Bimolecular membranes are formed from two lipid monolayers at an air-water interface by the apposition of their hydrocarbon chains when an aperture in a Teflon partition separating two aqueous phases is lowered through the interface. Lipid bilayers, which are thought to be the basic structural element of cell membranes, account for many of their properties. They can be assembled from lipids either as small vesicles (1) or as single planar structures that separate two aqueous phases (2). Both models complement each other, and each has its own advantages and shortcomings. The spherical bilayers allow flux measurements with relative ease, and the absence of hydrocarbon solvent may be a factor aiding the incorporation of membrane proteins for functional reconstitutions (3-5). However, their inner compartment is small and inaccessible to chemical manipulation and electrical measurements. In planar bilayers, both compartments are easily accessible, but their mode of formation and the presence of hydrocarbon solvent may be responsible for reported failures to incorporate large membrane proteins. In addition, their electrical capacity is considerably lower than that of cell membranes, implying a different structure or thickness of the dielectric region.For these reasons the formation of planar bilayers without the aid of a hydrocarbon solvent would be desirable. We report here the formation of planar bilayers separating two aqueous phases, in the absence of hydrocarbon solvent, by the hydrophobic apposition of two lipid monolayers at an airwater interface, by a modification of the method used by Takagi, Azuma, and Kishimoto (6) to form "rhodopsin membranes." It will be shown that the electrical capacity of these bilayers exactly matches that of biological membranes, and that the system allows the formation of asymmetric membranes; eventually, this technique may aid in the incorporation of membrane proteins into the lipid bilayer. MATERIALS AND METHODSThe following chemicals were used: glyceroldioleate ( The membranes were formed initially with a modified version of the apparatus described by Takagi (9) (see Fig. la Fig. la). The septum is sealed with silicone grease to the walls of the trough and insulates the two water compartments electrically. It can be rmoved by a motor at a preset speed downwards, so that the aperture moves from above to below the water surface. The troughs and septum were made from Teflon. The aperture in the thin Teflon film was formed either by an electrically heated platinum wire, which was ground to a sharp point, or by a punch made from a tuberculin-syringe needle by beveling its wall. In a simplified version of this method, the thin Teflon film with the aperture was clamped between two halves of a trough and kept stationary. The membrane was formed by filling the two compartments with water or saline to below the aperture and, after spreading a lipid monolayer on each side, raising first one, then the other water level slowly above the aperture by gravity flow.It is important that th...

No abstract

Alamethicin, monazomycin, or EIM induce electrical excitability in lipid bilayers. The voltage-dependent gating displays all the characteristics observed in excitable cells and its basic features can be quantitatively described by the Hodgkin-Huxley equations.A common molecular mechanism of membrane excitation has been postulated.It assumes that in the absence of an electrical field the channel-forming molecules lie at the surface of the membrane. An applied potential tilts them from the surface into the hydrocarbon region of the bilayer. Once in this position the molecules diffuse laterally and form aggregates which act as channels for the flow of ions.ellipsoid with t w o glutamic residues at one end, and a metal ion in four-o r five-fold coordination with peptide carbonyl oxygens at the other. An applied field pulls the cationic end through the membrane t o the other side, while the glutamic residues hold the other end attached t o the original surface. The molecules now span the membrane and aggregate, forming oligomeric channels in which most of the peptide carbonyls face toward the center, and the methyl groups outward. Monomers and dimers do not conduct and an individual channel can have different conductance values depending on the number of monomers in the aggregate and the resulting channel diameter. A quantitative description of this process matches observed gating kinetics, gating currents, and the single channel conductance increments. Without additional assumptions, inactivation follows directly from the aggregation process because with proper rate constants, the average degree of polymerization and therefore number of open channels goes through a maximum in time.In the case of alamethicin we assume that the molecule forms an elongatedThe model may also apply t o the excitation process of higher cells.

No abstract

No abstract

No abstract

Single stable bimolecular lipid and proteolipid membranes having the inert physical properties of cell membrane can be reconstituted in saline solution. After adsorption of appropriate molecules, these membranes become electrically excitable.

No abstract