Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Am J Physiol Renal Physiol. 2012 Sep 15;303(6):F800-11. doi: 10.1152/ajprenal.00703.2011. Epub 2012 Jul 11.
Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the β- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP(2). Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.
磷脂酰肌醇磷酸（PIPs）已知可调节上皮钠通道（ENaC）。脂质结合测定和共免疫沉淀表明，非洲爪蟾 ENaC 的β-和γ-亚基的氨基末端结构域可以直接结合磷脂酰肌醇 4,5-二磷酸（PIP（2）），磷脂酰肌醇 3,4,5-三磷酸（PIP（3））和磷脂酸（PA）。类似的测定表明，各种 PIP 可以强烈结合天然肉豆蔻酰化丙氨酸丰富的 C 激酶底物（MARCKS），但对 MARCKS 的突变体形式结合较弱或根本不结合。共聚焦显微镜显示 MARCKS 与 PIP（2）共定位。共聚焦显微镜还表明，MARCKS 在 PMA 诱导的 MARCKS 磷酸化或离子霉素诱导的细胞内钙增加后，从顶膜重新分布到细胞质。荧光共振能量转移研究表明，当 PKC 活性和细胞内钙浓度较低时，2F3 细胞中的 ENaC 和 MARCKS 彼此靠近。用 PMA 处理的非洲爪蟾 2F3 细胞的跨上皮电流测量和用 PKC 抑制剂处理的非洲爪蟾 2F3 细胞的单通道膜片钳研究改变了非洲爪蟾 ENaC 的活性，这表明 MARCKS 在调节非洲爪蟾 ENaC 活性中起重要作用。
