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...

The Na ؉ :K ؉ :2Cl ؊ cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl ؊ ]i. The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloridesensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.ion transport ͉ loop of Henle ͉ protein serine-threonine kinases ͉ hypertension ͉ diuretics