WNK3 modulates transport of Cl ^- in and out of cells: Implications for control of cell volume and neuronal excitability

Abstract

The regulation of Cl ؊ transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl ؊ influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl ؊ efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl ؊ ͞volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1͞2 in diverse Cl ؊ -transporting epithelia and in neurons expressing ionotropic GABA A receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl ؊ influx via NKCC1, and that it inhibits Cl ؊ exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl ؊ via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl ؊ ͞volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.cell volume ͉ GABA ͉ ion transport ͉ protein serine-threonine kinases

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).…”

“…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. The WNK3 gene encodes four putative alternatively spliced variants that are generated by the combination of two mutually exclusive exons 18 (18a and 18b), with the presence or absence of exon 22.…”

The role and the mechanism of γ-aminobutyric acid during central nervous system development

“…KCC family includes KCC1, KCC2, KCC3 and KCC4. KCC2 only exists in the nervous system and is the only one who can cotransport K + and Clunder isotonic conditions [19] . KCC2 increases the rate of Clextrusion, thus leading to a reduction of [Cl -] i and a consequent negative shift in E GABA .…”