Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport
Chao-Ling Yang, Xiaoman Zhu, Zhaohong Wang, Arohan R. Subramanya, David H. Ellison
Chao-Ling Yang, Xiaoman Zhu, Zhaohong Wang, Arohan R. Subramanya, David H. Ellison
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Article Cardiology

Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport

Abstract

With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt.

Authors

Chao-Ling Yang, Xiaoman Zhu, Zhaohong Wang, Arohan R. Subramanya, David H. Ellison

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The amino-terminal kinase domain of WNK4 is not required for NCC inhibit...
The amino-terminal kinase domain of WNK4 is not required for NCC inhibition but is required for WNK1 interaction. (A) 22Na uptake by oocytes injected with cRNA encoding NCC and several WNK4 constructs, with or without WNK1. The WNK4 constructs are diagrammed below. Note that carboxyterminal WNK4 constructs (WNK4-[168–1222] and WNK4-[445–1222]) inhibited Na uptake compared with NCC alone, but a kinase-domain WNK4 construct (WNK4-[1–444]) did not. WNK1 blocked the effect of a WNK4 construct that contains both the kinase domain and the carboxyl terminus (WNK4-[168–1222]) but did not affect the inhibition by carboxyterminal constructs (WNK4-[445–1222]). *P < 0.05 versus NCC alone. cc, coiled coil domains. (B) Immunoprecipitation of the amino-terminal WNK4 domain by WNK1. Oocytes were injected with HA-WNK4 amino- and carboxyterminal constructs and with His-WNK1-(1–555). Only the amino-terminal WNK1 precipitated the amino-terminal WNK4 construct. Bottom blots confirm protein expression of all constructs. A faint band indicates that a much weaker association between the carboxyl terminus of WNK4 and the amino terminus of WNK1 could be detected. (C) Immunoprecipitation of WNK4 fragments by WNK1 kinase domain alone. The left 2 panels show expression of both protein constructs. The right panel shows that myc-WNK1-(218–491) precipitated only WNK4 constructs that contain the kinase domain. Shown is 1 of 3 similar experiments.