The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex
Chao-Ling Yang, … , Xiaoman Zhu, David H. Ellison
Chao-Ling Yang, … , Xiaoman Zhu, David H. Ellison
Published November 1, 2007
Citation Information: J Clin Invest. 2007;117(11):3403-3411. https://doi.org/10.1172/JCI32033.
View: Text | PDF
Research Article

The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex

Abstract

The pathogenesis of essential hypertension remains unknown, but thiazide diuretics are frequently recommended as first-line treatment. Recently, familial hyperkalemic hypertension (FHHt) was shown to result from activation of the thiazide-sensitive Na-Cl cotransporter (NCC) by mutations in WNK4, although the mechanism for this effect remains unknown. WNK kinases are unique members of the human kinome, intimately involved in maintaining electrolyte balance across cell membranes and epithelia. Previous work showed that WNK1, WNK4, and a kidney-specific isoform of WNK1 interact to regulate NCC activity, suggesting that WNK kinases form a signaling complex. Here, we report that WNK3, another member of the WNK kinase family expressed by distal tubule cells, interacts with WNK4 and WNK1 to regulate NCC in both human kidney cells and Xenopus oocytes, further supporting the WNK signaling complex hypothesis. We demonstrate that physiological regulation of NCC in oocytes results from antagonism between WNK3 and WNK4 and that FHHt-causing WNK4 mutations exert a dominant-negative effect on wild-type (WT) WNK4 to mimic a state of WNK3 excess. The results provide a mechanistic explanation for the divergent effects of WT and FHHt-mutant WNK4 on NCC activity, and for the dominant nature of FHHt in humans and genetically modified mice.

Authors

Chao-Ling Yang, Xiaoman Zhu, David H. Ellison

×

Figure 1

WNK3 stimulates but does not phosphorylate NCC.

Options: View larger image (or click on image) Download as PowerPoint
WNK3 stimulates but does not phosphorylate NCC. (A) Full-length WNK3-sti...
(A) Full-length WNK3-stimulated Na uptake by Xenopus oocytes (expressed as percentage of Na uptake by oocytes injected with NCC alone), whereas kinase-dead WNK3 D294A downregulated Na uptake, compared with NCC alone. n = 4. (B) GST-WNK3 2–420 phosphorylated itself and the substrate histone, whereas GST-WNK3 2–586 was inactive. WNK3 2–420 also phosphorylated the kinase-inactive GST-WNK3 2–586 and GST-WNK1 1–491 D368A. WNK3 did not phosphorylate NCC, within either the N terminus (GST-NCC 1–136) or the C terminus (GST-NCC 600-1,001). Top: kinase assay; bottom: Coomassie-stained gel. Results are representative of experiments performed in triplicate. (C) The C terminus of WNK3 (WNK3 421–1,743) increased NCC activity in a manner similar to full-length WNK3. The NCC stimulation was inhibited by hydrochlorothiazide (HCTZ). Neither WNK3 alone nor WNK3 421–1,743 alone stimulated Na uptake in the absence of NCC. WNK3 2–420 had no effect on NCC activity (data not shown); n = 5. (D) WNK3 constructs were all expressed at the protein level (Western blot of Xenopus oocyte lysate). (E) WNK3 421–1,743, but not the kinase domain, WNK3 2–420, increased the abundance of NCC at the plasma membrane of oocytes, as detected by immunocytochemistry (results are representative of experiments performed in triplicate). Original magnification, ×400. Western blot of oocyte lysate showed no effect on total NCC. (F) Comparison of the domain structure of WNK1 and constructs employed in the present experiments. AID, autoinhibitory domain; CCD, coiled-coil domain. Sequences of autoinhibitory domains of WNK kinases are compared. Key phenylalanines shown to be essential for autoinhibition of WNK1 (24) are highlighted in blue.