Department of Physiology, Zunyi Medical College, Zunyi, China.
Department of Pharmacology, New York Medical College, Valhalla, New York.
Am J Physiol Renal Physiol. 2018 Oct 1;315(4):F986-F996. doi: 10.1152/ajprenal.00059.2018. Epub 2018 Jun 13.
Kir4.1/5.1 heterotetramer participates in generating the negative cell membrane potential in distal convoluted tubule (DCT) and plays a critical role in determining the activity of Na-Cl cotransporter (NCC). Kir5.1 contains a phosphothreonine motif at its COOH terminus (AA249-252). Coimmunoprecipitation showed that Nedd4-2 was associated with Kir5.1 in HEK293 cells cotransfected with Kir5.1 or Kir4.1/Kir5.1. GST pull-down further confirmed the association between Nedd4-2 and Kir5.1. Ubiquitination assay showed that Nedd4-2 increased the ubiquitination of Kir4.1/Kir5.1 heterotetramer in the cells cotransfected with Kir4.1/Kir5.1, but it has no effect on Kir4.1 or Kir5.1 alone. Patch-clamp and Western blot also demonstrated that coexpression of Nedd4-2 but not Nedd4-1 decreased K currents and Kir4.1 expression in the cells cotransfected with Kir4.1 and Kir5.1. In contrast, Nedd4-2 fails to inhibit Kir4.1 in the absence of Kir5.1 or in the cells transfected with the inactivated form of Nedd4-2 (Nedd4-2C821A). Moreover, the mutation of TPVT motif in the COOH terminus of Kir5.1 largely abolished the association of Nedd4-2 with Kir5.1 and abolished the inhibitory effect of Nedd4-2 on K currents in HEK293 cells transfected with Kir4.1 and Kir5.1 mutant (Kir5.1T249A). Finally, the basolateral K conductance in the DCT and Kir4.1 expression is significantly increased in the kidney-specific Nedd4-2 knockout or in Kir5.1 knockout mice in comparison to their corresponding wild-type littermates. We conclude that Nedd4-2 binds to Kir5.1 at the phosphothreonine motif of the COOH terminus, and the association of Nedd4-2 with Kir5.1 facilitates the ubiquitination of Kir4.1, thereby regulating its plasma expression in the DCT.
Kir4.1/5.1 异四聚体参与产生远端卷曲小管 (DCT) 的负细胞膜电位，并在决定 Na-Cl 共转运蛋白 (NCC) 的活性方面发挥关键作用。Kir5.1 在其羧基末端 (AA249-252) 含有一个磷酸苏氨酸基序。共免疫沉淀显示，在共转染 Kir5.1 或 Kir4.1/Kir5.1 的 HEK293 细胞中，Nedd4-2 与 Kir5.1 相关。GST 下拉进一步证实了 Nedd4-2 与 Kir5.1 之间的关联。泛素化测定表明，Nedd4-2 增加了共转染 Kir4.1/Kir5.1 的细胞中 Kir4.1/Kir5.1 异四聚体的泛素化，但对单独的 Kir4.1 或 Kir5.1 没有影响。膜片钳和 Western blot 也表明，Nedd4-2 的共表达而不是 Nedd4-1 的共表达降低了共转染 Kir4.1 和 Kir5.1 的细胞中的 K 电流和 Kir4.1 表达。相比之下，在没有 Kir5.1 或在转染 Nedd4-2 失活形式 (Nedd4-2C821A) 的细胞中，Nedd4-2 未能抑制 Kir4.1。此外，Kir5.1 羧基末端 TPVT 基序的突变在很大程度上消除了 Nedd4-2 与 Kir5.1 的关联，并消除了 Nedd4-2 对共转染 Kir4.1 和 Kir5.1 突变体 (Kir5.1T249A) 的 HEK293 细胞中 K 电流的抑制作用。最后，与相应的野生型同窝仔相比，肾脏特异性 Nedd4-2 敲除或 Kir5.1 敲除小鼠的 DCT 基底外侧 K 电导和 Kir4.1 表达显著增加。我们的结论是，Nedd4-2 结合到 Kir5.1 的羧基末端磷酸苏氨酸基序上，Nedd4-2 与 Kir5.1 的结合促进了 Kir4.1 的泛素化，从而调节其在 DCT 中的血浆表达。
