Ion transport through channels formed in lipid bilayers by Staphylococcus aureus alpha-toxin

Staphylotoxin channel appears to be predominantly anion-selective with non-linear and asymmetric current-voltage characteristics (CVC) at neutral pH. Increased salt concentrations induce linearity and asymmetry of CVC and loss of selectivity. At lower pH both the channel conductivity and anion selectivity increase. Higher temperatures raise the channel conductivity in parallel with the changes in electrical conductivity of the salt solution, but do not change selectivity. Experimental dependences are described obtained by approximation of electrical diffusion and considering the interactions of penetrating ions with fixed charges at the entrances and the channel energy profile.     

The attractive forces between polar lipid bilayers.

Long-range attractive forces between lipid bilayers are not well described by the Lifshitz theory of Van der Waals forces between macroscopic media. It is shown that when correlations between polar headgroups are taken into account, the predicted forces take a qualitatively different form consistent with the measured data.     

Short-range interactions between lipid bilayers measured by X-ray diffraction

Interactions between lipid bilayers are critical in many biological processes in which membrane surfaces come close together. Recent X-ray diffraction analyses of bilayers subjected to known osmotic pressures have provided critical information on the magnitude of both the repulsive and the attractive forces that exist between phospholipid and glycolipid membranes.     

Electrical properties of ionic channels formed by Helix pomatia hemocyanin in planar lipid bilayers

Helix pomatia hemocyanin forms ion-conducting channels in planar lipid bilayer membranes when added at mg/ml concentration. These channels have several original features. They fluctuate between one conducting and some poorly conducting states and fluctuations can be grouped in bursts. Different channels can have widely different conductance amplitudes. Both channel conductance and burst lifetime are dependent on the applied voltage. Fluctuations within a burst show a complex kinetic behaviour which has been explained developing a multistate model. The model calls for one single open state and six different closed states. Transitions are allowed only between one of the closed states and the open one and obey first order kinetics. This model is able to fit all our experimental curves obtained in single channel experiments.     

Pores formed in lipid bilayers and in native membranes by nodularin,a cyanobacterial toxin

Nodularin (NODLN), a cyclic pentapeptide hepatotoxin from the cyanobacterium Nodularia spumigena, induces pores in bilayers of diphytanoyl lecithin (DPhL) and in locust muscle membrane. NODLN increases the surface pressure of a DPhL monolayer; except when the surface pressure of the monolayer is high when the toxin causes a reduction of this parameter. NODLN pores exhibit many open conductance states; the higher state probabilities increasing when the transmembrane pressure is increased. The results from these studies are discussed in terms of two models for a NODLN pore, a torroidal model and a barrel-stave model. The edge energy of the NODLN pore of 1.4× 10^–12 J/m is determined.Abbreviations NODLN Nodularin - MCYST-LR Microcystin-LR - ADDA 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid - DPhL diphytanoyl lecithin Correspondence to: A. G. Petrov     

Molecular dynamics simulations of charged and neutral lipid bilayers: treatment of electrostatic interactions

Molecular dynamics (MD) simulations complement experimental methods in studies of the structure and dynamics of lipid bilayers. The choice of algorithms employed in this computational method represents a trade-off between the accuracy and real calculation time. The largest portion of the simulation time is devoted to calculation of long-range electrostatic interactions. To speed-up evaluation of these interactions, various approximations have been used. The most common ones are the truncation of long-range interactions with the use of cut-offs, and the particle-mesh Ewald (PME) method. In this study, several multi-nanosecond cut-off and PME simulations were performed to establish the influence of the simulation protocol on the bilayer properties. Two bilayers were used. One consisted of neutral phosphatidylcholine molecules. The other was a mixed lipid bilayer consisting of neutral phosphatidylethanolamine and negatively charged phosphatidylglycerol molecules. The study shows that the cut-off simulation of a bilayer containing charge molecules generates artefacts; in particular the mobility and order of the charged molecules are vastly different from those determined experimentally. In the PME simulation, the bilayer properties are in general agreement with experimental data. The cut-off simulation of bilayers containing only uncharged molecules does not generate artefacts, nevertheless, the PME simulation gives generally better agreement with experimental data.     

Analysis of the anisotropy decay of trans-parinaric acid in lipid bilayers.

An analysis is presented of the complex anisotropy behavior of trans-parinaric acid in single component DEPC lipid bilayers. It is shown that a model involving two species with distinct lifetime and motional behavior is required, and is adequate, to explain the observed data. In particular, the observed increase in the anisotropy at long times demonstrates the presence of a species with a long fluorescence lifetime that has a high anisotropy. The time dependence of the anisotropy for these two environments is treated using both a purely mathematical sum of exponentials and a constrained fit based on an approximate solution of the anisotropic diffusion problem. In this latter model the anisotropy is described in terms of the second and fourth rank order parameters, (P2) and (P4), and a single dynamical parameter, D1, the perpendicular diffusion coefficient for this uniaxial probe. The parameters of both models are accurately determined from the fits to the data when two environments coexist and an association is made between lifetime components and distinct rotational sites. The values of the parameters obtained demonstrate the "solid-like" and "fluidlike" nature of these two coexisting environments.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

Comparison between orientational and conformational orders in fluid lipid bilayers.

The orientational order as determined by 2H NMR and the infrared frequencies of the C--H stretching modes of the methylene groups have been measured for several systems (POPC, POPC/cholesterol and POPE), all in the fluid phase, and then were compared; this work reveals an unexpected linear correlation between them. This experimental result shows that both measurements are essentially sensitive to a common motion, most likely trans/gauche isomerisation. This new correlation with those already found in the literature suggest that several measurements related to the hydrophobic core of the fluid bilayer describe different aspects of a universal behavior. The correlation presented here does not extend to the lipid in gel phase where slower motions affect the NMR lineshape.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

Liquid-crystalline phases of cholesterol/lipid bilayers as revealed by the fluorescence of trans-parinaric acid.

The presence of two liquid-crystalline phases, alpha and beta, in mixed bilayers of dimyristoylphosphatidylcholine/cholesterol was detected by the changes in the distribution of the fluorescence lifetimes of t-PnA, as analyzed by the Maximum Entropy Method. The formation of the liquid-ordered beta-phase, in the 30-40 degrees C temperature range as a function of cholesterol concentration (0-40 mol%), could be related quantitatively to the relative amplitude of a long lifetime component of the probe (10-14 ns). Based on this evidence, the phase behavior of mixtures of the unsaturated lipid palmitoyloleoylphosphatidylcholine and cholesterol was determined using the same technique, for cholesterol concentrations in the 0-50 mol% range, between 10 and 40 degrees C. It was found that two liquid-crystalline phases are also formed in this system, with physical properties reminiscent of the alpha- and beta-phases formed with saturated lipids. However, in this case it was determined that, for temperatures in the physiological range, the alpha- and beta-phases coexist up to 40 mol% cholesterol. This finding may be of significant biological relevance, because it supports the long held notion that cholesterol is responsible for the lipid packing heterogeneity of several natural membranes rich in unsaturated lipid components.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Voltage-dependent conductance induced by alamethicin-phospholipid conjugates in lipid bilayers.

Alamethicin, a linear 20-amino acid antibiotic, forms voltage-dependent channels in lipid bilayer membranes. We show here that alamethicin-phospholipid conjugates can be prepared by photolysis of unilamellar vesicles containing alamethicin and a phosphatidylcholine analogue with a carbene precursor at the end of the C-2 fatty acyl chain. This result indicates that at least a portion of the alamethicin molecule is in contact with the hydrocarbon moiety of the membrane in the absence of an applied voltage. Furthermore, the alamethicin-phospholipid photoproduct is able to induce a voltage-gated conductance similar to that of natural alamethicin. The importance of these results in terms of mechanisms for channel gating is discussed.     

Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha-toxin in planar lipid bilayers

Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha-toxin in planar lipid membranes is a heptamer.     

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

Summary The E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl^– over K⁺) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pH's higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.     

Variation in hydration forces between neutral phospholipid bilayers: evidence for hydration attraction

It is now generally recognized that hydration forces dominate close interactions of lipid hydrophilic surfaces. The commonality of their characteristics has been reasonably established. However, differences in measured net repulsion, particularly evident when phosphatidylethanolamine (PE) and phosphatidylcholine (PC) bilayers are compared, suggest there exists a variety of behavior wider than expected from earlier models of hydration and fluctuation repulsion balanced by van der Waals attraction. To find a basis for this diverse behavior, we have looked more closely at measured structural parameters, degrees of hydration, and interbilayer repulsive forces for the lamellar phases of the following lipids: 1-palmitoyl-2-oleoyl-PE (POPE), egg PE, transphosphatidylated egg PE (egg PE-T), mono- and dimethylated egg PE-T (MMPE and DMPE), 1-stearoyl-2-oleoyl-PC (SOPC), and mixtures of POPE and SOPC. POPE and SOPC bilayers differ not only in their maximum degrees of hydration but also in the empirical hydration force coefficients and decay lengths that characterize their interaction. When mixed with POPE, SOPC effects sudden and disproportionate increases in hydration. POPE, egg PE, and egg PE-T differ in their degree of hydration, molecular area, and hydration repulsion. A single methylation of egg PE-T almost completely converts its hydration and bilayer repulsive properties to those of egg PC; little progression of hydration is seen with successive methylations. In order to reconcile these observations with the conventional scheme of balancing interbilayer hydration and fluctuation-enhanced repulsion with van der Waals attraction, it is necessary to relinquish the fundamental idea that the decay of hydration forces is a constant determined by the properties of the aqueous medium. Alternatively, one can retain that fundamental idea if one recognizes the possibility that polar group hydration has an attractive component to it. In the latter view, that attractive component originates from interbilayer hydrogen-bonded water bridges between apposing bilayer surfaces, arising from correlation of zwitterionic or other complementary polar groups or from factors that affect polar group solubility. The same Marcelja and Radic formalism that accounts so well for the repulsive component also leads to an estimate of the attractive one. We suggest that the full range of degrees of hydration and of interbilayer spacings observed for different neutral bilayers results in part from variable contributions of the attractive and repulsive hydration components.(ABSTRACT TRUNCATED AT 400 WORDS)