Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl ؊ dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a ''mirror image'' of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCTmediated Na ؉ influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ( 22 Nainflux reduced 50%, P < 1 ؋ 10 ؊9 , surface expression reduced 75%, P < 1 ؋ 10 ؊14 in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.protein serine-threonine kinases ͉ hypertension ͉ thiazide-sensitive Na-Cl cotransporter ͉ ion transport ͉ medical genetics H ypertension is the most common disease in industrialized societies, affecting Ͼ20% of the adult population and contributing to morbidity and mortality from stroke, myocardial infarction, renal failure, and congestive heart failure (1). Its pathogenesis is largely unknown, resulting in empiric pharmacologic therapy. In recent years, genetic approaches investigating rare Mendelian forms of high and low blood pressure have provided fundamental insight into mechanisms that contribute to blood pressure variation (2). These have demonstrated the causal role of inherited variation in renal salt homeostasis in blood pressure variation, with mutations in many genes known to play a role in mediating or regulating renal salt reabsorption resulting in altered blood pressure.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man database no. 145260) is an autosomal dominant disease featuring hypertension with hyperkalemia despite normal glomerular filtration rate; renal tubular acidosis is a variable associated finding. The clinical features of this disease are chloride dependent and are also corrected with thiazide diuretics, specific a...

Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.

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