Stockbridge Randy B, Tsai Ming-Feng
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts, USA.
Methods Enzymol. 2015;556:385-404. doi: 10.1016/bs.mie.2014.12.028. Epub 2015 Mar 21.
Ion channels are membrane proteins that facilitate passive ion movements down the electrochemical gradients across biological membranes. They regulate a wide range of important physiological functions, from neuronal excitation, muscle contraction, substance absorption in epithelial tissues, to innate immunity. In recent years, the membrane transport field has seen tremendous advances in membrane protein purification and structural determination. This paves the way for investigators to reconstitute ion channels into biochemically defined lipid systems free of other biological constituents and to study channel functions within a structural context. The goal of this chapter is to guide readers through the process of producing proteoliposomes, designing liposome assays for initial characterization of channel function, and performing electrical recordings for in-depth mechanistic investigation.
离子通道是膜蛋白,可促进离子顺着电化学梯度被动穿过生物膜。它们调节着广泛的重要生理功能,从神经元兴奋、肌肉收缩、上皮组织中的物质吸收到先天免疫。近年来,膜运输领域在膜蛋白纯化和结构测定方面取得了巨大进展。这为研究人员将离子通道重构到不含其他生物成分的生物化学定义脂质体系中,并在结构背景下研究通道功能铺平了道路。本章的目的是指导读者完成制备蛋白脂质体的过程、设计用于通道功能初步表征的脂质体检测方法以及进行电记录以进行深入的机制研究。