Kir5.1 Regulates Nedd4-2-Mediated Ubiquitination of Kir4.1 in Distal Nephron
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 co-transporter (NCC). Kir5.1 contains a phosphothreonine-motif at its c-terminus (AA249-252). Co-immunoprecipitation 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 co-transfected 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 co-expression 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 C-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 WT littermates. We conclude that Nedd4-2 binds to Kir5.1 at phosphothreonine-motif of the c-terminus and the association of Nedd4-2 with Kir5.1 facilitates the ubiquitination of Kir4.1 thereby regulating its plasma expression in the DCT.
Wang, M., Su, X., Wu, P., Gao, Z., Wang, W., Staub, O., & Lin, D. (2018). Kir5.1 Regulates Nedd4-2-Mediated Ubiquitination of Kir4.1 in Distal Nephron. American Journal of Physiology.Renal Physiology, 315 (4), F986-F996. https://doi.org/10.1152/ajprenal.00059.2018