Rapid reconstitution of a transmembrane protein into supported planar lipid membranes

In hepatocytes from control rats, the ureogenic action of epinephrine is mainly mediated through alpha 1-adrenoceptors and the effect is independent of the presence of extracellular calcium. In hepatocytes from adrenalectomized rats, both alpha 1- and beta-adrenoceptors are involved in the action of epinephrine. Furthermore, the alpha 1-adrenergic-mediated stimulation of ureogenesis in these cells is dependent on the presence of extracellular calcium. Our results indicate that glucocorticoids modulate the calcium dependency of alpha 1-adrenergic effects and are consistent with our suggestion that two pathways are involved in the transduction of the alpha 1-adrenergic signal.     

Pulse radiolysis at planar lipid membranes doped with ion carriers or pore formers

The effect of 14 MeV electrons on ion transport through planar lipid membranes was investigated. The membranes were formed in the presence of well defined ion carriers or pore forming substances. In the presence of the ion carriers valinomycin or nonactin or in the presence of the pore formers nystatin or amphotericin B, irradiation produced a transient increase of the membrane conductance followed by a long lasting decrease. The effects are interpreted on the basis of a time-dependent chemical modification of the membrane structure caused by exposure to high energy radiation. The pore former gramicidin A shows an exponential inactivation with increasing dose. At pH 3 and in the presence of oxygen the pore is highly sensitive to radiation ($\text{D}{}_{37}\text{≈ 10}\text{Gy}$) whereas at pH 9.5 a considerably lower radiation sensitivity ($\text{D}{}_{37}\text{≈ 1000}\text{Gy}$), was found. In the absence of oxygen, gramicidin A is virtually insensitive to irradiation. This is considered an evidence that the inactivation of this ion channel is primarily caused by the perhydroxyl radical HO₂.     

The effect of lipid demixing on the electrostatic interaction of planar membranes across a salt solution

We study the effect of lipid demixing on the electrostatic interaction of two oppositely-charged membranes in solution, modeled here as an incompressible two-dimensional fluid mixture of neutral and charged mobile lipids. We calculate, within linear and nonlinear Poisson-Boltzmann theory, the membrane separation at which the net electrostatic force between the membranes vanishes, for a variety of different system parameters. According to Parsegian and Gingell, contact between oppositely-charged surfaces in an electrolyte is possible only if the two surfaces have exactly the same charge density (sigma(1) = -sigma(2)). If this condition is not fulfilled, the surfaces can repel each other, even though they are oppositely charged. In our model of a membrane, the lipidic charge distribution on the membrane surface is not homogeneous and frozen, but the lipids are allowed to freely move within the plane of the membrane. We show that lipid demixing allows contact between membranes even if there is a certain charge mismatch, /sigma(1)/ not equal /sigma(2)/, and that in certain limiting cases, contact is always possible, regardless of the value of sigma(1)/sigma(2) (if sigma(1)/sigma(2) < 0). We furthermore find that of the two interacting membranes, only one membrane shows a major rearrangement of lipids, whereas the other remains in exactly the same state it has in isolation and that, at zero-disjoining pressure, the electrostatic mean-field potential between the membranes follows a Gouy-Chapman potential from the more strongly charged membrane up to the point of the other, more weakly charged membrane.     

Mitotic membrane helps to focus and stabilize the mitotic spindle

During mitosis, microtubules (MTs), aided by motors and associated proteins, assemble into a mitotic spindle. Recent evidence supports the notion that a membranous spindle matrix aids spindle formation; however, the mechanisms by which the matrix may contribute to spindle assembly are unknown. To search for a mechanism by which the presence of a mitotic membrane might help spindle morphology, we built a computational model that explores the interactions between these components. We show that an elastic membrane around the mitotic apparatus helps to focus MT minus ends and provides a resistive force that acts antagonistically to plus-end-directed MT motors such as Eg5.     

A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes

Abstract A new method of pore size determination is presented. The results of applying this simple method to ion channels formed by staphylococcal α-toxin and its N-terminal fragment as well as to cholera toxin channels are shown. The advantages and the difficulties of this method are discussed. It was found that (i) the mobility of ions in solutions depends only on the percentage of concentration of added non-electrolytes and practically not on their chemical nature (sugars or polyglycols) and molecular size; (ii) the proportional change of both ion channel conductance and bulk solution conductivity by low M . non-electrolytes may be used as an indication of a diffusion mechanism of ion transport through channels; (iii) the slope of the dependence of the ion channel conductance on the bulk conductivity of solutions containing different concentrations of non-electrolyte is a good measure of channel permeability for non-electrolytes.     

The dynamic stress responses to area change in planar lipid bilayer membranes

The viscoelastic properties of planar phospholipid (dimyristoylphosphatidylcholine) bilayer membranes at 308 K are studied, many of them for the first time, using the nonequilibrium molecular dynamics simulation (NEMD) method for membrane area change. First, we present a unified formulation of the intrinsic three-dimensional (3D) and apparent in-plane viscoelastic moduli associated with area change based on the constitutive relations for a uniaxial system. The NEMD simulations of oscillatory area change process are then used to obtain the frequency-domain moduli. In the 4-250 GHz range, the intrinsic 3D elastic moduli of 20-27 kbar and viscous moduli of 0.2-9 kbar are found with anisotropy and monotonic frequency dispersion. In contrast, the apparent in-plane elastic moduli (1-9 kbar) are much smaller than, and the viscous moduli (2-6 kbar) comparable to, their 3D counterparts, due to the interplay between the lateral and normal relaxations. The time-domain relaxation functions, separately obtained by applying stepwise strains, can be fit by 4-6 exponential decay modes spanning subpicosecond to nanosecond timescale and are consistent with the frequency-domain results. From NEMD with varying strain amplitude, the linear constitutive model is shown to be valid up to 6 and 20% area change for the intrinsic 3D elastic and viscous responses, respectively, and up to 20% area change for the apparent in-plane viscoelasticity. Inclusion of a gramicidin A dimer (approximately 1 mol %) yields similar response properties with possibly smaller (<10%) viscous moduli. Our results agree well with available data from ultrasonic experiments, and demonstrate that the third dimension (thickness) of the planar lipid bilayer is integral to the in-plane viscoelasticity.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition

Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.     

The diffusion of glycine and N-substituted glycines across bimolecular lipid membranes

Free glycine was shown to diffuse very slowly across synthetic bimolecular lipid membranes, whereas several N-substituted derivatives of glycine were found to penetrate the membranes more readily. Pyridoxal, formaldehyde and acetaldehyde were shown to enhance the diffusion of glycine across the membranes, presumably the result of imine formation between the aldehyde and the α-amino group of glycine. Several N-substituted glycines were synthesized and their rates of efflux from liposomes were found to be related to their water-chloroform partition coefficients. This is the first demonstration of carrier-mediated diffusion of amino acids across bimolecular lipid membrane.     

The movement of ion pairs across bilayer lipid membranes.

If a polyhalide concentration gradient exists across a bilayer lipid membrane (BLM), ion pair movement occurs. The term ion pair indicates a lipid soluble complex of cation and anion with stoichiometry dictated by the respective charges. In a mixture of metal halide (MX_n, X = I, Cl, Br) and iodine, the ion pair is of the form M(I₂X)_n. The flux of ion pairs was monitored by measuring the flow of metal ions or polyhalide ions across the BLM. The flux of ion pairs across the BLM depended on cation crystal radius, fluidity of the membrane, strength of the ion pair complex and on the osmotic gradient (i.e., there exists a coupling between water and ion pair fluxes). The relationship between ion pairing and the electrical conductivity of BLM is briefly discussed.     

A laser-T-jump study of the adsorption of dipolar molecules to planar lipid membranes

Phloretin and structurally related neutral molecules adsorb to the interface of lipid membranes and modify the electric dipole potential of the membrane/water interface. The adsorption process has been studied using a laser-T-jump relaxation technique in combination with an analysis of nonactin mediated potassium transport (see part I, Awiszus and Stark 1988).Deviations from the Langmuir isotherm were observed for most of the substance. The discrepancies were most pronounced at large surface densities, whereas good agreement was found at low concentrations in many cases.The partition coefficient in the limit of low concentrations was compared with that of octanol/water bulk phases. No correlation was found. The individual values of the two partition coefficients differed by more than three orders of magnitude. The contribution, b, of a single adsorbed molecule to the dipole potential could not be predicted from the dipole moment, _L , of the molecule measured in the bulk phase. Different values of b were found at identical values of _L .The study shows the limitations of the use of bulk phase data to predict molecular properties in lipid membranes.     

Interaction of the pore-forming protein equinatoxin II with model lipid membranes: A calorimetric and spectroscopic study.

The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry. EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition. This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10. For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule. The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements. Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG). The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII. The second transition involves the formation of alpha-helical structure. Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association.     

The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.     

Transport of potassium ions across planar lipid membranes by the antibiotic,grisorixin: I. The equilibrium state and self-diffusion K+ fluxes

Summary ⁴²K⁺ tracer flux and steady-state conductance measurements were carried out with bilayer lipid membranes containing grisorixin, a carboxylic polyether antibiotic. When the membranes are placed between two bulk aqueous solutions of identical composition, the exchange or self-diffusion transmembrane flux of potassium is measured by a method which allows the characterization of the bilayer K⁺ permeability at the equilibrium state. The K⁺ self-diffusion flux increases with the pH in the range pH 6 to pH 9 and reaches a constant value for values above 9. This can be directly related to the increase of the surface concentration of the 11 complex formed by K⁺ and the deprotonated polyether at both bilayer membrane interfaces. The transport model initially proposed by Pressman and coworkers (Proc. Natl. Acad. Sci. USA 58:1949–1956, 1967) is again taken into consideration in the quantitative analysis of the flux data. The transmembrane transport of K⁺ results from the translocation of its neutral complex with grisorixin and the association-dissociation of the antibiotic with either potassium or conditions by a translocation process of the acidic grisorixin. Using the data of some previous studies for mixed ionophorelipid monolayers at the air/water interface and the present results for the self-diffusion flux measurements, it was possible to propose an evaluation of the more important parameters characterizing the transport; namely, the total surface concentration of grisorixin, the interfacial pK and the translocation rate constant of its potassium neutral complex. The method proposed could be extended easily to other carboxylic polyethers, which would lead to an interesting comparison of their ionophoric properties using model membrane systems.     

Oligomerization and pore formation by equinatoxin II inhibit endocytosis and lead to plasma membrane reorganization

Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence cross-correlation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca(2+) entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II.     

Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) approximately 5.1 +/- 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) approximately 0.5 and a molar ratio of bound peptide-to-lipid of approximately 1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an "electroporation-like" mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures.