Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases.
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 chloride-sensitive 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.
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan, 14000 Mexico City, Mexico.
José
J
Ponce-Coria
Pedro
P
San-Cristobal
Kristopher T
KT
Kahle
Norma
N
Vazquez
Diana
D
Pacheco-Alvarez
Paola
P
de Los Heros
Patricia
P
Juárez
Eva
E
Muñoz
Gabriela
G
Michel
Norma A
NA
Bobadilla
Ignacio
I
Gimenez
Richard P
RP
Lifton
Steven C
SC
Hebert
Gerardo
G
Gamba
D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D009865:Oocytes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D002712:Chlorides; C578714:SLC12A1 protein, human; C578715:Slc12a1 protein, mouse; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D013912:Threonine; D017346:Protein Serine-Threonine Kinases