Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties

Abstract

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...

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Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

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