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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Only full-length WNK1 can inhibit WNK4 effects. (A) 22Na uptake by oocyt...
Only full-length WNK1 can inhibit WNK4 effects. (A) 22Na uptake by oocytes injected with cRNA encoding NCC, WNK4-(168–1222), WNK4-(D318A)-(168–1222), and several WNK1 constructs, as indicated. The WNK1 constructs are diagrammed below. Note that a putative kinase-deficient WNK4 construct inhibited Na uptake as effectively as a wild-type product. Only the full-length WNK1 construct suppressed the effect of WNK4 on NCC. *P < 0.05 versus NCC alone. (B) Blot showing that all WNK1 constructs were expressed by the oocytes at the protein level. (C) Schematic representation of major results. The carboxyl terminus of WNK4 associates with the carboxyl terminus of NCC. This interaction inhibits NCC activity (–). The 47 terminal WNK4 amino acids (shaded dark gray) function as a negative regulatory signal region but are not essential for binding. The amino-terminal domain of WNK1 associates with the amino-terminal domain of WNK4. This interaction inhibits WNK4 activity in a kinase-dependent manner. The functional effect requires full-length WNK1 (dotted line). Although catalytic activity is required, it is not clear whether WNK1 phosphorylates WNK4 directly.