Chloride Sensing by WNK1 Involves Inhibition of Autophosphorylation

Abstract

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.

Caveolin-1 Deficiency Inhibits the Basolateral K+ Channels in the Distal Convoluted Tubule and Impairs Renal K+ and Mg2+ Transport

“…Therefore, the disruption of cav-1 also inhibited the basolateral Cl -conductance in the DCT1, which should increase the intracellular Cl -concentration, thereby inhibiting WNK autophosphorylation and WNK-SPAK interaction. 26,27 We previously demonstrated that the disruption of Kcnj10 decreased the expression of NCC and SPAK in Kcnj10 KO mice. 13 Therefore, we examined the expression of SPAK in WT and cav-1 KO mice.…”

Réabsorption du sel et sécrétion du potassium par le néphron distal

“…Plusieurs études avaient suggéré que l'activité des WNK était modulée par des changements de concentration ionique, notamment en chlorure et en potassium. Plus récemment, Piala et al ont montré que L-WNK1 est une kinase dont l'activité est sensible au chlorure [24]. Elle est en effet plus active lorsque la concentration en chlorure est faible.…”

“…Elle est en effet plus active lorsque la concentration en chlorure est faible. L'activation de L-WNK1 requiert la phosphorylation d'une sérine (S382) située dans la boucle d'activation du domaine kinase (activation loop) [25], qui est plus importante lorsque la concentration en chlorure est faible [24]. Des études de cristallographie ont mis en évidence que le chlorure se fixe au domaine kinase, maintenant la protéine dans une conformation inactive.…”

“…Un résidu leucine (L379) joue un rôle crucial dans la formation du site de fixation pour le chlorure dans le domaine kinase. En effet, sa mutation en phénylalanine diminue la sensibilité de L-WNK1 au chlorure [24]. La concentration en chlorure retrouvée dans les modèles cellulaires in vitro est plus élevée que celle observée dans les cellules du DCT (40-50 mM [7] versus 10-20 mM [26] (Figure 2).…”

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl− cotransporters

“…The full-length KCCs (39,40,60), as well as the wild-type and mutants of human L-WNK1 kinase cDNAs used in this work, were previously described (7,46,63). L-WNK1 gives rise to several distinct isoforms.…”