WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl ^- cotransporters required for normal blood pressure homeostasis

Abstract

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

Protein kinase WNK3 increases cell survival in a caspase-3-dependent pathway

“…While this manuscript was under revision, a role for WNK3 in the regulation of chloride transporters was reported Rinehart et al, 2005), which are involved in the maintenance of cell volume in response to osmotic stress. Such osmotic stress has been related to epithelial cell apoptosis in previous reports (Dmitrieva and Burg, 2005).…”

Similar effects of all WNK3 variants on SLC12 cotransporters

“…10). WNK3 activates NKCCs by increasing their phosphorylation, even when cells are incubated in hypotonic medium, where normally they are inactivated (9,10,20,21). In contrast, the coexpression of KCCs with WNK3 completely inhibits KCC function, even when cells are exposed to a hypotonic medium, in which the KCCs are normally active and dephosphorylated (2,22).…”

“…Interestingly, due to a D294A substitution, the catalytically inactive form of WNK3 not only loses its activating and inhibitory properties against the NKCCs and KCCs, respectively, but acquires the opposite properties. WNK3-D294A becomes a potent inhibitor of the Na ϩ -coupled chloride cotransporters, inducing dephosphorylation even in hypertonic conditions in which NKCCs are activated, while it becomes a potent activator of KCCs, even in isotonic conditions in which they are usually inactive (2,9,10,21). Under isotonic conditions, activation of the KCCs by the kinase-dead WNK3 can be reversed by calyculin or cyclosporine, implying the involvement of protein phosphatases in the process.…”

“…Interestingly, exon 18b appears to be exclusively expressed in the central nervous system (CNS), while WNK3-18a is present in epithelial tissues (8). All functional studies discussed above were performed using the WNK3-18a epithelial variant (2,9,10,21). However, a recent study suggested that the effects of WNK3-18a and WNK3-18b toward NCC are opposite; that is, activation by WNK3-18a and inhibition by WNK3-18b (7).…”

“…The mechanism by which WNKs affect blood pressure is, at least in part, due to their ability to modulate the activity of the renal thiazide-sensitive Na ϩ -Cl Ϫ cotransporter, NCC (5,6). As indicated above, WNK3 is an activator and WNK3-D294A is an inhibitor of NCC (21). In addition to several epithelial tissues, WNK3 is present in the brain, in which it is coexpressed with NKCC1/KCC2 in GABAergic neurons (9), positioning it as an important kinase for modulating neuronal activity.…”