The renal Na ؉ :Cl ؊ cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl ؊ influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl ؊ efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr 53 and Thr 58 . Elimination of these threonines together with serine residue Ser 71 completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.The renal Na ϩ :Cl Ϫ cotransporter (NCC 4 or TSC, gene symbol SLC12A3, locus identification number 6559) that is expressed at the apical membrane of the mammalian distal convoluted tubule represents the major salt transport pathway in this segment of the nephron (1-4). Its essential role in preserving the extracellular fluid volume and blood pressure has been established by the identification of inactivating mutations of the SLC12A3 gene as the cause of Gitelman's disease (5, 6), an inherited disorder featuring arterial hypotension, renal salt wasting, hypokalemic metabolic alkalosis, hypocalciuria, and hypomagnesemia. In addition, a defect in NCC regulation by serine/threonine kinases WNK1 and WNK4 has been implicated in the pathogenesis of a salt-dependent form of human hypertension known as pseudohypoaldosteronism type II (PHAII) (7,8), which features marked sensitivity to hydrochlorothiazide and a clinical picture that is a mirror image of Gitelman's disease (9). NCC is the pharmacological target of thiazide-type diuretics that are currently recommended by the Joint National Committee VII for the detection, evaluation, and treatment of high blood pressure as the first line treatment of arterial hypertension either as the unique drug or in combination with other antihypertensive agents (10). Despite the importance of NCC for cardiovascular and renal physiology, pharmacology, and pathophysiology, little is known about t...

WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased K ؉ secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin.hypertension ͉ ion transport ͉ protein serine-threonine kinases