Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.     

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.     

Stable and unstable lipid domains in ceramide-containing membranes

We applied x-ray diffraction, calorimetry, and infrared spectroscopy to lipid mixtures of palmitoyl-oleoyl phosphatidylcholine, sphingomyelin, and ceramide. This combination of experimental techniques allowed us to probe the stability and structural properties of coexisting lipid domains without resorting to any molecular probes. In particular, we found unstable microscopic domains (compositional/phase fluctuations) in the absence of ceramide, and macroscopically separated fluid and gel phases upon addition of ceramide. We also observed phase fluctuations in the presence of ceramide within the broad phase transition regions. We compare our results with fluorescence spectroscopy data and complement the previously reported phase diagram. We also obtained electron paramagnetic resonance data to assess the possible limitations of techniques employing a single label. Our study demonstrates the necessity of applying a combination of experimental techniques to probe local/global structural and fast/slow motional properties in complex lipid mixtures.     

Site-directed spin-labeling analysis of reconstituted Mscl in the closed state

The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O(2) or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.     

Fluorescence correlation studies of lipid domains in model membranes

Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.     

Erythrocyte D-glucose transport activity in reconstituted model membranes of different lipid composition

The stereospecific influx of D-glucose into liposomes formed on sonication of different glyco- and phospholipids with transport proteins from human erythrocyte ghosts solubilized with Triton x-100 was measured as an index of their total D-glucose transport activity. Specific D-glucose transport increased when acidic phospho- and glycolipids (especially sulfatide) were added to the phosphatidylcholine bilayers of the model membranes while cholesterol strongly inhibited the process. The modulation of D-glucose transport activity and its possible correlation with the lipid composition and the chemico-physical state of the erythrocytes is discussed.     

The lipid environment of the nicotinic acetylcholine receptor in native and reconstituted membranes

Detailed knowledge of the membrane framework surrounding the nicotinic acetylcholine receptor (AChR) is key to an understanding of its structure, dynamics, and function. Recent theoretical models discuss the structural relationship between the AChR and the lipid bilayer. Independent experimental data on the composition, metabolism, and dynamics of the AChR lipid environment are analyzed in the first part of the review. The composition of the lipids in which the transmembrane AChR chains are inserted bears considerable resemblance among species, perhaps providing this evolutionarily conserved protein with an adequate milieu for its optimal functioning. The effects of lipids on the latter are discussed in the second part of the review. The third part focuses on the information gained on the dynamics of AChR and lipids in the membrane, a section that also covers the physical properties and interactions between the protein, its immediate annulus, and the bulk lipid bilayer.     

Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

Pump and displacement currents of reconstituted ATP synthase on black lipid membranes

Summary Purified ATP synthase (F ₀ F ₁) fromRhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P _i ). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and P _i were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2 sec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 sec were obtained. The kinetics of the charge movements were slowed down byF ₀ specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding toF ₁ is prevented by the catalytic site-blocking agent NBD-Cl. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.     

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.     

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.     

Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes

Lipids segregate with each other into small domains in biological membranes, which can facilitate the associations of particular proteins. The segregation of cholesterol and sphingomyelin (SPM) into domains known as rafts is thought to be especially important. The formation of rafts was studied by using planar bilayer membranes that contained rhodamine-phosphatidylethanolamine (rho-DOPE) as a fluorescent probe, and wide-field fluorescence microscopy was used to detect phase separation of the probe. A fluorescently labeled GM(1), known to preferentially partition into rafts, verified that rho-DOPE faithfully reported the rafts. SPM-cholesterol domains did not form at high temperatures but spontaneously formed when temperature was lowered to below the melting temperature of the SPM. Saturated acyl chains on SPMs therefore promote the formation of rafts. The domains were circular (resolution > or = 0.5 microm), quickly reassumed their circular shape after they were deformed, and merged with each other to create larger domains, all phenomena consistent with liquid-ordered (l(o)) rather than solid-ordered (s(o)) domains. A saturated phosphatidylcholine (PC), disteoryl-PC, could substitute for SPM to complex with cholesterol into a l(o)-domain. But in the presence of cholesterol, a saturated phosphatidylethanolamine or phosphatidylserine yielded s(o)-domains of irregular shape. Lipids with saturated acyl chains can therefore pack well among each other and with cholesterol to form l(o)-domains, but domain formation is dependent on the polar headgroup of the lipid. An individual raft always extended through both monolayers. Degrading cholesterol in one monolayer with cholesterol oxidase first caused the boundary of the raft to become irregular; then the raft gradually disappeared. The fluid nature of rafts, demonstrated in this study, may be important for permitting dynamic interactions between proteins localized within rafts.     

Characterization of plasma membrane domains enriched in lipid metabolites.

A subpopulation of plasma membrane vesicles enriched in membrane lipid metabolites has been isolated from petals of carnation flowers and leaves of canola seedlings. This was achieved by immunopurification from a microsomal membrane preparation using region-specific antibodies raised against a recombinant polypeptide of the plasma membrane H(+)-ATPase. The properties of this subpopulation of vesicles were compared with those of purified plasma membrane isolated by partitioning in an aqueous dextran-polyethylene glycol two-phase system. The lipid composition of the immunopurified vesicles proved to be clearly distinguishable from that of phase-purified plasma membrane, indicating that they represent a unique subpopulation of plasma membrane vesicles. Specifically, the immunopurified vesicles are highly enriched in lipid metabolites, including free fatty acids, diacylglycerol, triacylglycerol and steryl and wax esters, by comparison with the phase-purified plasma membrane. These findings can be interpreted as indicating that lipid metabolites generated within the plasma membrane effectively phase-separate by moving laterally through the plane of the membrane to form discrete domains within the bilayer. It is also apparent that these domains, once formed, are released as vesicles into the cytosol, presumably by microvesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.     

Effect of ethanol on tracheal potassium channels reconstituted into bilayer lipid membranes.

We examined the effect of ethanol on single potassium channels derived from plasma membranes of bovine tracheal smooth muscles. The observed potassium channels had a conductance of 296 +/- 31 pS (mean +/- S.D.) in symmetrical 250 mmol/l KCl solutions, and exhibited a voltage- and Ca2+-dependence similar to BKCa channels. Ethanol at 50, 100 and 200 mM concentrations increased the probability of open potassium channels to 112 +/- 5, 127 +/- 7 and 121 +/- 13% (mean +/- S.E.M.), respectively. It is suggested that increased activity of the BKCa channels by ethanol hyperpolarizes the plasma membrane and thus may contribute to relaxation of tracheal smooth muscle.     

TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.     

Fluorescence correlation studies of lipid domains in model membranes (Review)

Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.