Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Am J Physiol Renal Physiol. 2010 Dec;299(6):F1308-19. doi: 10.1152/ajprenal.00423.2010. Epub 2010 Sep 22.
The KCNQ1 K(+) channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQ1 plasma membrane expression and that AMPK regulates epithelial Na(+) channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct (mpkCCD(c14)) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized mpkCCD(c14) cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane.
KCNQ1 K(+) 通道在调节多种生理功能方面发挥着关键作用，包括心脏兴奋性、心血管张力和体内电解质稳态。代谢传感器 AMP 激活的蛋白激酶 (AMPK) 已被证明可以调节越来越多的离子转运蛋白。为了确定 AMPK 是否调节 KCNQ1，我们研究了 AMPK 激活对非洲爪蟾卵母细胞和集合管上皮细胞中 KCNQ1 电流的影响。AMPK 激活降低了非洲爪蟾卵母细胞中的 KCNQ1 电流和通道表面表达，但 AMPK 并未在体外使 KCNQ1 磷酸化，表明存在间接调节机制。最近已经表明，泛素蛋白连接酶 Nedd4-2 抑制 KCNQ1 质膜表达，并且 AMPK 通过 Nedd4-2 调节上皮钠通道，因此我们研究了 Nedd4-2 在 AMPK 依赖性调节 KCNQ1 中的作用。在共表达显性负性或组成性激活的 Nedd4-2 突变体或 Nedd4-2 相互作用缺陷的 KCNQ1 突变体的卵母细胞中，通道抑制被 AMPK 阻断，这表明 Nedd4-2 参与了 AMPK 对 KCNQ1 的调节。KCNQ1 在小鼠极化肾皮质集合管 (mpkCCD(c14)) 细胞和大鼠肾脏中表达于基底外侧膜。用 AMPK 激活剂 AICAR（2 mM）或二甲双胍（1 mM）处理可使顶端通透的极化 mpkCCD(c14)细胞中的基底外侧 KCNQ1 电流减少。此外，AICAR 处理离体大鼠肾切片诱导 AMPK 激活和 KCNQ1 从集合管主细胞的基底外侧膜的细胞内重分布。AICAR 处理还诱导从肾切片匀浆中免疫沉淀的 KCNQ1 的泛素化增加。这些结果表明，AMPK 通过促进 Nedd4-2 依赖性通道泛素化和从质膜回收来抑制 KCNQ1 活性。
