酸敏感离子通道亚基组装的荧光共振能量转移分析
Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel.
作者信息
Gao Ying, Liu Shuang-Shuang, Qiu Shuang, Cheng Wei, Zheng Jie, Luo Jian-Hong
机构信息
Department of Neurobiology, Institute for Neuroscience, Zhejiang University School of Medicine, Hangzhou 310058, China.
出版信息
Biochem Biophys Res Commun. 2007 Jul 20;359(1):143-50. doi: 10.1016/j.bbrc.2007.05.077. Epub 2007 May 22.
Acid-sensing ion channels (ASICs) are believed to be homo- or heteromeric complexes, which have been verified by classical methods such as co-immunoprecipitation or electrophysiological assays. However, the exact subunit combinations of ASICs in living cells have not been established yet. Here, we apply assays based on fluorescence resonance energy transfer (FRET) between GFP color mutants CFP and YFP to investigate ASIC assembly directly in living cells. Homomerization as well as heteromerization of different combinations of ASIC subunits were found. In addition, our results suggest the formation of heteromeric 1a/2a channels of stoichiometry consisting of at least two 1a subunits and two 2a subunits. Similar stoichiometry was observed from heteromeric 1a/2b and 2a/2b channels. Our results imply that these heteromeric ASIC channels contain at least four subunits.
酸敏感离子通道（ASICs）被认为是同聚体或异聚体复合物，这已通过共免疫沉淀或电生理分析等经典方法得到证实。然而，活细胞中ASICs的确切亚基组合尚未确定。在此，我们应用基于绿色荧光蛋白（GFP）颜色突变体CFP和YFP之间荧光共振能量转移（FRET）的分析方法，直接在活细胞中研究ASIC的组装。我们发现了ASIC亚基不同组合的同聚化以及异聚化现象。此外，我们的结果表明形成了化学计量比为至少两个1a亚基和两个2a亚基的异聚体1a/2a通道。在异聚体1a/2b和2a/2b通道中也观察到了类似的化学计量比。我们的结果表明，这些异聚体ASIC通道至少包含四个亚基。
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