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.     

Acyl chain interdigitation in saturated mixed-chain phosphatidylcholine bilayer dispersions

The molecular packing of various fully hydrated mixed-chain phosphatidylcholines was studied by X-ray diffraction and electron microscopy. All of the mixed-chain phosphatidylcholines under study were shown to adopt a lamellar or bilayer form in aqueous media. The bilayer thickness of these mixed-chain phosphatidylcholines was determined from the lamellar repeat distance in the small-anglé X-ray diffraction region by controlled swelling experiments. At T greater than Tm, the bilayer thickness of C(18):C(12)PC and C(18):C-(10)PC is found to be comparable to that of C(14):C(14)PC. In contrast, the bilayer thickness of these highly asymmetric phosphatidylcholines is considerably less than that of the symmetric C(14):C(14)PC at temperatures below Tm. Moreover, the wide-angle X-ray diffraction patterns taken at T less than Tm consist of at least two sharp reflections at 4.2 and 4.6 A. These X-ray diffraction data suggest that these highly asymmetric mixed-chain phospholipids, in excess water, form mixed interdigitated bilayers in the gel state and that the acyl chain packing in the gel-state bilayer is not hexagonal. The freeze-fracture planes of these mixed-chain phosphatidylcholines are discontinuous at T less than Tm, supporting the conclusion drawn from X-ray diffraction data that these highly asymmetric phosphatidylcholines form interdigitated bilayers at temperatures below Tm. The molecular packing of fully hydrated C(18):C(14)PCs in bilayers is distinctively different from that of C(18):C(10)PCs or C(18):C(10)PCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

Shapes of bilayer vesicles with membrane embedded molecules

The interdependence of the lateral distribution of molecules which are embedded in a membrane (such as integral membrane proteins) and the shape of a cell with no internal structure (such as phospholipid vesicles or mammalian erythrocytes) has been studied. The coupling of the lateral distribution of the molecules and the cell shape is introduced by considering that the energy of the membrane embedded molecule at a given site of the membrane depends on the curvature of the membrane at that site. Direct interactions between embedded molecules are not considered. A simple expression for the interaction of the membrane embedded molecule with the local membrane curvature is proposed. Starting from this interaction, the consistently related expressions for the free energy and for the distribution function of the embedded molecules are derived. The equilibrium cell shape and the corresponding lateral distribution of the membrane embedded molecules are determined by minimization of the membrane free energy which includes the free energy of the membrane embedded molecules and the membrane elastic energy. The resulting inhomogeneous distribution of the membrane embedded molecules affects the cell shape in a nontrivial manner. In particular, it is shown that the shape corresponding to the absolute energy minimum at given cell volume and membrane area may be elliptically non-axisymmetric, in contrast to the case of a laterally homogeneous membrane where it is axisymmetric.     

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides

The amphipathic helix, in which hydrophobia and hydrophilic residues are grouped on opposing faces, is a structural mot if found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed. © 1994 John Wiley & Sons, Inc.     

Physical properties of lipid bilayer membranes: relevance to membrane biological functions

Over the last 25 years one of us (WKS) has been investigating physical properties of lipid bilayer membranes. In 1991 a group led by WKS was organized into the Laboratory of Structure and Dynamics of Biological Membranes, the effective member of which is AW. Using mainly the electron paramagnetic resonance (EPR) spin-labeling method, we obtained unexpected results, which are significant for the better understanding of the functioning of biological membranes. We have developed a new pulse EPR spin-labeling method for the detection of membrane domains and evaluation of lipid exchange rates. This review will be focused on our main results which can be summarized as follows: (1) Unsaturation of alkyl chains greatly reduces the ordering and rigidifying effects of cholesterol although the unsaturation alone gives only minor fluidizing effects, as observed by order and reorientational motion, and rather significant rigidifying effects, as observed by translational motion of probe molecules; (2) Fluid-phase model membranes and cell plasma membranes are not barriers to oxygen and nitric oxide transport; (3) Polar carotenoids can regulate membrane fluidity in a way similar to cholesterol; (4) Formation of effective hydrophobic barriers to the permeation of small polar molecules across membranes requires alkyl chain unsaturation and/or the presence of cholesterol; (5) Fluid-phase micro-immiscibility takes place in cis-unsaturated phosphatidylcholine-cholesterol membranes and induces the formation of cholesterol-rich domains; (6) In membranes containing high concentrations of transmembrane proteins a new lipid domain is formed, with lipids trapped within aggregates of proteins, in which the lipid dynamics is diminished to the level of gel-phase.     

Catalytic properties and conformation of hydrophobized alpha-chymotrypsin incorporated into a bilayer lipid membrane

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT-PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody-independent protection of the middle ear.     

Interaction of gramicidin S analogs with lipid bilayer membrane.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.     

Concentration-dependent behavior of nisin interaction with supported bilayer lipid membrane

Nisin is a positively charged antibacterial peptide that binds to the negatively charged membranes of gram-positive bacteria. The initial interaction of the peptide with the model membrane of negatively charged DPPG (dipalmitoylphosphatidylglycerol) was studied by cyclic voltammetry and a.c. impedance spectroscopy. Nisin could induce pores in the supported bilayer lipid membrane, thus, it led to the marker ions Fe(CN)(6)(3-/4-) crossing the lipid membrane and giving the redox reaction on the glassy carbon electrode (GCE). Experimental results suggested that the pore formation on supported bilayer lipid membrane was dependent on the concentration of nisin and it included three main concentration stages: low, middling, high concentration.     

Phase behavior of a model bilayer membrane with coupled leaves

We discuss the thermodynamic behavior of a bilayer composed of two coupled leaves and derive the Gibbs Phase Rule for such a system. A simple phenomenological model of such a system is considered in which the state of the bilayer is specified by the relative number of ordering lipids in the outer leaf, and in the inner leaf. Two cases are treated. In the first, both inner and outer leaves could undergo phase separation when uncoupled from one another. The bilayer can exist in four different phases, and can exhibit three-phase coexistence. In the second case, an outer layer which can undergo phase separation by itself is coupled to an inner leaf which cannot. We find that when the coupling is weak, the bilayer can exist in only two phases, one in which the outer layer is rich in ordering lipids and the inner leaf is somewhat richer in them than when uncoupled, and another in which the outer layer is poor in ordering lipids and the inner leaf is poorer in them than when uncoupled. Increasing the coupling increases the effect on the inner leaf composition due to small changes in those of the outer leaf. For sufficiently large coupling, a phase transition occurs and the bilayer exhibits four phases as in the first case considered. Our results are in accord with several observations made recently.     

Critical point for membrane bilayer formation

Unilamellar liposomes often are employed in investigations of lipid-protein interactions and the delivery of drugs in therapies for disease. Also, related lipid-containing nanoparticles have been developed as elements of a new class of mRNA vaccines. We show that only unilamellar films form in equilibrium lipid dispersions, at temperature values {T*} that depend on the identities of the lipids (e.g., T* ≈ 29 °C for DMPC). Thermodynamic analysis confirms that films at air-water surfaces can be used to monitor the properties of the lipid vesicles that form in the dispersion. When T > T*, critical exponents describing film properties as T approaches T* are μ ≈ 1.4 and ν ≈ 0.7, which are close to values for the interfacial tension and the correlation length of density fluctuations at fluid interfaces. These results, and observations that within the bilayer the lateral diffusion of fluorescent lipid probes demonstrates increases at T*, suggest that unilamellar vesicles at T* are a transition state between two different multilamellar structures. We generalize the thermodynamic arguments to explain the linkage between lipid structures in the surface and bulk dispersion within more complex samples, showing that dispersions containing total lipid extracts of cell membranes have properties similar to those in dispersions containing single lipids. Information from various independent studies indicates that T* noted for bilayer membranes of a population of cells is identical to the temperature at which the growth or gestation of the cells occurs in vivo. Examples include whole-cell lipid extracts obtained from bacteria, and poikilothermic and homeothermic animals.     

Deuterium order parameters in relation to thermodynamic properties of a phospholiped bilayer. A statistical mechanical interpretation.

The physical properties of bilayers of dipalmitoyl-3-sn-phosphatidylcholine are analyzed in terms of a statistical model proposed by Marcelja (S. Marcelja (1974), Biochim. Biophys. Acta 367, 165). The model is used to calculate the segmental order parameters of the hydrocarbon chains, the transition temperature of the crystalline leads to liquid crystalline phase transition, the entropy change of the transition, the bilayer thickness, and the linear thermal expansion coefficient. The theoretical predictions are in excellent agreement with experimental results obtained by deuterium magnetic resonance, differential scanning calorimetry, and X-ray diffraction. The model yields the probabilities of trans and gauche conformations and also those of more specific conformational defects like kinks or jogs.     

Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts

1. Extracts of the human erythrocyte membrane have been prepared by solubilization with Triton X-100 and analysed by electrophoresis and gel filtration techniques. 2. The extracts have been incorporated asymmetrically into lecithincholesterol-n-decane planar bilayers. 3. The electrical characteristics and glucose permeabilities of the bilayers have been measured. 4. The extracts increased the electrical conductance of the bilayers and also markedly enhanced the D-glucose permeability but not the L-glucose permeability. 5. The enhanced D-glucose permeability was inhibited by monosaccharide transport inhibitors. 6. The results support the claim that a monosachharide facititated diffusion system has been set-up in vitro which has many of the characteristics of the transport system in the human erythrocyte membrane. 7. The data indicates that the trans membrane polypeptides of band 3 of the electrophoretogram of the erythrocyte membrane proteins (notation of Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2616) are implicated in D-glucose transport, although the possibility that relatively minor component of the membrane could be responsible for glucose transport cannot be eliminated.     

Amphiphile-induced tubular budding of the bilayer membrane

Amphiphile-induced tubular budding of the erythrocyte membrane was studied using transmission electron microscopy. No chiral patterns of the intramembraneous particles were found, either on the cylindrical buds, or on the tubular nanoexovesicles. In agreement with these observations, the tubular budding may be explained by in-plane ordering of anisotropic membrane inclusions in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane.