Polymerization in black lipid membranes

A variety of different lipids containing dienoyl groups in the side chains were tested for membrane formation using the planar lipid bilayer approach. One of these lipids formed stable bilayers which could be polymerized using UV-illumination. The influence of the polymerization was studied in monolayers, lipid vesicles and planar bilayers. The stability of the lipid bilayer membranes was increased by polymerization. Thus, the lifetime of the membranes increased from about 1 h to 4–5 h or longer. Furthermore, the specific conductance of unmodified membranes and of carrier-mediated transport is reduced. The transport of lipophilic ions was investigated as a function of polymerization using the charge-pulse method. The absorption of dipicrylamine (DPA^-) is not affected. The translocation of this compound and of tetraphenylborate (B(Ph) ₄ ^- ) showed a strong decrease with polymerization time. The influence of polymerization on the membrane structure may be explained on the basis of a strong viscosity increase in the lipid bilayer membrane.     

Fluctuation and relaxation analysis of monazomycin-induced conductance in black lipid membranes

Summary Fluctuation and relaxation analyses were performed on monazomycin-induced conductance of lipid bilayer membranes. With both methods a slow (sec) and a fast (msec) current component are apparent; however, the amplitude of the slow, voltage-dependent process is greater than that of the fast component in the step relaxation experiment and less in the fluctuation experiment. The fluctuation analysis showed principally a rapid voltage-dependent process which appears to be related to the multistate character of the conducting channel. The experimental results are interpreted in terms of a simplified kinetic model which is used to calculate relaxation and noise amplitudes.     

Curvature-electric effect in black lipid membranes

Upon periodical bending of a BLM, by means of oscillating hydrostatic pressure with sound frequency, the generation of an a.c. electric current with the same frequency can be observed under short circuit conditions. Previously, this phenomenon was attributed by us to a displacement current due to the oscillating flexoelectric polarization. The latter is proportional to the membrane curvature and depends on the lipid dipole moment and surface charge.The theory of this effect is outlined here. Earlier results concerning dipolar and quadrupolar contributions to the total current are presented and new expressions about charge contributions are derived for the two basic regimes of free and blocked lateral lipid exchange.Further, a systematic study of the frequency dependence of the amplitude and phase of the curvature-electric signal from a bacterial phosphatidylethanolamine/n-decane BLM is reported. Constant membrane curvature at each vibration frequency was assured by a calibration of the capacitance current observed with a small transmembrane voltage.The frequency dependence of the curvature-electric current amplitude was characterized by two regions: low frequency plateau and high frequency slope, the boundary between them being about 160 Hz. Such behaviour suggested a switching of the mechanism of membrane polarization from free to blocked lateral lipid exchange. Frequency dependence of the phase shift was characterized by low frequency and high frequency plateaus and a gradual transition between them. From phase measurements on initially curved membranes the sign of the membrane flexo-coefficient was found to be negative.The influence of some modifiers of the surface charge and surface dipole, as well as of the membrane conductivity, upon the value of the effect was studied. Surface charge was separately measured by the internal field compensation method under an ionic strength gradient. The membrane flexoelectric coefficient was evaluated and compared to the theoretical predictions. A conclusion was drawn that under the present experimental conditions the main contribution to the effect comes from the curvatureinduced shift of the surface charge equilibrium.Presented at the Tenth International Liquid Crystal Conference, 15–21 July 1984, York, UK     

Interaction of liposomes with black lipid membranes

Liposomes labelled with fluorescent pigments were allowed to interact with black lipid films. The transfer of label from the liposomes to the film was studied by fluorescence and photoelectric measurements. With the neutral lipid lecithin, in contrast to negatively charged phosphatidylinositol, a rapid transfer was observed. The results are discussed with respect to fusion of liposomes with black lipid films.     

Thickness fluctuations in black lipid membranes.

Because a black lipid membrane is compressible, there will be spontaneous fluctuations in its thickness. Qualitative arguments are given that the preferred configuration of the membranes is flat and that thickness fluctuations are smaller in amplitude than the differences in mean thickness observed using different hydrocarbon solvents. Fluctuations with short characteristic lengths will not be large as a result of the large amounts of oil-water contact these would entail. Quantitative analysis based on an extension of the treatment for soap films, predicts that the root mean square (rms) amplitude for fluctuations of wavelength longer than approximately 10 nm is negligible for glyceryl monooleate membranes with squalene (less than 3%) but may be approximately 20% with n-decane. rms fluctuations of 20% would lead to a discrepancy between the rms thickness of the core and the mean reciprocal thickness of only 6%.     

Interaction of saponin and digitonin with black lipid membranes and lipid monolayers

The effects of the plant glycosides saponin as well as digitonin on the electrical conductance of black lipid membranes and the effect of these agents on the surface pressure of lipid monofilms was investigated. Both saponin and digitonin induced channel-like fluctuations in planar bilayers made either of diphytanoylphosphatidylcholine ( DPhPC ) or of DPhPC and cholesterol 2: 1 (w/w). In cholesterol-free bilayers the amount needed to induce an increase in conductance was 0.3-1 mg/ml for saponin and about 0.2 mg/ml for digitonin. In contrast, in cholesterol-containing bilayers the concentration needed to induce pores was about 10 micrograms/ml for both saponin and digitonin. In cholesterol-containing membranes the fluctuating pores induced by saponin were about 3-times more permeable to K+ than to Cl- and the macroscopic current showed an ohmic behaviour. Surface pressure experiments demonstrate that both glycosides could penetrate into lipid monofilms of pure DPhPC spread at the air/water interface with an initial surface pressure of 30 mN/m. The increase in surface pressure was considerably enhanced in cholesterol-containing films. It is assumed that the channel-like fluctuations induced by saponin as well as digitonin, in both cholesterol-free and cholesterol-rich bilayers are due to the formation of micellar structures within the lipid lattice. Probably the penetration of the glycosides into the lipid bilayer is considerably enhanced by the presence of cholesterol.     

Concentration of black spots and the effect of detergents on black lipid membranes

Effect of water-soluble detergents, such as triton X-100, saponin and trimethyl octadecylammonium bromide on the concentration of black spots, tension and stability of black lipid membranes was studied. Changes in mechanic stability of the lipid bilayer are discovered, which are in a good correlation with the litic effect of true detergents on cell membranes.     

Incorporation of ionic channels from yeast plasma membranes into black lipid membranes.

Recently, patch-clamping of yeast protoplasts has revealed the presence of plasma membrane K+ channels (Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culberston, and C. Kung. 1986. Science (Wash. DC). 233:1195-1197). In this work we show that fusion of purified plasma membranes into planar bilayers allows the study of the yeast channels. The main cationic conductances detected were of 64 and 116 pS, however, larger and smaller conductances have been observed. The two main conductances were sensitive to the K+ channels blockers tetraethylammonium (TEA+) and Ba2+. Bionic experiments indicated that both conductances were K+ selective.     

Charge transfer mediated by nigericin in black lipid membranes

Nigericin, in the concentration range (10^–6M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K⁺ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K⁺. This charged complex accounts for the uncoupling effect observed in intact mitochondria.A preliminary communication of this work was presented at the International School on Mitochondria: Biogenesis, Structure and Function. Mexico City, June 1975.     

Repulsive stabilization in black lipid membranes. A hydrodynamic model

A linear stability analysis is performed for a black lipid membrane. The hydrodynamic model consists of a viscous hydrocarbon film sandwiched between two aqueous phases. Attractive forces (van der Waals and electrical) and repulsive forces (steric) are expressed as body forces in the equations of fluid motion in the three phases. The steric repulsion due to overlap of the hydrocarbon chains of the lipids at small film thicknesses is described via an exponentially decaying interaction potential. The dispersion equation displays two modes of vibrations: the bending mode with the two Film surfaces transversely in phase, and the squeezing mode with the two surfaces 180 degrees out of phase. For symmetrical films, these two modes are uncoupled, and the squeezing mode (with thickness variations) is stabilized by the repulsive interactions. For nonsymmetrical films (different surface tensions, surface charges, etc.). these two modes are coupled and the asymmetry induces a shift of the marginal stability curve to shorter wavelengths.     

Anionic detergents as divalent cation ionophores across black lipid membranes

Summary Three ionic detergents commonly used in membrane-bound protein isolation and reconstitution experiments, SDS, cholate, and DOC, are shown to act as divalent cation ionophores when incorporated into black lipid membranes made from either oxidized cholesterol or a mixture of phosphatidylcholine and cholesterol (PC/cholesterol=51 mg). At a concentration greater than or equal to 1 m, SDS shows large selectivity differences between cations and anions and among the different cations tested (Ba²⁺, Ca²⁺, Sr²⁺, Mg²⁺, and Mn²⁺). Deoxycholate and cholate at concentrations greater than 4×10^–4 m and 10^–3 m, respectively, also act as divalent cation ionophores. The selectivity sequence measured for these two detergents is evidence for a strong ionic interaction between the divalent cation, and the anionic charged groups on the detergent. In the case of cholate, the conductance depends on the third or fourth power of the cholate concentration and shows a linear dependence on CaCl₂ concentration. The conductance for deoxycholate depends on the sixth or seventh power of the DOC concentration and is also linearly dependent on the CaCl₂ concentration. In an oxidized cholesterol black lipid membrane in the presence of 5mm CaCl₂, small concentrations of LaCl₃ (<1 m) inhibit the ionophoric activity of each of the detergents tested. Evidence is presented to show that this inhibitory effect is a nonspecific effect on oxidized cholesterol BLM's, and is not due to a direct effect of La³⁺ on detergent-mediated transport.     

Charge transfer mediated by nigericin in black lipid membranes.

Nigericin, in the concentration range (10(-6) M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K+ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K+. This charged complex accounts for the uncoupling effect observed in intact mitochondria.