A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin

The cationic amphipathic alpha-helical antibiotic peptide, pleurocidin, from the winter flounder Pleuronectes americanus associates strongly with anionic membranes where it is able to translocate across the membrane, cause dye leakage from vesicles and induce pore like channel conductance. To investigate the mechanism of pleurocidin antibiotic activity in more detail we have applied a variety of spectroscopic techniques to study the interaction of pleurocidin with model membranes. At neutral pH the peptide inserts into membranes containing anionic lipids and, as shown by proton-decoupled 15N solid-state NMR spectroscopy of macroscopically oriented samples, is aligned parallel to the membrane surface. 2H solid-state NMR spectroscopy of chain deuterated phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids in mixed membranes shows that pleurocidin interacts with both the zwitterionic PE and anionic PG but disrupts the lipid acyl chain order of the anionic PG lipids more effectively. At acidic pH the three histidine residues of pleurocidin become protonated and positively charged which does not alter the membrane disrupting effect nor the location of the peptide in the membrane. The results are interpreted in terms of a structural model for pleurocidin inserted into anionic lipid membranes and the implications of our data are discussed in terms of a general mechanism for the antibiotic activity.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Calorimetric and fluorescence characterization of interactions between enkephalins and liposomal and synaptic plasma membranes containing gangliosides

The interactions of the opioid peptide [D-Ala2]methionine-enkephalinamide with dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing gangliosides GM1, Gd1a, and Gt1b and synaptic plasma membranes selectively enriched with dimyristoylphosphatidylcholine (DMPC) and ganglioside Gd1a have been investigated by using high-sensitivity differential scanning calorimetry. In the absence of gangliosides, the addition of enkephalinamide in concentrations of up to 10(-3) M does not induce any appreciable change in the heat capacity function of DPPC. In the presence of gangliosides, however, changes in the heat capacity function were observed with as little as micromolar concentrations of the enkephalinamide; the same is true for DMPC-Gd1a-enriched synaptic membranes. The magnitude and the nature of the enkephalinamide effect depend on the type of ganglioside studied. For DPPC vesicles containing ganglioside GM1 only a slight broadening in the heat capacity function and a small upward shift in the transition temperature were observed. For DPPC vesicles containing ganglioside Gd1a the effect was more dramatic; enkephalinamide concentrations as low as 10(-5) M caused the appearance of two well-defined peaks in the heat capacity function in contrast to the one peak observed in the absence of enkephalinamide. In the case of DPPC vesicles containing ganglioside Gt1b the enkephalinamide effect was seen at concentrations of 10(-4) M or higher. Synaptic plasma membranes were isolated from bovine brain, selectively enriched with exogenous lipid, and their thermotropic behavior was characterized by steady-state fluorescence spectroscopy and differential scanning calorimetry. This lipid enrichment results in the appearance of a membrane phase transition otherwise absent in the intact membrane preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine

Amphotericin B (AmB) is a popular antibiotic applied in treatment of deep-seated mycotic infections. The mode of action of AmB is based upon interactions with biomembranes but exact binding properties of the antibiotic to the lipid membranes still remain obscure. Effect of incorporation of AmB into egg yolk phosphatidylcholine membranes in the concentration range from 0.01 to 5 mol% on structural and dynamic properties of lipid bilayers was studied with application of small-angle neutron scattering, X-ray diffractometry and Fourier-transform infrared spectroscopy (FTIR). The results of the experiments show that AmB is located predominantly in the headgroup region of the membranes at concentrations below 1 mol%. The process of AmB aggregation, at concentrations above 1 mol%, is associated with ordering effect within the acyl chain region and therefore indicates incorporation of AmB into the hydrophobic membrane core.     

Exposure of phosphatidylinositol transfer proteins to sphingomyelin-cholesterol membranes suggests transient but productive interactions with raft-like, liquid-ordered domains

Both isoforms of rat phosphatidylinositol transfer protein (PITP) mediate the intermembrane transfer of sphingomyelin (CerPCho). In the plasma membrane, CerPCho often segregates with cholesterol into microdomains such as lipid rafts and caveolae. To test the hypothesis that PITP exhibits a preference for CerPCho- and cholesterol-rich membranes, we prepared unilamellar vesicles containing variable amounts of these two lipids. We also used CerPCho species with different acyl composition and treated vesicles with agents known to sequester and remove cholesterol. We observed that the beta isoform of rat PITP was more sensitive to membrane cholesterol than was the alpha isoform, as shown by increases in specific activities of lipid transfer of 2-6-fold. A relatively high membrane content of cholesterol (mole fraction > 0.4) was required to elicit such enhancements. Treatment of cholesterol-rich membranes with a series of beta cyclodextrins demonstrated that, upon depletion of cholesterol from participating membranes, the PITPbeta activity changes were fully reversible. We finally noted that the mechanism by which cholesterol enhances the activity of PITPbeta appeared to involve a decreased affinity of the protein for the membrane surface, in a manner that was independent of vesicle size and membrane microviscosity. We conclude that PITPbeta interacts transiently but productively with the liquid-ordered phase formed by CerPCho and cholesterol and discuss the possibility of PITP interactions in vivo with sphingolipid- and cholesterol-rich membrane microdomains.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Chain-Length Heterogeneity Allows for the Assembly of Fatty Acid Vesicles in Dilute Solutions

A requirement for concentrated and chemically homogeneous pools of molecular building blocks would severely restrict plausible scenarios for the origin of life. In the case of membrane self-assembly, models of prebiotic lipid synthesis yield primarily short, single-chain amphiphiles that can form bilayer vesicles only at very high concentrations. These high critical aggregation concentrations (cacs) pose significant obstacles for the self-assembly of single-chain lipid membranes. Here, we examine membrane self-assembly in mixtures of fatty acids with varying chain lengths, an expected feature of any abiotic lipid synthesis. We derive theoretical predictions for the cac of mixtures by adapting thermodynamic models developed for the analogous phenomenon of mixed micelle self-assembly. We then use several complementary methods to characterize aggregation experimentally, and find cac values in close agreement with our theoretical predictions. These measurements establish that the cac of fatty acid mixtures is dramatically lowered by minor fractions of long-chain species, thereby providing a plausible route for protocell membrane assembly. Using an NMR-based approach to monitor aggregation of isotopically labeled samples, we demonstrate the incorporation of individual components into mixed vesicles. These experiments suggest that vesicles assembled in dilute, mixed solutions are depleted of the shorter-chain-length lipid species, a finding that carries implications for the composition of primitive cell membranes.     

Low pH-Induced Pore Formation by the T Domain of Botulinum Toxin Type A is Dependent upon NaCl Concentration

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.     

The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.     

Sorting of streptavidin protein coats on phase-separating model membranes

Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.     

Lipopolysaccharide-Induced Dynamic Lipid Membrane Reorganization: Tubules,Perforations, and Stacks

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na⁺ leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca²⁺ gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions.     

Cholesterol reduces membrane electroporation and electric deformation of small bilayer vesicles

Electric fields, similar in the order of magnitude of the natural membrane fields of cellular lipid/protein membranes, and chemical relaxation spectrometry can be used as tools to quantify the rigidifying effect of cholesterol in membranes. Small unilamellar vesicles of radius a=50+/-3 nm, prepared form phosphatidylcholine, phosphatidylserine and phosphatidyl-glycerol in the molar ratio 1:1:1 and containing the optical lipid probe molecule 2-(3-diphenyl-hexatrienyl) propanoyl)-1-palmitoyl-sn-glycerol-3-phosphocholine (beta-DPH pPC), serve as examples for curved lipid membranes. The data of electrooptical turbidity and absorbance relaxations at the wavelength lambda=365 nm are analysed in terms of membrane bending rigidity kappa and membrane stretching modulus K. Both kappa and K increase with increasing mole fraction x of cholesterol up to x=0.5. The cholesterol induced denser packing of the lipids reduces the extent of both membrane electroporation (ME) and electroelongation of the vesicles. Further on, cholesterol in the lipid phase and sucrose in the aqueous suspension reduce the extent of membrane undulation and electro-stretching.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Do proteins facilitate the formation of cholesterol-rich domains?

Both biological and model membranes can exhibit the formation of domains. A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given. Some of these domains are enriched in cholesterol. The segregation of lipids into cholesterol-rich domains can occur in both pure lipid systems as well as membranes containing peptides and proteins. Peptides and proteins can promote the formation of cholesterol-rich domains not only by preferentially interacting with cholesterol and being sequestered into these regions of the membrane, but also indirectly as a consequence of being excluded from cholesterol-rich domains. The redistribution of components is dictated by the thermodynamics of the system. The formation of domains in a biological membrane is a consequence of all of the intermolecular interactions including those among lipid molecules as well as between lipids and proteins.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chain-Length Heterogeneity Allows for the Assembly of Fatty Acid Vesicles in Dilute Solutions

A requirement for concentrated and chemically homogeneous pools of molecular building blocks would severely restrict plausible scenarios for the origin of life. In the case of membrane self-assembly, models of prebiotic lipid synthesis yield primarily short, single-chain amphiphiles that can form bilayer vesicles only at very high concentrations. These high critical aggregation concentrations (cacs) pose significant obstacles for the self-assembly of single-chain lipid membranes. Here, we examine membrane self-assembly in mixtures of fatty acids with varying chain lengths, an expected feature of any abiotic lipid synthesis. We derive theoretical predictions for the cac of mixtures by adapting thermodynamic models developed for the analogous phenomenon of mixed micelle self-assembly. We then use several complementary methods to characterize aggregation experimentally, and find cac values in close agreement with our theoretical predictions. These measurements establish that the cac of fatty acid mixtures is dramatically lowered by minor fractions of long-chain species, thereby providing a plausible route for protocell membrane assembly. Using an NMR-based approach to monitor aggregation of isotopically labeled samples, we demonstrate the incorporation of individual components into mixed vesicles. These experiments suggest that vesicles assembled in dilute, mixed solutions are depleted of the shorter-chain-length lipid species, a finding that carries implications for the composition of primitive cell membranes.     

The effect of long-chain bases on polysialic acid-mediated membrane interactions

Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.     

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.     

Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein.

We have investigated the intervesicular transfer of galactosylceramide between unilamellar bilayer vesicles composed of differing sphingomyelin and phosphatidylcholine molar ratios. To monitor glycolipid transfer from donor to acceptor vesicles, we used a fluorescence resonance energy transfer assay involving anthrylvinyl-labeled galactosylceramide (AV-GalCer) and perylenoyl-labeled triglyceride. The transfer was mediated by glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycolipids. The initial transfer rate and the total accessible pool of glycolipid in the donor vesicles were both measured. An increase in the sphingomyelin content of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles decreased the transfer rate in a nonlinear fashion. Decreased transfer rates were clearly evident at sphingomyelin mole fractions of 0.22 or higher. The pool of AV-GalCer available for GLTP-mediated transfer also was smaller in vesicles containing high sphingomyelin content. In contrast, AV-GalCer was more readily transferred from vesicles composed of POPC and different disaturated phosphatidylcholines. Our results show that GLTP acts as a sensitive probe for detecting interactions of glycosphingolipids with neighboring lipids and that the lateral mixing of glycolipids is probably affected by the matrix lipid composition. The compositionally driven changes in lipid interactions, sensed by GLTP, occur in membranes that are either macroscopically fluid-phase or gel/fluid-phase mixtures. Gaining insights into how changes in membrane sphingolipid composition alter accessibility to soluble proteins with affinity for membrane glycolipids is likely to help increase our understanding of how sphingolipid-enriched microdomains (i.e., "rafts" and caveolae) are formed and maintained in cells.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.