Kir5.1 regulates Nedd4-2-mediated ubiquitination of Kir4.1 in distal nephron

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.

Keywords: EAST/SeSAME syndrome; Kcnj10; Kcnj16; NCC; Nedd4-1.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Ion Transport / physiology
  • Kidney Tubules, Distal / metabolism
  • Membrane Potentials / physiology
  • Mice, Knockout
  • Nedd4 Ubiquitin Protein Ligases / metabolism*
  • Nephrons / metabolism*
  • Potassium Channels, Inwardly Rectifying / metabolism*
  • Ubiquitination*


  • Kcnj10 (channel)
  • Kir5.1 channel
  • Potassium Channels, Inwardly Rectifying
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4l protein, mouse