The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.

Abstract-We identified a new kindred with the familial syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II or Gordon's syndrome) containing an affected father and son. Mutation analysis confirmed a single heterozygous G to C substitution within exon 7 (1690GϾC) that causes a missense mutation within the acidic motif of WNK4 (564DϾH). We confirmed the function of this novel mutation by coexpressing it in Xenopus oocytes with either the NaCl cotransporter (NCCT) or the inwardly rectifying K-channel (ROMK). Wild-type WNK4 inhibits 22 Na ϩ flux in Xenopus oocytes expressing NCCT by Ϸ90% (PϽ0.001), whereas the 564DϾH mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced Ͼ50% inhibition of steady-state current through ROMK at a ϩ20-mV holding potential (PϽ0.001). The 564DϾH mutant produced further inhibition with steady-state currents to some 60% to 70% of those seen with the wild-type WNK4. Using fluorescent-tagged NCCT (enhanced cyan fluorescent protein-NCCT) and ROMK (enhanced green fluorescent protein-ROMK) to quantify the expression of the proteins in the oocyte membrane, it appears that the functional effects of the 564DϾH mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564DϾH causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564DϾH, is functionally active and highlights further how switching charge on a single residue in the acid motif of WNK4 affects its interaction with the thiazide-sensitive target NCCT and the potassium channel ROMK.

The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D>A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression.J Am Soc Nephrol 17: 1867 -1874, 2006 . doi: 10.1681 T he WNK (with no lysine kinase) kinases WNK1 and WNK4 are widely expressed in mammalian transporting epithelia (1,2), and expression studies in Xenopus oocytes suggest that they are able to modify the expression of several co-transporters and ion channels (3,4). The details of the interaction are best understood for WNK4, which reduces surface expression of the thiazide-sensitive NaCl co-transporter (NCCT; gene symbol SLC12A3) in Xenopus oocytes (5-8). This effect of WNK4 depends on its serine-threonine (S/T) kinase activity as well as a highly conserved downstream acidic motif (EPEEPEADQH). Mutations that cause charge-changing amino acid substitutions within this motif abolish the inhibitory effect of wild-type WNK4 and cause the phenotype of hypertension and hyperkalemia that characterizes Gordon syndrome (pseudohypoaldosteronism type 2 [PHA2]; OMIM #145260) (9). WNK1 mutations also can cause this phenotype, but published data suggest that WNK1 protein is effective only in regulating NCCT trafficking when coexpressed with WNK4 (5,10). This suggests that the WNK may form a multimeric complex with NCCT and that protein-protein interactions between WNK1 and WNK4 are key to the functionality of WNK1.It is not known whether this paradigm of WNK1-WNK4 interaction extends to the effects of WNK on other transporters or ion channels. Lifton's laboratory has shown, for example, that WNK4 also inhibits expression of the Na-K-Cl cotransporter SLC1...