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首页> 外文期刊>Cellular Physiology and Biochemistry >A FRET-Based Approach for Quantitative Evaluation of Forskolin-Induced Pendrin Trafficking at the Plasma Membrane in Bronchial NCI H292 Cells
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A FRET-Based Approach for Quantitative Evaluation of Forskolin-Induced Pendrin Trafficking at the Plasma Membrane in Bronchial NCI H292 Cells

机译：基于FRET的定量评估毛喉素诱导的Pendrin在支气管NCI H292细胞质膜中运输的方法

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biBackground /i/bHuman pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important. biMethods /i/bTrafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions. biResults /i/bFRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition. biConclusion /i/bTrafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable.
机译：背景 人pendrin（SLC26A4，PDS）是一种完整的膜蛋白，可作为电子中性阴离子交换剂。 Pendrin蛋白功能丧失的突变会导致Pendred综合征，该疾病以感觉神经性耳聋和部分碘化物组织缺陷为特征，可能导致甲状腺甲状腺肿。此外，Pendrin上调可能在多种疾病的发病机理中起作用，包括支气管哮喘和慢性阻塞性肺疾病（COPD）。因此，在活细胞的环境中监测质膜的丰度和联苯戊醚的运输至关重要。 方法 通过荧光共振能量转移（FRET）监测Pendrin向质膜的转运，该现象是在靠近两个荧光团（FRET供体和受体）之间发生的一种物理现象。空间接近度。由于能量转移的效率与供体与受体之间距离的六次方成反比，因此FRET对供体与受体之间距离的微小变化极为敏感，因此是确定蛋白质与蛋白质相互作用的有力工具。 结果 FRET研究表明，福司可林诱导的cAMP产生与质膜上pendrin表达的显着增加有关，这与细胞内pH的降低平行。 Pendrin转位至膜的过程通过Rho-GTPase抑制伴随着肌动蛋白细胞骨架的部分解聚。 结论 贩运到质膜对于Pendrin活性的调节至关重要。因此，非常需要用于监视和量化此现象的可靠工具。

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