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Abstract：
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.
摘要：
Kir4.1/5.1异四聚体参与在远曲小管（DCT）中产生负细胞膜电位，并在确定Na-Cl协同转运蛋白（NCC）的活性中起关键作用。Kir5.1在其COOH末端含有磷酸苏氨酸基序(AA249-252)。免疫共沉淀显示Nedd4-2在共转染Kir5.1或Kir4.1/Kir5.1的HEK293细胞中与Kir5.1相关。GST下拉进一步证实了Nedd4-2和Kir5.1之间的关联。泛素化实验表明，Nedd4-2增加了与Kir4.1/Kir5.1共转染的细胞中Kir4.1/Kir5.1异源四聚体的泛素化，但对Kir4.1或Kir5.1没有影响。膜片钳和Westernblot还表明，Nedd4-2而非Nedd4-1的共表达降低了与Kir4.1和Kir5.1共转染的细胞中的K电流和Kir4.1表达。相比之下,Nedd4-2在不存在Kir5.1的情况下或在用灭活形式的Nedd4-2(Nedd4-2C821A)转染的细胞中不能抑制Kir4.1。此外，在Kir4.1和Kir5.1突变体(Kir5.1T249A)转染的HEK293细胞中,Kir5.1的COOH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K电流的抑制作用.最后,在肾脏特异性Nedd4-2敲除或Kir5.1敲除小鼠中,与相应的野生型同窝小鼠相比,DCT和Kir4.1表达的基底外侧K电导显著增加.我们得出结论，Nedd4-2在COOH末端的磷酸苏氨酸基序与Kir5.1结合，Nedd4-2与Kir5.1的结合促进了Kir4.1的泛素化，从而调节了其在DCT中的血浆表达。
