Formation of bimolecular membranes from lipid monolayers.

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. Formation of the membrane is monitored by an increase of the electrical capacity, as measured with a voltage clamp. Electrical resistance of the unmodified membrane is analogous to that of conventional planar bilayers (black lipid membranes) prepared in the presence of a hydrocarbon solvent, i.e., 106f108 ohm cm2; the resistance can be lowered to values of 103 ohm cm2 by gramicidin, an antibiotic that modifies the conductance only when the membranes are of bimolecular thickness. In contrast to the resistance, there is a significant difference between the capacity of bilayers made from monolayers and that of hydrocarbon-containing bilayers made by phase transition; the average values are 0.9 and 0.45 AF cm-2, respectively. The value of 0.9 uF cm-2 approximates that of biological membranes. Assuming a dielectric constant of 2.1 for the hydrocarbon region, the dielectric thickness, as calculated from a capacity of 0.9 1F cm 2, is 22 A. This value is 6-10 A smaller than the actual thickness of the hydrocarbon region of bilayers and cell membranes, as determined by x-ray diffraction. The difference may be due to a limited penetration of water into the hydrocarbon region near the ester groups that would lower the electrical resistance of this region and reduce the dielectric thickness. Asymmetric membranes have been formed by adjoining two lipid monolayers of different chemical composition. 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 METHODS The following chemicals were used: glyceroldioleate (Armak Chemicals Division, Philadelphia, Pa. and Applied Science Laboratories, Inc., State College, Pa.), oleyoyl acid phosphate (Hooker Chemical Co., Niagara Falls, N.Y.), egg lecithin, bovine cardiolipin, and plant phosphatidylinositol (Applied Science Laboratories, Inc.), gramicidin and cholesterol (Sigma Chemical Co., St Louis, Mo). Analytical grade reagents were used throughout. White-matter lipid from brain was extracted according to Mueller et al. (7) or by the method of Leuzinger and Schneider (8). The membranes were formed initially with a modified version of the apparatus described by Takagi (9) (see Fig. la). It consists essentially of two monolayer troughs (18 X 12 cm) separated by a vertically movable septum containing in its center a hole of 1.0 cm (diameter) covered by a thin Teflon (tetrafluoroethylene) film (25 Mm thick), obtained from Yellow Springs Instruments, Yellow Springs, Ohio (Membrane Kit no. 5937); this film contains a small aperture (see 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 the film containing the aperture have a hydrophobic surface; this favors contact with the lipid hydrocarbon chains. Of several materials tested, only Teflon and 3561 $ To whom reprint requests should be addressed. D ow nl oa de d by g ue st o n F eb ru ar y 8, 2 02 0 3562 Biochemistry: Montal and Mueller