Renal-Electrolyte Div., Dept. of Medicine, S976.1 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261.
Am J Physiol Renal Physiol. 2013 Oct 1;305(7):F943-56. doi: 10.1152/ajprenal.00303.2013. Epub 2013 Jul 17.
The vacuolar H(+)-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) inhibited V-ATPase-dependent H(+) secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3(-)-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.
液泡型 H(+)-ATP 酶（V-ATPase）在肾集合管的闰细胞中促进管腔酸化和非挥发性酸的排泄。我们之前的研究表明，V-ATPase 细胞质 V1 区的 A 亚基（ATP6V1A）可在体外和肾细胞中被代谢传感器 AMP 激活的蛋白激酶（AMPK）磷酸化。在此，我们通过实验证明，用 AMPK 激活剂 5-氨基咪唑-4-甲酰胺-1-β-D-核糖呋喃糖苷（AICAR）处理兔离体灌注集合管，可抑制酸负荷后闰细胞中 V-ATPase 依赖的 H(+)分泌。我们通过质谱鉴定出 Ser-384 是 V-ATPase A 亚基的一个主要 AMPK 磷酸化位点，这一结果通过比较野生型 A 亚基（WT-A）与 Ser-384 到 Ala 突变的 A 亚基（S384A-A）在体外和完整的 HEK-293 细胞中 AMPK 依赖性磷酸化标记得到证实。与表达 WT-A 的 HEK-293 细胞相比，表达 S384A-A 的细胞中 HCO3(-) 含培养基的稳态酸化更为明显。此外，用 AICAR 处理表达 WT-A 亚基的克隆 C 兔闰细胞可减少 V-ATPase 依赖的细胞外酸化，而在表达磷酸化缺陷的 S384A-A 突变体的细胞中这种作用被阻断。最后，S384A-A 突变体的表达可阻止 AICAR 在克隆 C 细胞中引起的 V-ATPase 细胞质重分布。总之，AMPK 对 A 亚基 Ser-384 的直接磷酸化是肾闰细胞中 V-ATPase 的一种新的调节机制。AMPK 对 V-ATPase 的调节可能将 V-ATPase 的活性与细胞代谢状态联系起来，这与肾和其他组织中的缺血性损伤有关。
