Klaerke Dan A, Tejada Maria de Los Angeles, Christensen Vibeke Grøsfjeld, Lassen Mette, Pedersen Per Amstrup, Calloe Kirstine
Department of Veterinary and Animal Sciences, Section for Anatomy, Biochemistry, and Physiology, University of Copenhagen, Frederiksberg, Denmark.
Department of Biology, Cell Biology and Physiology, University of Copenhagen, Copenhagen, Denmark.
Curr Protoc Pharmacol. 2018 Jun;81(1):e37. doi: 10.1002/cpph.37. Epub 2018 May 4.
Detergent-solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar "black lipid membranes" formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step-by-step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single-channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added. © 2018 by John Wiley & Sons, Inc.
经去污剂增溶的纯化离子通道可重构到脂质双层中用于电生理分析。传统上，离子通道被插入到囊泡中，随后与由溶解在诸如癸烷等疏水溶剂中的脂质形成的平面“黑色脂质膜”融合。本文提供了将纯化的离子通道蛋白重构到巨型单层囊泡（GUV）中的分步指南。该程序导致形成可用于平面双层形成和单通道电流电生理表征的脂质体。通过使用预先形成的GUV，可以省去膜溶剂。与传统制剂相比，由GUV形成的脂质双层提供了更类似于天然细胞膜的环境。还描述了一种替代方案，该方案需要从GUV生产平面脂质双层，并在其上添加去污剂中的蛋白质。©2018约翰威立父子公司。
