A New Kindred With Pseudohypoaldosteronism Type II and a Novel Mutation (564D>H) in the Acidic Motif of the WNK4 Gene

Abstract

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.

Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways

“…This is consistent with the idea that phosphorylation of Thr60 is a key trigger for membrane translocation of NCC, hence leading to enhanced salt transport. These results are also consistent with other studies reporting that the WNK pathway regulates the membrane localisation of NCC (Cai et al, 2006;Golbang et al, 2005;Wilson et al, 2003). By contrast, we have no evidence that the SPAK-OSR1 pathway controls the membrane localisation of NKCC2, as hypotonic low-chloride conditions or mutation of the identified key phosphorylation sites on NKCC2 failed to influence plasma membrane localisation (Fig.…”

The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway

“…Consistent with this idea, several reports have correlated NCC activity with the level of this enzyme at the plasma membrane (Cai et al, 2006;Golbang et al, 2005;Wilson et al, 2003;Yang et al, 2003). It is also possible that the phosphorylation of ion co-transporters by SPAK and OSR1 induces a conformational change that directly activates their ability to transport ions.…”

“…In WNK4, PHAII-causing mutations are point mutations that lie in a highly conserved acidic region of the protein that is located outside the kinase domain ( Fig. 1) (Golbang et al, 2005;Wilson et al, 2001). These mutations are not known to alter the expression or intrinsic activity of WNK4 and the molecular mechanism by which they affect WNK4 function is unknown.…”

WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl - cotransporters required for normal blood pressure homeostasis

“…In contrast to WNK4's inhibitory effect on NCC in oocytes (6,7,12), coexpression of WNK3 with NCC led to a dramatic Ͼ3-fold increase in NCC activity, as measured by metolazonesensitive 22 Na ϩ uptake (P Ͻ 10 Ϫ6 , Fig. 2B).…”

“…Wild-type WNK4 is an inhibitor of NCC, NKCC1, the K ϩ channel ROMK1 (or Kir1.1; encoded by KCNJ1), and an activator of paracellular Cl Ϫ flux; some of these effects are kinase-dependent, whereas others are independent of WNK4's catalytic activity (6)(7)(8)(9)(10)(11)(12). Importantly, disease-causing missense mutations in WNK4 cluster within a highly conserved acidic domain (5) and have sharply divergent effects on these downstream targets; PHAII mutations eliminate inhibition of NCC and increase paracellular Cl Ϫ permeability, whereas simultaneously increasing inhibition of ROMK1 (6)(7)(8)(9)(10)(11)(12). Together, these effects can account for the observed increase in NaCl reabsorption and the decrease in K ϩ secretion seen in affected subjects.…”