Yoo Dana, Kim Bo Young, Campo Cristina, Nance Latreece, King Amanda, Maouyo Djikolngar, Welling Paul A
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2003 Jun 20;278(25):23066-75. doi: 10.1074/jbc.M212301200. Epub 2003 Apr 8.
The Kir1.1 (ROMK) subtypes of inward rectifier K+ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney.
内向整流钾离子通道的Kir1.1（ROMK）亚型介导钾分泌，并调节肾脏中氯化钠的重吸收。皮质集合管顶端膜上ROMK通道的密度与生理需求协同精确调节。尽管Kir1.1中三个磷酸化位点之一的丝氨酸-44（Ser-44）的蛋白激酶A依赖性磷酸化也是一个典型的血清糖皮质激素调节激酶（SGK-1）磷酸化位点，控制着活性通道的数量，但尚不清楚这是否涉及激活已经存在于质膜上的休眠通道，还是将新通道招募到细胞表面。在这里，我们探索其机制，并测试ROMK的SGK-1磷酸化是否调节细胞表面表达。通过点突变（Kir1.1，S44A）去除磷酸化位点，显著减弱了非洲爪蟾卵母细胞中的宏观电流密度。通过针对外部表位标记形式的Kir1.1的抗体结合测量，Kir1.1 S44A的表面表达受到抑制，这与宏观电流的降低平行。相反，磷酸化模拟突变Kir1.1 S44D增强了表面表达和宏观电流密度。体外磷酸化分析表明，丝氨酸-44是SGK-1磷酸化的底物，野生型通道与SGK-1共表达将通道密度增加到与磷酸化模拟突变相同的水平。此外，磷酸化位点的突变完全消除了SGK-1的刺激作用。总之，Kir1.1的SGK-1磷酸化驱动其在质膜上的表达。由于SGK-1是一种早期醛固酮诱导基因，我们的结果提示了醛固酮依赖性调节肾脏分泌性钾通道的一种可能分子机制。
