Asymmetric Lipid Bilayers and Potassium Channels Embedded Therein in the Contact Bubble Bilayer

Cell membranes are highly intricate systems comprising numerous lipid species and membrane proteins, where channel proteins, lipid molecules, and lipid bilayers, as continuous elastic fabric, collectively engage in multi-modal interplays. Owing to the complexity of the native cell membrane, studying the elementary processes of channel-membrane interactions necessitates a bottom-up approach starting from forming simplified synthetic membranes. This is the rationale for establishing an in vitro membrane reconstitution system consisting of a lipid bilayer with a defined lipid composition and a channel molecule. Recent technological advancements have facilitated the development of asymmetric membranes, and the contact bubble bilayer (CBB) method allows single-channel current recordings under arbitrary lipid compositions in asymmetric bilayers. Here, we present an experimental protocol for the formation of asymmetric membranes using the CBB method. The KcsA potassium channel is a prototypical model channel with huge structural and functional information and thus serves as a reporter of membrane actions on the embedded channels. We demonstrate specific interactions of anionic lipids in the inner leaflet. Considering that the local lipid composition varies steadily in cell membranes, we `present a novel lipid perfusion technique that allows rapidly changing the lipid composition while monitoring the single-channel behavior. Finally, we demonstrate a leaflet perfusion method for modifying the composition of individual leaflets. These techniques with custom synthetic membranes allow for variable experiments, providing crucial insights into channel-membrane interplay in cell membranes.