The Effect of WNK4 on the Na⁺:Cl^‐ Cotransporter is modulated by Intracellular Chloride

Abstract

IntroductionThe effect of WNK4 on the renal Na+:Cl‐ cotransporter, NCC is controversial since several studies in vitro and in vivo have shown negative or positive effects. We hypothesize that both effects coexist and are modulated by the intracellular chloride concentration ([Cl‐]i). Piala et al. (2014) showed that L‐WNK1 is a Cl‐‐sensitive kinase and identified two leucine residues forming the Cl‐‐binding site. These leucines are conserved in WNK isoforms.Materials and MethodsWe used the functional expression system of Xenopus oocytes and HEK293 cells. NCC activity was measured by 22Na+ uptake and/or amino acid threonine phosphorylation in the absence or presence of wild type or mutant WNK4, in control conditions, or after [Cl‐]i depleting maneuvers. WNK4 autophosphorylation was also assessed in these conditions.ResultsAt baseline [Cl‐]i ∼ 45 mM, WNK4 autophosphorylation was undetectable and NCC activity was either inhibited or unaffected. [Cl‐]i reduction (25‐30 mM) promoted autophosphorylation transforming WNK4 to an activator of NCC in a kinase‐dependent manner. The L322F substitution transformed WNK4 into a constitutively autophosphorylated kinase that activated NCC without chloride depletion.ConclusionWNK4 is Cl‐ sensitive kinase exerting differential effects on NCC depending on the [Cl‐]i. It is possible that small changes in [Cl‐]i in distal convoluted tubule cells may have a crucial role determining WNK4 effect on NCC, suggesting that diverse physiological processes could converge in the Cl‐modulation of WNK's.

The Post-Translational Modification Networking in WNK-Centric Hypertension Regulation and Electrolyte Homeostasis

“…Specifically, WNKs’ homo- or heterodimer formation induces the conformational change of WNK kinase, which in turn facilitates autophosphorylation at the T-loop activation kinase domain, leading to WNK activation [ 28 ]. Thus, mutations at the T-loop activation kinase domain, such as D321A or D321K-K186D, ablate WNK kinase activity [ 32 ]. In addition, mutations of catalytic homologous serine, including S335A of WNK4 and S382A of WNK1, eliminate their phosphorylation activity.…”

“…In addition, it was reported that chloride ions bound to the chloride-binding pocket located in the kinase domain inhibited the autophosphorylation and activation of WNK [ 95 ]. Studies indicated that WNK4 L322F mutation in the chloride pocket desensitizes WNK4′s binding affinity to Cl − ions, therefore resulting in the increased autophosphorylation of WNK4, even in the presence of a higher chloride concentration [ 32 ]. WNK1, WNK3, and WNK4 each express distinct chloride-binding affinities, in which chloride binds most efficiently to WNK4 and least efficiently to WNK3 [ 27 , 95 ].…”

“…Under high intracellular [Cl − ] conditions, WNK4 responds swiftly to the chloride change and assembles into inactive dimers with other WNK monomers ( Figure 3 ). On the contrary, WNK3 is insensitive to chloride inhibition, remaining potent to phosphorylate SPAK kinase and promote surface channels [ 32 ]. Also, Eduardo RA et al co-injected KS-WNK1 with WNK4 into Xenopus oocytes, and the resultant WNK4/KS-WNK1 dimer had a lower Cl − affinity, blunting the inhibitory effect of chloride on the activating T-loop autophosphorylation on WNK4 S335 [ 27 ].…”

(Pro)renin Receptor Deletion in Distal Convoluted Tubule 1 Produces Salt-Sensitive Hypertension

“…4 The intracellular chloride concentration ([Cl − ] i ) regulates the autophosphorylation of WNK4. 5 The binding of Hypertension. 2021;78:1039-1041.…”

An in vivo protein landscape of the mouse DCT during high dietary K+ or low dietary Na+ intake

“…Recently it was also demonstrated using a transgenic mouse model expressing a Cl − sensor that raising extracellular K + transiently increases the intracellular Cl − concentration in the DCT, and that blockade of Kir4.1/Kir5.1 or inhibition of the Cl − channel ClC-K2 increases intracellular Cl − concentrations (33). Hence, diet induced changes in the extracellular K + concentration are thought to alter the basolateral plasma membrane potential via Kir4.1/Kir5.1, resulting in altered intracellular Cl − levels and ultimately modulation of the Cl − sensitive lysine deficient protein kinase (WNK) 1 and 4, affecting the WNK-SPAK/OSR1 kinase signaling pathway and NCC activity (3,28). Considering this, the high K + induced increase in Kir4.1 and Kir5.1 expression in the DCT is surprising.…”