Effect of bilayer cholesterol content on reconstituted human erythrocyte sugar transporter activity

The influence of altered bilayer cholesterol content on the catalytic activity of the human red cell hexose transporter was examined by reconstitution of the transport protein (band 4.5) into bilayers of large unilamellar vesicles formed from dipalmitoyl lecithin and varying amounts of cholesterol. The physical state of the bilayers was characterized by differential scanning calorimetry. The major findings are as follows: changes in bilayer phase behavior occur at membrane cholesterol levels of 15 to 20 mol % and 30 to 40 mol %; and the catalytic activity of the reconstituted transporter (Vmax/transporter) correlates with bilayer phase behavior. In crystalline bilayers, this is seen as an abrupt, stimulation of activity at 15 mol % cholesterol (which is reversed at 17.5 mol %) and a gradual acceleration of activity between 30 to 40 mol % cholesterol. In fluid bilayers (where activity is high), activity is unaffected by 10, 20, and 30 mol % cholesterol. However, 12.5 and 17.5 mol % cholesterol reduce activity by 100-fold. These studies demonstrate that small changes in bilayer cholesterol content result in drastic alterations in transporter activity. Transporter sensitivity to cholesterol is a complex rather than monotonic function of bilayer cholesterol content and appears to be primarily determined by bilayer composition rather than by bilayer "fluidity."     

Reconstituted human erythrocyte sugar transporter activity is determined by bilayer lipid head groups

The effects of bilayer lipid head group on human erythrocyte passive sugar transport protein activity were examined by reconstituting the transporter into bilayers of large unilamellar vesicles (LUVs) formed from lipid classes of identical (or similar) acyl chain composition. Two reconstituted transport parameters were measured as a function of temperature. These were Km and turnover number [Tn = Vmax per reconstituted D-glucose-sensitive cytochalasin B binding site (transport molecule)]. Tn for sugar transport was found to be almost entirely a function of the properties of the bulk lipid composition of the reconstituted LUVs. It was found to be independent of both reconstituted transporter density and small amounts (less than or equal to 3%) of endogenous red cell lipids. With the dimyristoylphospholipids, Tn increases at all temperatures in the order phosphatidylcholine less than phosphatidylglycerol less than phosphatidic acid less than phosphatidylserine (at 50 degrees C, Tn for transport in dimyristoylphosphatidylcholine is 100-fold lower than Tn for transport in dimyristoylphosphatidylserine). Similar results are found with egg yolk derived lipids. Only dimyristoyl- and dipalmitoylphosphatidylcholine bilayers are incapable of supporting detectable transport activity at temperatures below the bilayer phase transition, and only the phosphatidylcholines show a clear increase in Tn during the bilayer melt. All other bilayer systems studied (phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and sphingomyelin) support a small or negligible increase in Tn during the bilayer melt, the major change in transport being restricted to altered Km. With the disaturated phosphatidylglycerols (C14-C18), Tn and the activation energy (Ea) for reconstituted transport increase with acyl chain carbon number. Similar results are found with the phosphatidylcholines. Transport in bilayers formed from egg yolk sphingomyelin (a lipid containing a sphingosine rather than a glycerol backbone) is characterized by very high Km and low Tn parameters. Moreover, protein-mediated transport in sphingomyelin bilayers "spikes" during the bilayer phase transition. These and previous findings [Carruthers, A., & Melchior, D.L. (1984) Biochemistry 23, 6901-6911; Connolly, T.J., Carruthers, A., & Melchior, D. L. (1985) Biochemistry 24, 2865-2873] indicate that those bilayer factors influencing reconstituted sugar transporter activity are, in order of importance, lipid head group greater than lipid acyl chain length and saturation/unsaturation greater than lipid backbone greater than bilayer "fluidity".     

Lecithin:cholesterol acyltransferase regulation. Effect of fluidity of dimyristoylphosphatidylcholine vesicles

The regulation of lecithin:cholesterol acyltransferase by changes in phospholipid bilayer fluidity was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity of dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles was decreased by the addition of up to 20% (mol/mol) cholesterol and increased by the addition of up to 10% (mol/mol) lysoDMPC. When both cholesterol and lysoDMPC are present in the bilayer, their individual effects on fluidity are altered. These changes can be explained by complex formation between cholesterol and phospholipid as in the model of Presti et al. (Presti, F.C., Pace, R.J. and Chan, S.I. (1982) Biochemistry 21, 3831-3335). Lecithin:cholesterol acyltransferase activity with these vesicles as substrates was measured to determine whether activity can be modulated by the fluidity changes of the bilayer on which the enzyme acts. When 10% lysoDMPC, a known lecithin:cholesterol acyltransferase inhibitor, is added to the vesicles, inhibition of activity is observed. When 7.5% lysoDMPC is added to vesicles which contain either 5 or 10% cholesterol, lecithin:cholesterol acyltransferase activity increases. This increase in lecithin:cholesterol acyltransferase activity due to vesicle-fluidity increase is sufficient to overcome the decrease in activity due to lecithin:cholesterol acyltransferase inhibition. This is the first report of the ability of lysoDMPC to increase lecithin:cholesterol acyltransferase activity.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence

In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.     

Transport of 8-anilino-1-naphthalenesulfonate as a probe of the effect of cholesterol on the phospholipid bilayer structures.

The transport of 8-anilino-1-naphthalenesulfonate in dimyristoyl-L-alpha-lecithin bilayers has been found to be extremely sensitive to the crystalline state of the phospholipid dispersions. Thus this reaction may be used for probing the membrane structures. In binary mixtures of cholesterol and phospholipid the fluorescence enhancement of the dye completely disappears when the mole fraction of cholesterol reaches 33%. At temperatures below and above the phase transition of the lipid bilayers, the rate of the probe transport increases significantly in the binary mixtures. It reaches a maximum at 17 mol % of cholestero. The rate at this cholesterol content approaches the maximum value obtained for the probe transport in pure phospholipis, e.i., the rate at the midpoint of the phase transition. These observations indicate that the effect of cholesterol in the phospholipid dispersion is to maintain the bilayer structure close to the melting temperature of the lipid phase transition. In other words, cholesterol may be an effective buffer for membrane crystalline state when its concentration is near 17 mol %.     

Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes

The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.     

The effect of membrane composition and alcohols on the insulin-sensitive reconstituted monosaccharide-transport system of rat adipocyte plasma membranes.

The monosaccharide transporter from the plasma membranes of rat adipocytes and insulin-stimulated adipocytes has been reconstituted in sonicated liposomes. The stereospecific D-glucose uptake by liposomes made from a range of phospholipids and incorporating fatty acids has been investigated. D-Glucose uptake is correlated with an increase in lipid fluidity as a consequence of the addition of fluidizing fatty acids, changes in phospholipid acyl chain length and temperature. Benzyl alcohol and ethyl alcohol, which are generally considered to increase bilayer fluidity, decrease stereo-specific D-glucose uptake in both whole adipocytes and reconstituted liposomes. It is suggested that, although these alcohols may affect D-glucose transport by lipid-mediated fluidity changes, they also interact directly with the transporter resulting in inhibition of transport.     

Cholesterol-sphingomyelin interactions: a molecular dynamics simulation study

Stearoylsphingomyelin (SSM) bilayers containing 0, 22, and 50 mol % cholesterol (Chol) and a pentadecanoyl-stearoylphosphatidylcholine (15SPC) bilayer containing 22 mol % Chol were molecular dynamics simulated at two temperatures (37 degrees C and 60 degrees C). 15SPC is the best PC equivalent of SSM. The Chol effect on the SSM bilayer differs significantly from that on the 15SPC bilayer. At the same temperature and Chol content, H-bonding of Chol with SSM is more extensive than with 15SPC. SSM-Chol H-bonding anchors the OH group of Chol in the lower regions of the SSM-Chol bilayer interface. Such a location strengthens the influence of Chol on the SSM chains. In effect, the phase of the SSM-Chol bilayer containing 22 mol % Chol at 37 degrees C is shifted from the gel to the liquid-ordered phase, and the bilayer displays similar properties below and above the main phase-transition temperature for a pure SSM bilayer of approximately 45 degrees C. In contrast, due to a higher location, Chol is not able to change the phase of the 15SPC-Chol bilayer, which at 37 degrees C remains in the gel phase. Chol affects both the core and interface of the SSM bilayer. With increasing Chol content, the order of SSM chains and hydration of SSM headgroups increase, whereas polar interactions between lipids decrease.     

Membrane composition modulates thiopental partitioning in bilayers and biomembranes

The nonspecific interaction of thiopental with erythrocyte ghosts, synaptic membranes, microsomes and mitochondria has been measured at 25°C and pH 6.6. In cholesterol-depleted erythrocyte ghosts the partition coefficient decreases with increasing cholesterol content. In sonicated liposomes made from egg lecithin and cholesterol the partition coefficient also decreases with increasing cholesterol content. The dependence of the partition coefficient on cholesterol content in the biological membranes, on average, parallels that in the lipid bilayers. The partition coefficient in lipid bilayers made from lipids extracted from erythrocyte ghosts was comparable to that in the corresponding egg lecithin/cholesterol bilayer. The partition coefficients of all the biomembranes are consistently lower than those in the corresponding egg lecithin/cholesterol bilayer, the free energy of transfer between biomembrane and corresponding bilayer being ?1 kcal/mol.     

Effects of physical states of phospholipids on the incorporation and cytochalasin B binding activity of human erythrocyte membrane proteins in reconstituted vesicles

Proteoliposomes were reconstituted from a Triton extract of human erythrocyte membrane proteins and a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios. With mixtures of egg PC and soybean PE, the protein/lipid ratio of the reconstituted vesicles was maximal at 25% PC and 75% PE, the composition which is known to have a maximum bilayer disruption (highest occurrence of lipidic particles seen by freeze-fracture electron microscopy). With mixtures of 1-palmitoyl-2-oleoyl-PC and dilinoleoyl-PE, which gave vesicles with few isolated lipidic particles at room temperature, the effect was less pronounced. The specific activity of the cytochalasin B (CB) binding protein in the reconstituted vesicles, on the other hand, was increased monotonically up to severalfold as the PC content was increased in the egg PC/soybean PE mixture. A similar increase was observed when soybean PE was partially substituted by dimyristoyl-PC, cholesterol, or transphosphatidylated PE from egg PC. These findings indicate that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution whereas reduction of the bilayer fluidity facilitates the CB binding activity in the reconstituted vesicles.     

Placental alkaline phosphatase is efficiently targeted to rafts in supported lipid bilayers

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.     

Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics

The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of monounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transmembrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be important for the conformational mobility of the Na,K-ATPase changing the distribution of conformations. Lipid fluidity was important for several parameters of reconstitution, e.g., the amount of protein inserted and the orientation in the liposomes. The temperature dependence of the Na,K-ATPase as well of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATPase activity of Na,K-ATPase reconstituted with monounsaturated phospholipids. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnover. In the case of ATPase reconstituted without cholesterol, the minimum free energy of activation and the maximum turnover both shifted to longer acyl chain lengths of about 22.     

Effect of cholesterol on the physical state and function of lymphocyte membranes

Effect of gradual increase of cholesterol content in T-lymphocyte membranes on the structure and physical state of plasmic membrane lipids and activities of the membrane-bound enzymes was investigated. The increase in cholesterol content was shown to result in a two-phase change of luminescence parameters of the fluorescent probes dimethylaminochalcone and pyrene, which indicates heterogeneity of cholesterol in the membranes. With the growth of steroid content in the cell membranes, at first, we observed a sharp decrease in the lipid bilayer fluidity and inhibition of Na+, K+-ATPase activity, which at the molar ratio cholesterol/phospholipids 0.6 in thymocyte membranes, remains at the same level. With higher cholesterol concentrations ATPase activity did not change. The effect of cholesterol on ATPase activity was in a good agreement with the effect of membrane lipids on fluidity. It is suggested that two pools of cholesterol molecules exist in the membranes, differing in their effects of bilayer fluidity and functional activity of the membranes.     

Direct Measurement of the Structure of Reconstituted High-Density Lipoproteins by Cryo-EM

Early forms of high-density lipoproteins (HDL), nascent HDL, are formed by the interaction of apolipoprotein AI with macrophage and hepatic ATP-binding cassette transporter member 1. Various plasma activities convert nascent to mature HDL, comprising phosphatidylcholine (PC) and cholesterol, which are selectively removed by hepatic receptors. This process is important in reducing the cholesterol burden of arterial wall macrophages, an important cell type in all stages of atherosclerosis. Interaction of apolipoprotein AI with dimyristoyl (DM)PC forms reconstituted (r)HDL, which is a good model of nascent HDL. rHDL have been used as an antiathersclerosis therapy that enhances reverse cholesterol transport in humans and animal models. Thus, identification of the structure of rHDL would inform about that of nascent HDL and how rHDL improves reverse cholesterol transport in an atheroprotective way. Early studies of rHDL suggested a discoidal structure, which included pairs of antiparallel helices of apolipoprotein AI circumscribing a phospholipid bilayer. Another rHDL model based on small angle neutron scattering supported a double superhelical structure. Herein, we report a cryo-electron microscopy-based model of a large rHDL formed spontaneously from apolipoprotein AI, cholesterol, and excess DMPC and isolated to near homogeneity. After reconstruction we obtained an rHDL structure comprising DMPC, cholesterol, and apolipoprotein AI (423:74:1 mol/mol) forming a discoidal particle 360 Å in diameter and 45 Å thick; these dimensions are consistent with the stoichiometry of the particles. Given that cryo-electron microscopy directly observes projections of individual rHDL particles in different orientations, we can unambiguously state that rHDL particles are protein bounded discoidal bilayers.     

Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes

The penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers.     

Influence of the bilayer composition on the binding and membrane disrupting effect of Polybia-MP1, an antimicrobial mastoparan peptide with leukemic T-lymphocyte cell selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.     

Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations.

The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.     

Ceramide drives cholesterol out of the ordered lipid bilayer phase into the crystal phase in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol/ceramide ternary mixtures

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.     

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.