Determination of membrane protein transporter oligomerization in native tissue using spatial fluorescence intensity fluctuation analysis.
Determination of membrane protein transporter oligomerization in native tissue using spatial fluorescence intensity fluctuation analysis.
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DOI：
10.1371/journal.pone.0036215
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kurtz I
中科院分区：
文献类型：
--
作者：
Sergeev M;Godin AG;Kao L;Abuladze N;Wiseman PW;Kurtz I
膜转运蛋白存在于各种寡聚体状态之间的复杂动态平衡中，所述寡聚体状态包括单体、二聚体、二聚体的二聚体和更高级的寡聚体。鉴于其亚光学显微镜分辨率大小，膜转运蛋白的寡聚化状态难以在不需要组织破坏和间接生化方法的情况下进行定量。在这里，我们提出的应用程序的荧光测量技术相结合的荧光图像矩分析和空间强度分布分析（SpIDA），以确定在原位膜蛋白的寡聚化状态。作为模型系统，我们分析了在培养细胞和大鼠肾脏中的产电碳酸氢钠协同转运蛋白NBCe 1-A的寡聚状态。我们所描述的方法首次提供了研究膜转运蛋白在其天然状态下的寡聚状态的能力。
Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.
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