Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

The interaction of hydrophobic ions with lipid bilayer membranes.

Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and L?uger (J. Membrane Biol. 5:225, 1971). This medel visualizes as three distinct steps the interfacial absorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions absorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon absorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon absorption, of a highly ordered water structure which surrounds the hydrophic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead by attributed to a more complex effect equivalent to a reduction of membrane fluidity.     

Effect of hydrophobic mismatch on phase behavior of lipid membranes

We investigate the competing effects of hydrophobic mismatch and chain stretching on the morphology and evolution of domains in lipid membranes via Monte Carlo techniques. We model the membrane as a binary mixture of particles that differ in their preferred lengths, with the shorter particles mimicking unsaturated nonraft lipids and the longer particles mimicking saturated raft lipids. We find that phase separation can be induced upon increasing either the ratio J/kappa of the hydrophobic surface tension J to the compressibility modulus kappa. J/kappa determines the decay length for thickness changes. When this decay length is larger than the system size the membrane remains mixed. Furthermore, increasing the thickness relaxation time can induce transient phase separation.     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with d-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with D-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

A molecular model for lipid-protein interaction in membranes: the role of hydrophobic mismatch.

The interaction free energy between a hydrophobic, transmembrane, protein and the surrounding lipid environment is calculated based on a microscopic model for lipid organization. The protein is treated as a rigid hydrophobic solute of thickness dP, embedded in a lipid bilayer of unperturbed thickness doL. The lipid chains in the immediate vicinity of the protein are assumed to adjust their length to that of the protein (e.g., they are stretched when dP > doL) in order to bridge over the lipid-protein hydrophobic mismatch (dP-doL). The bilayer's hydrophobic thickness is assumed to decay exponentially to its asymptotic, unperturbed, value. The lipid deformation free energy is represented as a sum of chain (hydrophobic core) and interfacial (head-group region) contributions. The chain contribution is calculated using a detailed molecular theory of chain packing statistics, which allows the calculation of conformational properties and thermodynamic functions (in a mean-field approximation) of the lipid tails. The tails are treated as single chain amphiphiles, modeled using the rotational isometric state scheme. The interfacial free energy is represented by a phenomenological expression, accounting for the opposing effects of head-group repulsions and hydrocarbon-water surface tension. The lipid deformation free energy delta F is calculated as a function of dP-doL. Most calculations are for C14 amphiphiles which, in the absence of a protein, pack at an average area per head-group ao approximately equal to 32 A2 (doL approximately 24.5 A), corresponding to the fluid state of the membrane. When dP = doL, delta F > 0 and is due entirely to the loss of conformational entropy experienced by the chains around the protein. When dP > doL, the interaction free energy is further increased due to the enhanced stretching of the tails. When dP < doL, chain flexibility (entropy) increases, but this contribution to delta F is overcounted by the increase in the interfacial free energy. Thus, delta F obtains a minimum at dP-doL approximately 0. These qualitative interpretations are supported by detailed numerical calculations of the various contributions to the interaction free energy, and of chain conformational properties. The range of the perturbation of lipid order extends typically over few molecular diameters. A rather detailed comparison of our approach to other models is provided in the discussion.     

Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers

Vibrational Raman spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of synthetic DL-erythro-N-lignoceroylsphingosylphosphocholine [C(24):SPM], a racemic mixture of two highly asymmetric hydrocarbon chain length sphingomyelins. Raman spectral peak-height intensity ratios of vibrational transitions in the C-H stretching-mode region show that the C(24):SPM-H2O system undergoes two thermal phase transitions centered at 48.5 and 54.5 degrees C. Vibrational data for fully hydrated C(24):SPM are compared to those of highly asymmetric phosphatidylcholine dispersions. The Raman data are consistent with the plausible model that the lower temperature transition can be ascribed to the conversion of a mixed interdigitated gel state (gel II) to a partially interdigitated gel state (gel I) and that the higher temperature transition corresponds to a gel I----liquid-crystalline phase transition. The observation of a mixed interdigitated gel state (gel II) at temperatures below 48.5 degrees C implies that biological membranes may have lipid domains in which some of the lipid hydrocarbon chains penetrate completely across the entire hydrocarbon width of the lipid bilayer.     

Effect of local anesthetics on the bilayer membrane of dipalmitoylphosphatidylcholine: interdigitation of lipid bilayer and vesicle-micelle transition

The phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membrane were observed by means of differential scanning calorimetry (DSC) as a function of the concentration of local anesthetics, dibucaine (DC x HCl), tetracaine (TC x HCl), lidocaine (LC x HCl) and procaine hydrochlorides (PC x HCl). LC x HCl and PC x HCl depressed monotonously the temperatures of the main- and pre-transition of DPPC bilayer membrane. The enthalpy changes of both transitions decreased slightly with an increase in anesthetic concentration up to 160 mmol kg(-1). In contrast, the addition of TC x HCl or DC x HCl, having the ability to form a micelle by itself, induced the complex phase behavior of DPPC bilayer membrane including the vesicle-to-micelle transition. The depression of both temperatures of the main- and pre-transition, which is accompanied with a decrease in enthalpy, was observed by the addition of TC x HCl up to 21 mmol kg(-1) or DC x HCl up to 11 mmol kg(-1). The pretransition disappeared when these concentrations of anesthetic were added, and the interdigitated gel phase appeared above these concentrations. The appearance of the interdigitated gel phase, instead of the ripple gel phase, brings about the stabilization of the gel phase by 1.8-2.4 kcal mol(-1). In the concentration range of 70-120 mmol kg(-1) TC x HCl (or 40-60 mmol kg(-1) DC x HCl), the enthalpy of the main transition exhibited a drastic decrease, resulting in the virtual disappearance of the main transition. This process includes the decrease in vesicle size with increasing anesthetic concentration, resulting in the mixed micelle of DPPC and anesthetics. Therefore, in this range of anesthetic concentration, the DPPC vesicle solubilized an anesthetic which coexists with the DPPC-anesthetic mixed micelle. Above the concentration of 120 mmol kg(-1) TC x HCl (or 60 mmol kg(-1) DC x HCl), there exists the DPPC-anesthetic mixed micelle. Two types of new transitions concerned with the mixed micelle of DPPC and micelle-forming anesthetics were observed by DSC.     

Transport mechanism of hydrophobic ions through lipid bilayer membranes

Summary Evidence is presented that the transport of lipid-soluble ions through bilayer membranes occurs in three distinct steps: (1) adsorption to the membranesolution interface; (2) passage over an activation barrier to the opposite interface; and (3) desorption into the aqueous solution. Support for this mechanism comes from a consideration of the potential energy of the ion, which has a minimum in the interface. The formal analysis of the model shows that the rate constants of the individual transport steps can be determined from the relaxation of the electric current after a sudden change in the voltage. Such relaxation experiments have been carried out with dipicrylamine and tetraphenylborate as permeable ions. In both cases the rate-determining step is the jump from the adsorption site into the aqueous phase. Furthermore, it has been found that with increasing ion concentration the membrane conductance goes through a maximum. In accordance with the model recently developed by L. J. Bruner, this behavior is explained by a saturation of the interface, which leads to a blocking of the conductance at high concentrations.     

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.     

On the transport noise of hydrophobic ions in lipid bilayer membranes

The effect of transit time on the electrical transport noise of a closed one-barrier model at equilibrium as proposed by Kolb and Läuger [6] is studied using the master-equation approach. A transit time is the time for an ion to cross the energy barrier (membrane interior) when the energy of the ion reaches the barrier height. Both the time correlation function and the noise power spectrum are obtained as functions of the transit time of the ions. Possible effects of transit time on the time correlation function of transport of dipicrylamine ions in lipid bilayers as reported by Bruner and Hall [13] and on the noise power spectrum as reported by Kolb and Läuger [6] are discussed.     

Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes

Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T _m and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X _Cer = 0.05. These broad components had higher T _m values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X _Cer = 0.05–0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

Temperature and composition dependence of the interaction of delta-lysin with ternary mixtures of sphingomyelin/cholesterol/POPC

The kinetics of carboxyfluorescein efflux induced by the amphipathic peptide delta-lysin from vesicles of porcine brain sphingomyelin (BSM), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol (Chol) were investigated as a function of temperature and composition. Sphingomyelin (SM)/Chol mixtures form a liquid-ordered (L(o)) phase whereas POPC exists in the liquid-disordered (L(d)) phase at ambient temperature. delta-Lysin binds strongly to L(d) and poorly to L(o) phase. In BSM/Chol/POPC vesicles the rate of carboxyfluorescein efflux induced by delta-lysin increases as the POPC content decreases. This is explained by the increase of delta-lysin concentration in L(d) domains, which enhances membrane perturbation by the peptide. Phase separations in the micrometer scale have been observed by fluorescence microscopy in SM/Chol/POPC mixtures for some SM, though not for BSM. Thus, delta-lysin must detect heterogeneities (domains) in BSM/Chol/POPC on a much smaller scale. Advantage was taken of the inverse variation of the efflux rate with the L(d) content of BSM/Chol/POPC vesicles to estimate the L(d) fraction in those mixtures. These results were combined with differential scanning calorimetry to obtain the BSM/Chol/POPC phase diagram as a function of temperature.     

The interaction of calcium with gangliosides in bilayer membranes

We studied the binding of calcium to bilayer membranes formed from mixtures of phosphatidylcholine and mono-, di-, or trisialoganglioside by measuring its effect on the electrophoretic mobility of multilamellar vesicles and the conductance of planar bilayers. In 0.001 M monovalent salt solutions the surface potential of the membranes is large and micromolar concentrations of calcium have a significant effect on the mobility and conductance. In 0.1 M monovalent salt solutions the surface potential is small and millimolar concentrations of calcium are required to affect these parameters. The strong apparent binding of calcium we observed at low ionic strength could be due to the nonspecific accumulation of calcium in the electrical diffuse double layer. To distinguish between this nonspecific effect and binding of calcium to the membrane, we substituted dimethonium for calcium. Dimethonium is a divalent cation that screens negative charges but does not bind to lipids. We also examined the effect of replacing phosphatidylcholine by monoolein: calcium binds to phosphatidylcholine but not to monoolein. We describe our electrophoretic mobility results by combining the Poisson-Boltzmann and Navier-Stokes equations with the Langmuir adsorption isotherm. We conclude that calcium binds weakly to gangliosides with an intrinsic association constant of less than 100 M-1, which is similar to the association constant of calcium with phospholipids.