BK通道在细胞膜上的单分子定位及空间分布  

Single-Molecule Localization and Spatial Distribution of BK Channels in the Cell Membrane

作　　者：刘永锋[1] 侯盼盼[1] 夏程龙 王伟[1] 孔文娟[1] 丁久平[1] 

机构地区：[1]华中科技大学生命科学与技术学院,湖北武汉430074 [2]中国科学技术大学生命科学学院,安徽合肥230027

出　　处：《现代生物医学进展》2014年第8期1411-1414,共4页Progress in Modern Biomedicine

基　　金：国家重点基础发展规划项目(2010CB529804);国家自然科学基金项目(31170814)

摘　　要：目的:研究大电导、钙离子和电压激活的钾离子通道(BK通道)在HEK293细胞膜上的单分子定位及其总体空间分布情况。方法:分别用mEos2、Dronpa等荧光蛋白标记BK通道的α亚基和辅助性β2亚基,将这些质粒在HEK293细胞内瞬时转染以表达通道蛋白,然后用激光共聚焦荧光显微成像、全内反射荧光显微成像、光敏定位荧光成像等技术观察BK通道的亚细胞定位及单分子分布,并用电生理实验技术检测荧光蛋白对BK通道有影响。结果:激光共聚焦荧光显微成像和全内反射荧光显微成像技术只能在亚细胞水平定位通道蛋白,BK通道在细胞膜上聚集并形成不规则的蛋白簇,它的α亚基和β2亚基在细胞膜上完全共定位;光敏定位荧光成像技术成功定位BK通道蛋白簇里面的单分子,虽然α和β2亚基紧紧靠在一起,它们之间依然存在空间距离;BK通道的质膜表达和功能特性不受荧光蛋白的影响。结论:BK通道蛋白簇里面包含大量的α和β2亚基的蛋白单分子,它们紧密地聚集在一起,但是并没有完全共定位,在分子水平上揭示了BK通道α和β亚基功能耦合的结构基础,为以后研究大分子蛋白质间的相互作用机制提供了很好的分子模型,光敏定位荧光成像技术作为一种全新的单分子荧光成像手段,在基因表达、信号通路、蛋白质相互作用等许多重要生命活动的研究中发挥重要作用。Objective： To investigate the Single-molecule localization and spatial distribution of BK channels in cultured HEK293 cells. Methods： BKct and β2 subunits were labeled with fluorescence proteins, such as mEos2, Dronpa, TagGFP. Then the plasmids were transferred into HEK293 cells and cultured at 37℃ for 24 h. The cells were observed by laser scanning confocal microscopy （LSCM）, total internal reflection fluorescence microscopy （T1RFM） and photoactivated localization microscopy （PALM）. The effects of fluorescence proteins on BK channel were also investigated by patch clamp recording technique. Results： Fluorescence images obtained from LSCM and TIRFM microscopy revealed that BK channels assembled into irregular clusters and BK ct subunits were well co-localized with β2 subunits in HEK293 cells. While the PALM microscopy illustrated that there is spatial distance between BKα and β2 molecules. The localization and function properties of BK channel were not influenced by the fused fluorescence proteins. Conclusion： Each cluster of BK channel was assembled with many α andβ molecules but they were not fully co-localized. This result revealed the structural basis of BK channels at the molecule level and provided a model for studying the molecular mechanisms of protein interaction. Also PALM microscopy servers as a new tool in the research of many vital activities, such as gene expression, signaling pathways and protein-protein interaction.

关 键 词：BK通道 辅助性亚基 光敏定位荧光成像(PALM) 共定位 

分 类 号：Q73[生物学—分子生物学] Q248