Department of Anesthesiology and Reanimatology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.
Department of Molecular Neuroscience, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.
Methods Mol Biol. 2024;2796:1-21. doi: 10.1007/978-1-0716-3818-7_1.
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.
细胞膜是一个高度复杂的系统，包含许多脂质种类和膜蛋白。其中，通道蛋白、脂质分子和脂质双层作为连续的弹性膜，共同参与多种模式的相互作用。由于天然细胞膜的复杂性，研究通道-膜相互作用的基本过程需要从形成简化的合成膜开始，采用自下而上的方法。这就是建立由具有明确定义的脂质组成的脂质双层和通道分子组成的体外膜重组系统的基本原理。最近的技术进步促进了不对称膜的发展，接触气泡双层（CBB）方法允许在不对称双层中的任意脂质组成下记录单通道电流。在这里，我们介绍了使用 CBB 方法形成不对称膜的实验方案。KcsA 钾通道是一个具有巨大结构和功能信息的典型模型通道，可作为嵌入通道上膜作用的报告器。我们展示了内层中阴离子脂质的特定相互作用。考虑到细胞膜中局部脂质组成不断变化，我们提出了一种新的脂质灌注技术，允许在监测单通道行为的同时快速改变脂质组成。最后，我们展示了一种用于修饰单层脂质组成的叶层灌注方法。这些具有定制合成膜的技术允许进行可变实验，为细胞膜中通道-膜相互作用提供了重要的见解。
