Study of F-actin interaction with planar and liposomal bilayer phospholipid membranes

Interaction of the cytoskeletal protein F-actin with planar bilayer lipid membrane (BLM) induced formation of single ionic channels in both NaCl and KCl bathing solutions. We also recorded noiselike high-currentjumps with a mean conductivity of approximately 160 pS, which might represent the simultaneous opening and closing of several channels of lower conductivity. The ratio of cation to anion permeabilities (Pc/Pa) of the BLM with many channels in KCl was 26 +/- 2. Freeze-fracture electron microscopy revealed fibrillar-like structures on the hydrophobic surfaces of liposomal membranes. We also observed some structural features giving evidence for the penetration of F-actin fibers through an artificial phospholipid membrane. We suggest that the F-actin/lipids complexes can transmit electric signals in synaptic and other intercellular contacts.     

The nonelectrolyte permeability of planar lipid bilayer membranes

The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.     

Perturbation of the lipid bilayer of model membranes by synthetic signal peptides

The interaction of synthetic peptides corresponding to the signal sequences of Escherichia coli alkaline phosphatase: Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala-OCH₃, chicken lysozyme: Met-Lys-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys(Bzl)-Phe-Leu-Pro-Leu-Ala-Ala-Leu-Gly-OCH₂-C₆H₅ and variant of the chicken lysozyme signal sequence with a charged residue in the hydrophobic region: Lys-Leu-Leu-Ile-Ala-Leu-Val-Leu-Lys-Phe-Leu-Pro-Leu-Ala-Ala-Leu-Gly-OCH₃ with model membranes of brain phosphatidylserine (PS) and egg phosphatidylcholine (PC) have been investigated by 90° light scattering and fluorescence spectroscopy. Our results indicate that the association of signal peptides with model membranes results in extensive perturbation of the lipid bilayer so as to cause fusion of PS vesicles and aggregation of PC vesicles. The vesicles are also rendered permeable to hydrophilic molecules like carboxyfluorescein. The variant peptide with the lysine residue in the hydrophobic region also has the ability to perturb lipid bilayers of model membranes.     

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.     

A quartz cell for studying planar lipid bilayer membranes

A quartz chamber is proposed for use in experiments with planar lipid bilayer membranes. Membranes are formed in a hole made on the lateral wall of a fused quartz test tube, immersed in an electrolyte solution. The quartz cell is easy to clean, chemically inert and easily made. Membranes formed in this chamber had specific resistances higher than 10⁸ Ω·cm² and excellent mechanical stability.     

Gating properties of channels formed by colicin Ia in planar lipid bilayer membranes

Summary Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (>+10 mV) are applied to thecis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant () of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric aroundV _o (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction inn, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be itcis orrans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change inn. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open,F _(v). For a given voltage,F _(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino-acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Ion channels on synaptic vesicle membranes studied by planar lipid bilayer method.

An anion selective channel and three types of cation selective channels were found in planar lipid bilayers incorporating synaptic vesicles from rat brains. In asymmetric KCl solutions (cis: 300 mM/trans: 150 mM), the anion selective channel showed a single-channel conductance of 94 pS and was inactivated by negative voltages and by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid disodium salt (SITS). In the same solution, single-channel conductances of three types of cation selective channels were 250 pS (Type 1), 248 pS (Type 2), and 213 pS (Type 3), respectively. These channels resembled one another in single-channel conductances but were different in gating behaviors. Type 1 channel, which was most frequently observed, had a remarkable subconducting state (175 pS). Type 2 channel had a flickering state that increased as the potential became more positive, and a long inactive state that increased as the potentials were more negative. Type 3 channel, which was also sensitive to the potentials, had the open-channel probability increased as the potential became more positive.     

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.     

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides.

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.     

Colicin N forms voltage- and pH-dependent channels in planar lipid bilayer membranes

The protein antibiotic colicin N forms ion-permeable channels through planar lipid bilayers. Channels are induced when positive voltages higher than +60 mV are applied. Incorporated channels activate and inactivate in a voltage-dependent fashion. It is shown that colicin N undergoes a transition between an “acidic” and a “basic” channel form which are distinguishable by different voltage dependences. The single-channel conductance is non-ohmic and strongly dependent on pH, indicating that titratable groups control the passage of ions through the channel. The ion selectivity of colicin N channels is influenced by the pH and the lipid composition of the bilayer membrane. In neutral membranes the channel undergoes a transition from slightly cation-selective to slightly anion-selective when the pH is changed from 7 to 5. In lipid membranes bearing a negative surface charge the channel shows a more pronounced cation selectivity which decreases but does not reverse upon lowering the pH from 7 to 5. The high degree of similarity between the channel characteristics of colicin A and N suggests that the channels share common features in their molecular structure. Offprint requests to: F. Pattus     

Formation of planar bilayer membranes from lipid monolayers. A critique.

The formation of planar bilayer membranes from lipid monolayers as described by Montal and Mueller (Proc. Natl. Acad. Sci. 1972. 69:3561) is analyzed. Bilayers absolutely free of alkane solvents or other nonpolar hydrocarbons can be formed on polytetrafluoroethylene (PTFE) (e.g. Teflon) septa only if certain boundary conditions are satisfied. Measurements have been made of the contact angles between monolayer-coated water and PTFE in the presence and absence of alkane solvents. The measurement suggest that the boundary conditions for formation of stable bilayers can be satisfied only when a nonpolar solvent is present. We conclude that the bilayer must be surrounded by a torus of alkane solvent, petroleum jelly, or silicone grease depending upon the details of technique used to form the bilayer. The non-polar solvent used in the formation of the bilayer may or may not be present in the bilayer depending upon the water solubility and size of the solvent molecule relative to the size of the alkyl chain of the lipid. Detailed sketches describing the formation of bilayers from monolayers are presented.     

Photocurrents generated by bacteriorhodopsin on planar bilayer membranes

When purple-membrane fragments from Halobacterium halobium are added to one aqueous phase of a positively-charged black lipid membrane, the membrane becomes photoelectrically active. Under normal conditions the steady-state photo-current is extremely low, but increases considerably when the lipid bilayer is doped with proton-permeable gramicidin channels or with a lipophilic acid-base system. These findings indicate that the purple-membrane sheets are bound to the surface of the bilayer, forming a sandwich-like structure. The time-behaviour of the photocurrent may be interpreted on the basis of a simple equivalent circuit which contains the conductance and capacitance of the purple membrane in series with the conductance and capacitance of the lipid bilayer. From the dependence of the photocurrent on the polarization of the exciting light the average angle between the transition moment of the retinal chromophore and the plane of the bilayer was calculated to be about 28 degrees. Furthermore, it was shown that chromophore-free apomembrane binds to the lipid bilayer and that its photoelectrical activity can be restored in situ by adding all-trans-retinal to the aqueous phase.     

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.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Exchange diffusion of dopamine induced in planar lipid bilayer membranes by the ionophore X537A

The ionophore X537A causes a large increase in the [(14)C]dopamine (a catecholamine) permeability of planar bilayer membranes. Dopamine transport increases linearly with the ionophore concentration. At relatively high concentrations in the presence of dopamine, the ionophore omdices a conductance which is nearly ideally selective for the dopamine cation. However, the total dopamine flux as determined in tracer experiments is not affected by an electric field and is over 10(5) times larger than predicted from the estimated dopamine conductance. Increasing the dopamine concentration on the side containing radioactive dopamine (the cis side) saturates the dopamine transport. This saturation is relieved by trans addition of nonradioactive dopamine, tyramine, H(+), or K(+). With unequal concentrations of dopamine cis and trans (49 and 12.5 mM), the unidirectional dopamine fluxes are equal. Increasing H(+) cis and trans decreases dopamine transport. It is concluded that at physiological pH, the X537A-induced transport of dopamine occurs via an electrically silent exchange diffusion of dopamine cation with another cation (e.g., dopamine(+), H(+), or K(+)). X537A induces a Ca(++)-independent release of catecholamines from sympathetic nerves by interfering with intracellular storage within storage vesicles (R.W. Holz. 1975. Biochim. Biophys. Acta. 375:138-152). It is suggested that X537A causes an exchange of intravesicular catecholamine with a cytoplasmic cation (perhaps K(+) or H(+)) across the storage vesicle membrane.     

Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes

alpha-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores.