de Wet Heidi, Lippiat Jonathan D, Allen Marcus
Institute of Membrane and Systems Biology, University of Leeds, UK.
Methods Mol Biol. 2008;491:177-86. doi: 10.1007/978-1-59745-526-8_14.
A number of recent studies have described the activation of BK(Ca) channels by steroid hormones such as estrogen. The proposed mechanisms are diverse and include both the direct interaction with the ion channel subunits and the stimulation via receptor activation and cell signalling pathways. To investigate the activation of BK(Ca) channels by estrogen we devised a cell-free system by incorporating recombinant channels of known subunit composition into artificial bilayers and recorded single channel currents. This chapter describes the methods used to prepare purified membrane fractions from cultured cells and the construction of artificial phospholipids bilayers for the incorporation and recording of ion channels.
最近的一些研究描述了类固醇激素（如雌激素）对大电导钙激活钾通道（BK(Ca)通道）的激活作用。提出的机制多种多样，包括与离子通道亚基的直接相互作用以及通过受体激活和细胞信号通路的刺激。为了研究雌激素对BK(Ca)通道的激活作用，我们设计了一种无细胞系统，将已知亚基组成的重组通道整合到人工双层膜中，并记录单通道电流。本章介绍了从培养细胞中制备纯化膜组分的方法，以及用于整合和记录离子通道的人工磷脂双层膜的构建方法。
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