Random insertion of split-cans of the fluorescent protein venus into Shaker channels yields voltage sensitive probes with improved membrane localization in mammalian cells
Random insertion of split-cans of the fluorescent protein venus into Shaker channels yields voltage sensitive probes with improved membrane localization in mammalian cells
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DOI：
10.1016/j.jneumeth.2011.03.028
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发表时间：
2011-07-15
影响因子：
3
通讯作者：
Hughes, Thomas
中科院分区：
文献类型：
--
作者：
Jin, Lei;Baker, Bradley;Hughes, Thomas
FlaSh-YFP是一种荧光蛋白（FP）电压传感器，是Shaker钾通道与黄色荧光蛋白（YFP）的融合体，主要在哺乳动物细胞的内质网（ER）中表达，可能是由于错误折叠的单体。为了改善质膜表达，将FP分成两个非荧光的半部分。通过转座子反应将每一半随机插入Shaker单体中。含有5'半的Shaker亚基与含有3'半的Shaker亚基共表达。FP的再缀合需要Shaker亚基的四聚化。ER中捕获的错误折叠的单体不太可能四聚化和重建β-can结构，因此细胞内荧光可能会降低。这种分罐转座子方法产生了56个荧光探针，其中30个（54%）在质膜上表达，并能够光学报告膜电位的变化。对于100 mV去极化，来自这些新型FP-传感器的最大信号在Δ F/F中为-1.4%，开启时间常数为约15 ms，关闭时间常数为约200 ms。(C)2011爱思唯尔有限公司版权所有。
FlaSh-YFP, a fluorescent protein (FP) voltage sensor that is a fusion of the Shaker potassium channel with yellow fluorescent protein (YFP), is primarily expressed in the endoplasmic reticulum (ER) of mammalian cells, possibly due to misfolded monomers. In an effort to improve plasma membrane expression, the FP was split into two non-fluorescent halves. Each half was randomly inserted into Shaker monomers via a transposon reaction. Shaker subunits containing the 5' half were co-expressed with Shaker subunits containing the 3' half. Tetramerization of Shaker subunits is required for re-conjugation of the FP. The misfolded monomers trapped in ER are unlikely to tetramerize and reconstitute the beta-can structure, and thus intracellular fluorescence might be reduced. This split-can transposon approach yielded 56 fluorescent probes, 30 (54%) of which were expressed at the plasma membrane and were capable of optically reporting changes in membrane potential. The largest signal from these novel FP-sensors was a -1.4% in Delta F/F for a 100 mV depolarization, with on time constants of about 15 ms and off time constants of about 200 ms. This split-can transposon approach has the potential to improve other multimeric probes. (C) 2011 Elsevier B.V. All rights reserved.