Modulation of the phase transition behavior of phosphatidylethanolamine by cholesterol and oxysterols

Cholesterol lowers the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines up to a mole fraction of about 0.1. At cholesterol mole fractions above about 0.3, the effect of this sterol is to stabilize the bilayer phase. The relatively weak effects of cholesterol in altering the bilayer to hexagonal phase transition temperature can be explained on the basis of lateral phase separation. This is indicated by the horizontal liquidus line for the gel to liquid-crystalline transition in the phase diagram for mixtures of cholesterol with dielaidoylphosphatidylethanolamine (DEPE) as well as the fact that cholesterol does not greatly decrease the cooperativity of the bilayer to hexagonal phase transition. The enthalpy of this latter transition increased with increasing mole fractions of cholesterol. Two oxidation products of cholesterol are 5-cholesten-3 beta,7 alpha-diol and cholestan-3 beta,5 alpha,6 beta-triol. Compared with cholesterol, 5-cholesten-3 beta,7 alpha-diol had a greater effect in decreasing the bilayer to hexagonal phase transition temperature and broadening this transition. It is suggested that its effectiveness is due to its greater solubility in the DEPE. In contrast, cholestan-3 beta,5 alpha,6 beta-triol raises the bilayer to hexagonal phase transition temperature of DEPE. This is due to its larger and more hydrophilic head group. In addition, its length, being shorter than that of DEPE, would not allow it to pack efficiently in a hexagonal phase arrangement.We suggest that this same effect is responsible for cholesterol raising the bilayer to hexagonal phase transition temperature at higher mole fractions.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Mechanism of complement cytolysis and the concept of channel-forming proteins

Complement damages membranes via the terminal reaction sequence that leads to the formation of membrane-bound, macromolecular C5b-9(m) protein complexes. These complexes represent C5b-8 monomers to which varying numbers of C9 molecules can be bound. Complexes carrying high numbers of C9 (ca. 6/8-12/16?) exhibit the morphology of hollow protein channels. Because they are embedded within the lipid bilayer, aqueous transmembrane pores are generated that represent the primary lesions caused by complement in the target cell membrane. Many other proteins damage membranes by forming channels in a manner analogous to the C5b-9(m) complex. Two prototypes of bacterial exotoxins, Staphylococcus aureus alpha-toxin and streptolysin-O, are discussed in this context, and attention is drawn to the numerous analogies existing among these protein systems. Common to all is the process of self-association of the native proteins to form supramolecular complexes. This event is in turn accompanied by a unique transition of the molecules from a hydrophilic to an amphiphilic state.     

A new interpretation of eutectic behavior for distearoylphosphatidylcholine-cholesterol binary bilayer membrane

We investigated the thermotropic phase behavior of the distearoylphosphatidylcholine (DSPC)–cholesterol binary bilayer membrane as a function of the cholesterol composition (X_ch) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra, each of which has a single maximum, showed that the wavelength at the maximum intensity (λ_max) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L_β′ or L_β) and the liquid ordered (L_o) phases, ca. 470 nm for the ripple gel (P_β′) phase and ca. 490 nm for the liquid crystalline (L) phase, respectively. The transition temperatures were determined from the temperature dependences of the λ_max and endothermic peaks of the DSC thermograms. Both measurements showed that the pretransition disappears around X_ch = 0.035. The constructed temperature–X_ch phase diagram indicated that the phase behavior of the binary bilayer membrane at X_ch ≤ 0.15 is similar to that of general liquid–solid equilibrium for a binary system where both components are completely miscible in the liquid phase and completely immiscible in the solid phase. It was also revealed that the diagram has two characteristic points: a congruent melting point at X_ch = 0.08 and a peritectic-like point at X_ch = 0.15. The hexagonal lattice model was used for the interpretation of the phase behavior of the binary bilayer membrane. These characteristic compositions well correspond to the bilayer states in each of which cholesterol molecules are regularly distributed in the hexagonal lattice in a different way. That is, each composition of 0.035, 0.08 and 0.15 is nearly equal to that for the binary bilayer membrane which is entirely occupied with units, each composed of a cholesterol and 30 surrounding DSPC molecules within the next-next-next nearest neighbor sites (Unit (1:30): L_β(1:30)), with units, each of a cholesterol and 12 surrounding DSPC molecules within the next nearest sites (Unit (1:12): L_β(1:12)) or with units, each of a cholesterol and 6 surrounding DSPC molecules at the nearest neighbor sites (Unit (1:6): L_β(1:6)), respectively. Therefore, the eutectic behavior observed in the phase diagram was fully explainable in terms of a kind of phase separation between two different types of regions with different types of regular distributions of cholesterol. Further, the L_o phase was found in the higher X_ch-region (X_ch > 0.15). No endothermic peak over the temperature range from 10 to 80 °C at X_ch = 0.50 suggested that the single L_o phase can exist at X_ch > 0.50.     

Polymyxin B and Ca(2+) induce conductance fluctuations in the dipalmitoyl phosphatidic acid bilayer lipid membrane

Polymyxin B in micromolar concentrations induces current fluctuations in liquid crystalline bilayer lipid membranes from dipalmitoylphosphatidic acid identified as ion channels. The appearance of ion channels correlates with phase separation of the lipid in the presence of peptide polycations detected by differential scanning calorimetry. Ca2+ also induces the formation of ion channels in liquid crystalline bilayer lipid membranes from dipalmitoylphosphatidic acid followed by the phase transition of the phospholipid. The capacitive current, which indicates the possibility of structural transformations of bilayer-non-bilayer type (hexagonal phase II), precedes the formation of Ca(2+)-induced channels in bilayer lipid membranes from dipalmitoylphosphatidic acid.     

The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers

A set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12) have been used to examine gradients in fluorescence polarization, lifetime (tau F), relative quantum yield (phi rel) and positions of emission maxima (lambda max) through bilayers composed of synthetic phospholipids. The fluorophores of these probes report the environment at a graded series of depths from the surface to the centre of the bilayer structure. 1. Polarizations decrease as the fluorophore is moved deeper into the bilayer indicating greater rotational motion of the fluorophore in the hydrocarbon core of the bilayer. 2. The different responses of the probe diphenylhexatriene and the anthroyloxy fatty acids to the action of cholesterol on lipid bilayers are discussed in terms of the orientation of these probes in the bilayer and the types of anisotropic rotational motions which result in depolarization of fluorescence. 3. Stearic acid derivatives which have the fluorophore in the 6-, 9- and 12-positions along the acyl chain have a similar response to solvent polarity as measured by values of lambda max and phi rel in a variety of organic solvents. 4. The position of the emission maximum has little dependence on solvent viscosity, but viscosity does change the degree of vibrational structure seen in the emission spectrum. The vibrational structure itself may be used as an indication of the 'mciroviscosity' gradient in the transverse plane of the bilayer. 5. Values of lambda max, tau F and phi rel indicate that a gradient of polarity exists from the surface to the centre of the bilayer. For dipalmitoyl phosphatidylcholine in the crystalline phase, cholesterol acts to make this polarity gradient shallower.     

Specific bio-recognition reactions observed with an integrated Mach-Zehnder interferometer.

The combination of an integrated Mach-Zehnder-interferometer (iMZI) at the bottom of a fluidic microchannel system with supramolecular interfacial binding layers optimized for biosensing purposes is described. The model system used is based on the highly specific interaction of streptavidin to its 'ligand' biotin: a single monolayer of a correspondingly derivatized silane-compound is formed by a self-assembly procedure on top of the channel rib guiding the light through the channels. Injection of a streptavidin solution which leads to the formation of a protein monolayer of d = 2.8 nm in effective thickness results in a phase shift of the sample light relative to the reference channel of delta phi = 6 pi, in good agreement with the theoretical sensitivity of delta phi/delta df = 2.9 pi/nm for a protein layer (n = 1.45) calculated for the device.     

Effects of viral chemotherapeutic agents on membrane properties. Studies of cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly and amantadine

Cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly, and amantadine inhibit the dilution of fluorescently labeled lipids, as measured with the resonance energy exchange assay for membrane fusion. The fusion was studied using sonicated vesicles containing 1,2-dioleoyl-sn-glycero(3)phosphoethanolamine, egg (3-sn-phosphatidyl)choline, and cholesterol in a 1:1:1.3 molar ratio. All three antiviral agents inhibited myelin basic protein-induced membrane fusion when present at low concentrations in the membrane. The mechanism by which these agents affect membrane properties was investigated. The effect of these agents on the bilayer to hexagonal phase transition of 1,2-dielaidoyl-sn-glycero(3)phosphoethanolamine was determined using both differential scanning calorimetry and 31P NMR. Benzyloxycarbonyl-D-Phe-L-Phe-Gly is particularly effective in raising the bilayer to hexagonal phase transition temperature while cyclosporin promotes the greatest amount of broadening of the 31P NMR signal. Both effects are suggested to be related to the inhibitory activity of these substances on membrane fusion and possibly also to their antiviral activity.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H(II) phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H(II) phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H(II) phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H(II) phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H(II) phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine. Effect of n-alkanols

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.     

Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines

The bilayer to hexagonal phase transition temperatures of dielaidoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylethanolamine are 65.6 and 71.4 degrees C, respectively. Using high-sensitivity differential scanning calorimetry, I have shown that these transition temperatures are extremely sensitive to the presence of small amounts of other lipid components. For example, at a mole fraction of only 0.01, dilinolenin lowers the bilayer to hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine by 8.5 degrees C. Other diacylglycerols have similar effects on this transition temperature, although the degree of unsaturation of the acyl chains has some effect, with distearin being less potent. In comparison, the 20-carbon alkane eicosane lowers this transition temperature by 5 degrees C, while palmitoyl-lysolecithin raises it by 2.5 degrees C. Similar effects of these additives on the bilayer to to hexagonal phase transition temperature are observed with dielaidoylphosphatidylethanolamine. At these concentrations of additive, there is no effect on the gel-state to liquid-crystalline-state transition temperature. The observed shifts in the temperature of the bilayer to the hexagonal phase transition can be qualitatively interpreted in terms of the effects of these additives on the hydrophilic surface area and on the hydrophobic volume. Substances expanding the hydrophobic domain promote hexagonal phase formation and lower the bilayer to hexagonal phase transition temperature. The sensitivity of the bilayer to hexagonal phase transition temperature to the presence of additives is at least as great as that which has been observed for any other lipid phase transition.     

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions.

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL-HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of alpha-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.     

The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study.

1. The polymorphic phase behaviour of aqueous dispersions of phosphatidylethanolamines isolated from human erythrocytes, hen egg yolk and Escherichia coli have been investigated employing 31P NMR techniques. All species exhibit well defined, reversible bilayer to hexagonal (H11) phase transitions as the temperature is increased. The temperatures at which these transition take place (10, 25--30 and 55--60 degrees C for erythrocyte, egg yolk and E. coli phosphatidylethanolamine, respectively) are sensitive to the fatty acid composition, occurring at a temperature up to 10 degrees C above the high temperature end of the hydrocarbon phase transition as detected by differential scanning calorimetry. In some cases the bilayer to hexagonal (H11) transitions may also be detected employing calorimetric techniques. 2. The addition of equimolar concentrations of cholesterol to these naturally occurring phosphatidylethanolamines does not dramatically affect the bilayer-hexagonal (H11) transition temperature, producing changes of up to 10 degrees C. 3. 18 : 1t/18 : 1t phosphatidylethanolamine undergoes the bilayer to hexagonal (H11) phase transition as the temperature is increased through the interval 50--55 degrees C. Alternatively, hydrated 12 : 0/12 : 0 phosphatidylethanolamine remains in the bilayer phase at temperatures up to 90 degrees C (50 degrees C above the hydrocarbon phase transition temperature). 4. The presence of 100 mM NaCl or 10 mM CaCl2 in aqueous dispersions of egg yolk phosphatidylethanolamine does not alter the temperature-dependent polymorphic phase behaviour significantly. However, at 40 degrees C, increasing the p2H above 8.0 results in progressive inhibition of the hexagonal (H11) phase and the appearance of a phase possibly of cubic structure at p2H 9.0. At p2H 10.0 the bilayer phase is preferred. 5. It is suggested that in biomembranes containing phosphatidylethanolamine as a majority species (such as that of E. coli) the fatty acid composition may primarily reflect the need to maintain bilayer structure. Alternatively, it is pointed out that in mammalian membranes such as that of the erythrocyte, phosphatidylethanolamine tends to destabilize bilayer structure. The resulting possibility that transitory non-bilayer lipid configurations may occur may be directly related to many important properties of biological membranes.     

Changes in the electrical and visco-elastic properties of bilayer lipid membranes during interaction with proteins and lipoproteins

A method for simultaneous registration of planar bilayer lipid membrane (BLM) DC conductance G, capacitance C, surface potential difference delta phi and transversal elasticity module E is developed. C, delta phi and E are proportional to the amplitude of the first, second and third harmonics of capacitance current respectively. A comparative study of the interaction of BLM with very low density lipoproteins (VLDL), influenza virus matrix protein (M-protein) and yeast invertase was carried out. The kinetics of delta phi, E and G changes at different concentrations of VLDL, and dependence of delta phi and G on M-protein and invertase concentration was investigated. It is shown for VLDL invertase and M-protein that the changes in delta phi and E occur before the change in G. The method used permits to study peculiarities of individual stages of interaction between charge particles, supramolecular structures and lipid membranes.     

Acid-induced fusion of liposomes: studies with 2,3-seco-5 alpha-cholestan-2,3-dioic acid

The effect of 2,3-seco-5 alpha-cholestan-2,3-dioic acid on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine is markedly dependent on pH. Above pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid raises the temperature of this transition, i.e., it stabilizes the bilayer phase. At pH 6.56 there is little effect of this sterol derivative on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. However, below pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid markedly lowers the temperature of this transition. The promotion of hexagonal phase formation increases both with increasing mol fraction of this sterol derivative and with lower pH, particularly in the range between pH 6.56 and pH 5.0. Below about pH 6, 2,3-seco-5 alpha-cholestan-2,3-dioic acid also induces vesicle fusion as measured both by lipid mixing as well as by mixing of aqueous contents. For these assays vesicles made of phosphatidylethanolamine (made from egg phosphatidylcholine) and extruded through 0.2 micron pore membranes were used. At higher concentrations or at lower pH the 2,3-seco-5 alpha-cholestan-2,3-dioic acid induces some leakage of the contents of these vesicles. Nevertheless, with vesicles containing only 2 weight% sterol derivative, it was possible to demonstrate substantial mixing of aqueous contents of the vesicles over the pH range 3.5 to 5.5. Several of the properties of 2,3-seco-5 alpha-cholestan-2,3-dioic acid indicate that this compound may be useful in sensitizing vesicles to acid-induced fusion for the purpose of endocytic drug delivery.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Ion channels, induced by amphotericin B introduced unilaterally into lipid bilayers

Single ionic channels of approximately 10 pS in magnitude and approximately 100 ms duration (in 2 M KCl solution) were recorded when amphotericin B (AB) was added to one side of a lipid bilayer. Using blocking ions it has been shown that these channels are asymmetrical half-pores (similar to those postulated by Marty and Finkelstein) which are capable of forming long-living symmetrical pores if AB is added to both sides of the membrane.     

Effect of avidin on channel kinetics of biotinylated gramicidin

Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.     

Curcumin is a modulator of bilayer material properties

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is the major bioactive compound in turmeric (Curcuma longa) with antioxidant, antiinflammatory, anticarcinogenic, and antimutagenic effects. At low muM concentrations, curcumin modulates many structurally and functionally unrelated proteins, including membrane proteins. Because the cell membranes' lipid bilayer serves as a gate-keeper and regulator of many cell functions, we explored whether curcumin modifies general bilayer properties using channels formed by gramicidin A (gA). gA channels form when two monomers from opposing monolayers associate to form a conducting dimer with a hydrophobic length that is less than the bilayer hydrophobic thickness; gA channel formation thus causes a local bilayer thinning. The energetic cost of this bilayer deformation alters the gA monomer <--> dimer equilibrium, which makes the channels' appearance rate and lifetime sensitive to changes in bilayer material properties, and the gA channels become probes for changes in bilayer properties. Curcumin decreases bilayer stiffness, increasing both gA channel lifetimes and appearance rates, meaning that the energetic cost of the gA-induced bilayer deformation is reduced. These results show that curcumin may exert some of its effects on a diverse range of membrane proteins through a bilayer-mediated mechanism.     

Simultaneous optical and electrical recording of single gramicidin channels

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.