Mazzochi Christopher, Bubien James K, Smith Peter R, Benos Dale J
Department of Physiology and Biophysics and Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Biol Chem. 2006 Mar 10;281(10):6528-38. doi: 10.1074/jbc.M509386200. Epub 2005 Dec 14.
The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.
氨氯地平敏感的上皮钠通道(ENaC)的活性受F-肌动蛋白调节。然而,α-ENaC与肌动蛋白之间是否存在直接相互作用尚不清楚。我们使用共聚焦显微镜、免疫共沉淀和蛋白质结合研究相结合的方法,研究了肌动蛋白细胞骨架直接与α-ENaC羧基末端结合的假说。对稳定转染野生型大鼠α-、β-和γ-ENaC亚型的Madin-Darby犬肾细胞单层进行共聚焦显微镜观察,结果显示α-ENaC与顶端膜和顶端下细胞质中的皮质F-肌动蛋白细胞骨架共定位。在该细胞系的全细胞裂解物中,发现F-肌动蛋白与α-ENaC发生免疫共沉淀。凝胶覆盖试验表明F-肌动蛋白特异性结合α-ENaC的羧基末端。F-肌动蛋白共沉降研究进一步证实了F-肌动蛋白与α-ENaC羧基末端之间的直接相互作用。这是首次报道F-肌动蛋白与ENaC之间存在直接且特异性生化相互作用的研究。