NYMC Faculty Publications

Kir5.1 Regulates Nedd4-2-Mediated Ubiquitination of Kir4.1 in Distal Nephron

DOI

10.1152/ajprenal.00059.2018

Journal Title

American Journal of Physiology.Renal Physiology

First Page

F986

Last Page

F996

Document Type

Article

Publication Date

October 2018

Department

Pharmacology

Keywords

Kidney Tubules, Distal, Nedd4 Ubiquitin Protein Ligases, Potassium Channels, Inwardly Rectifying, Sodium Chloride Symporters, EAST/SeSAME syndrome, Kcnj10, Kcnj16, NCC, Nedd4-1

Disciplines

Medicine and Health Sciences

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 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.

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