caa a22 4500 530045885 CHVBK 20190303135355.0 cr unu---uuuuu 181024e20181001xx s 000 0 eng 10.1152/ajprenal.00059.2018 doi (SERVAL)BIB_177C962852AF 29897283 pmid 000447626000028 isiid Kir5.1 regulates Nedd4-2-mediated ubiquitination of Kir4.1 in distal nephron [Elektronische Daten] [M.X. Wang, X.T. Su, P. Wu, Z.X. Gao, W.H. Wang, O. Staub, D.H. Lin] 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. Wang M.X. aut Su X.T. aut Wu P. aut Gao Z.X. aut Wang W.H. aut Staub O. aut Lin D.H. aut American journal of physiology. Renal physiology 315/4(2018-10-01), F986-F996 315:4<F986-F996 2018 315 1 article serval SERVAL 700 1- Wang M.X. aut SERVAL 700 1- Su X.T. aut SERVAL 700 1- Wu P. aut SERVAL 700 1- Gao Z.X. aut SERVAL 700 1- Wang W.H. aut SERVAL 700 1- Staub O. aut SERVAL 700 1- Lin D.H. aut SERVAL 773 0- American journal of physiology. Renal physiology 315/4(2018-10-01), F986-F996 315:4<F986-F996 2018 315 BK010053 XK010053 XK010000 SERVAL SERVAL SERVAL article