期刊
JOURNAL OF GENERAL PHYSIOLOGY
卷 126, 期 5, 页码 429-438出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200509368
关键词
-
类别
资金
- NINDS NIH HHS [R01 NS021501, R01-NS21501, P01 NS042202, P01-NS42202] Funding Source: Medline
The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer ( FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the alpha subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The success rate of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据