, the release was close to 8%. Next, we prepared lipid monolayers with PC/SM mixtures to determine whether this behavior could be observed in other model membranes. The initial surface pressure (π0) was set at 20 mN/m, and we measured the increase in surface pressure (Δπ) after injection of 1 μm Eqt-II into the aqueous subphase (Fig. 1A). The insertion followed the same pattern observed in LUV: 1) maximum values for Δπ (between 12 and 14 mN/m) were observed when the molar fractions of PC and SM were similar; 2) when one of the lipids predominated, Δπ was reduced to 8–9 mN/m, and 3) for 100% PC and 100% SM monolayers, Δπ was practically the same (5.3 and 5.5 mN/m, respectively). We also measured the “critical pressure” (πc) for different PC/SM mixtures (i.e. the initial surface pressure (π0) above which no Δπ is observed after injection of Eqt-II into the subphase). In all of the lipid compositions tested, the higher the π0, the smaller the Δπ, because tighter lipid packing prevented protein insertion (Fig. 1B). The critical pressures were calculated by linear fitting of the experimental Δπ versus π0 (initial surface pressure) values and extrapolation to Δπ = 0. Fig. 1C shows the πc values obtained for different SM/PC mixtures. Again, the highest πc values (around 36 mN/m) were observed in mixtures approximately equimolar and decreased when one of the lipids was predominant. This means that at intermediate SM molar fractions there are more binding sites available for Eqt-II, and more protein molecules are able to penetrate the monolayer. The mixtures producing maximum penetration were also associated with the maximum values of protein-induced leakage from LUV (Fig. 1A). The gel to liquid-crystal phase transition temperatures (Tm) for PC and SM are –5 °C (39Untracht S.H. Shipley G.G. J. Biol. Chem. 1977; 252: 4449-4457Abstract Full Text PDF PubMed Google Scholar) and 38 °C (40Shipley G.G. Avecilla L.S. Small D.M. J. Lipid Res. 1974; 15: 126-131Abstract Full Text PDF Google Scholar), respectively. A detailed phase diagram of egg PC and bovine brain SM has been published (39Untracht S.H. Shipley G.G. J. Biol. Chem. 1977; 252: 4449-4457Abstract Full Text PDF PubMed Google Scholar). According to those data, at our experimental temperature (25 °C), pure egg PC exists in the fluid lamellar phase. The addition of SM gives rise to a (PC + SM) fluid lamellar phase plus a pure SM gel phase. Phase separation is clear above 33 mol % SM, and above 90 mol % SM only the gel phase occurs. Our data (Fig. 1) show maximum protein insertion and maximum bilayer permeabilization at SM mol % between 30 and 70 (i.e. in the phase diagram region where phase separation predominates). In addition, binary phase diagrams at 23 °C of palmitoyloleoyl phosphatidylcholine and palmitoyl sphingomyelin mixtures also showed gel/fluid coexistence at palmitoyl sphingomyelin proportions between 30 and 70 mol % (41Almeida R.F.M. Fedorov A. Prieto M. Biophys. J. 2003; 85: 2406-2416Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). Thus, it seems that the coexistence of gel and fluid lipid phases favors the degree of protein insertion and the extent of vesicle permeabilization. The Effect of Temperature—We studied the effect of temperature on the EqT-II-induced release of fluorescent solutes encapsulated in PC/SM (1:1) LUV (Fig. 2). The highest percentages of leakage were observed between 11 and 25 °C. At temperatures higher than 25 °C, the toxin activity started to decrease, a trend that was even more pronounced above 30 °C. Above 40 °C, the leakage was reduced to 13%. One possible explanation for this behavior would be a potential thermal destabilization of the protein. In a control experiment, we measured the fluorescence of 7.9 μm ANS in the presence of 2.6 μm Eqt-II as a function of temperature (data not shown). Between 16 and 54 °C, the ANS fluorescence remained low and constant, an indication that in this temperature interval the folding of the protein was compact (42Stryer L. J. Mol. Biol. 1965; 13: 482-495Crossref PubMed Scopus (1329) Google Scholar). Therefore, the decrease in EqT-II activity shown in Fig. 2 was not due to protein denaturation. However, a partial phase diagram for mixtures of SM and PC of natural origin (43Ruiz-Argüello M.B. Veiga M.P. Arrondo J.L.R. Goñi F.M. Alonso A. Chem. Phys. Lipids. 2002; 114: 11-20Crossref PubMed Scopus (58) Google Scholar) reveals that, above 32 °C, only the fluid phase exists. The egg PC/bovine brain SM phase diagram (39Untracht S.H. Shipley G.G. J. Biol. Chem. 1977; 252: 4449-4457Abstract Full Text PDF PubMed Google Scholar) also suggests that at 37 °C complete miscibility of these two lipids occurs. In addition, the phase diagram for the equimolar mixture of palmitoyl sphingomyelin/palmitoyloleoyl phosphatidylcholine shows that above 35 °C only the liquid-disordered phase exists (41Almeida R.F.M. Fedorov A. Prieto M. Biophys. J. 2003; 85: 2406-2416Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). Thus, in our opinion, the temperature-dependent decrease in protein activity results from changes in the membrane structure associated with an increased lipid miscibility and the disappearance of coexisting lipid phases. Interaction of Eqt-II with PC/Cholesterol Mixtures—Actinoporins can also permeabilize PC-cholesterol membranes (14Caaveiro J.M.M. Echabe I. Gutiérrez-Aguirre I. Nieva J.L. Rodríguez-Arrondo J.L. González-Mañas J.M. Biophys. J. 2001; 80: 1343-1353Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16De los Ríos V. Mancheño J.M. Lanio M.E. Oñaderra M. Gavilanes J.G. Eur. J. Biochem. 1998; 252: 284-289Crossref PubMed Scopus (101) Google Scholar). In Fig. 3A, we represent the kinetics of Eqt-II-induced leakage of ANTS/DPX encapsulated in LUV made of PC-cholesterol (70:30). At mammalian physiological temperatures, there was almost no leakage. However, at lower temperatures, the release increased, and at 4 °C it was 22%. Fixing the temperature at 4 °C, the extent of leakage increased with the cholesterol content of the model membrane (Fig. 3B). Thus, the effect of cholesterol is temperature- and concentration-dependent. In lipid monolayers formed at an initial pressure of 20 mN/m, the increase in surface pressure after injecting 1 μm Eqt-II into the subphase depends linearly on the amount of cholesterol (Fig. 4A). We also observed a small increment in the critical pressures (Fig. 4B). The effects of cholesterol are not as conspicuous as those of SM but might be related with a similar feature (i.e. the formation of different lipid phases within the model membrane). At low temperatures, cholesterol is likely to interact with the phospholipid to form an Lo lipid phase in coexistence with the bulk fluid phase (44Thewalt J.L. Bloom M. Biophys. J. 1992; 63: 1176-1181Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 45London E. Curr. Opin. Struct. 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Molecular interactions between SM and cholesterol induce Lo-Ld phase coexistence in SM/PC/cholesterol mixtures (47Veiga M.P. Arrondo J.L.R. Goñi F.M. Alonso A. Marsh D. Biochemistry. 2001; 40: 2614-2622Crossref PubMed Scopus (133) Google Scholar). We have tested the effect of Eqt-II on model membranes having two different lipid compositions, namely SM-PC-cholesterol (50:35:15) and SM-PC-cholesterol (50:15:35). The results obtained were compared with those observed on the SM/PC (50:50) mixture. Fig. 5A shows the kinetics of Eqt-II-induced release of ANTS/DPX encapsulated in LUV. In the lipid mixtures containing cholesterol, the leakage was larger than in the SM/PC (50:50) control mixture. Moreover, release in the cholesterol-containing vesicles was less dependent on protein concentration (Fig. 5B). Therefore, the presence of cholesterol within the SM-containing model membrane renders it much more susceptible to the lytic activity of Eqt-II. Eqt-II inserted to a similar extent in SM-PC-cholesterol (50: 35:15) and SM-PC (50:50) monolayers, but the rate of insertion was faster in the cholesterol-containing mixture. In monolayers containing SM-PC-cholesterol (50:15:35), both the degree of protein penetration and the rate of the process increased as compared with the cholesterol-free monolayer (Fig. 6A). The critical pressure values also changed in the presence of cholesterol (Fig. 6B). Whereas the πc values for SM-PC-cholesterol (50:35:15) and SM-PC (50:50) were nearly the same (36.8 and 37.2 mN/m, respectively), the πc for SM-PC-cholesterol (50:15: 35) was 46.8 mN/m. At any given initial surface pressure value, more protein was able to insert into the SM-PC-cholesterol (50:15:35) monolayer than in the cholesterol-free films. This fact was particularly evident when the initial pressure approached 30 mN/m, a value that is thought to be close to the lateral packing of phospholipids in membranes (48Demel R.A. Geu
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