Similar effects of all WNK3 variants on SLC12 cotransporters

Abstract

kinase 3 (WNK3) is a member of a subfamily of serine/threonine kinases that modulate the activity of the electroneutral cation-coupled chloride cotransporters. WNK3 activates NKCC1/2 and NCC and inhibits the KCCs. Four splice variants are generated from the WNK3 gene. Our previous studies focused on the WNK3-18a variant. However, it has been suggested that other variants could have different effects on the cotransporters. Thus, the present study was designed to define the effects of all WNK3 variants on members of the SLC12 family. By RT-PCR from a fetal brain library, exons 18b and 22 were separately amplified and subcloned into the original WNK3-18a or catalytically inactive WNK3-D294A to obtain all four potential combinations with and without catalytic activity (18a, 18aϩ22, 18b, and 18bϩ22). The basal activity of the cotransporters and the effects of WNK3 isoforms were assessed in Xenopus laevis oocytes coinjected with each of the WNK3 variant cRNAs. In isotonic conditions, the basal activity of NCC and NKCC1/2 were increased by coinjection with any of the WNK3. The positive effects occurred even in hypotonic conditions, in which the basal activity of NKCC1 is completely prevented. Consistent with these observations, when expressed in hypotonicity, all KCCs were active, but in the presence of any of the WNK3 variants, KCC activity was completely reduced. That is, NKCC1/2 and NCC were inhibited, even in hypertonicity, while KCCs were activated, even in isotonic conditions. We conclude that the effects of all WNK3 variants toward SLC12 proteins are similar.

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

“…Ϫ cotransport activity of all four wild-type KCCs (KCC1-KCC4) and the endogenous Na ϩ -K ϩ -2Cl Ϫ (NKCC1) was assessed using the heterologous expression system of Xenopus laevis oocytes following our standard procedures (10,36,37,39). Briefly, mature oocytes were injected with water alone or containing 0.2 g/l of KCCs cRNA alone or together with 0.1 g/l of the various isoforms and mutants of L-WNK1 kinase cRNA.…”

“…Two days after injection, the activity of the KCCs was determined by measuring the Cl Ϫ -dependent 86 Rb ϩ uptake under hypotonic conditions (110 mosmol/kgH2O). The activity of endogenous NKCC1 was determined by bumetanide-sensitive 86 Rb ϩ uptake under hypotonic (155 mosmol/ kgH2O) conditions, which is known to promote inhibition of the cotransporter (16,27 (10,36,37,39). Tracer activity was determined for each oocyte by ␤-scintillation counting.…”

“…To generate the transient cell lines, HEK-293 cells underwent passages every 2-3 days to maintain the cells in their exponential growth phase. Cells were culture on 10-cm-diameter dishes in Dulbecco's modified Eagle medium (DMEM) supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 10% FBS (vol/ vol), and 2 mM L-glutamine in a humidified atmosphere containing 95% air-5% CO 2 at 37°C (9,36 (10,36). To reach the isotonic conditions for HEK-293 cells (ϳ300 mosmol/kgH2O), mediums were supplemented with 1.7 g/100 ml sucrose.…”

“…(36,37,39) and that wild-type WNK3 prevents their cell swelling-induced activation. Likewise catalytically inactive WNK3-D294A is able to bypass the tonicity requirements to regulate KCCs under isotonicity (10,12,16). Here, we analyzed the effect of L-WNK1 on the K ϩ -Cl Ϫ cotransporters and whether its effect requires the kinase catalytic activity.…”

“…Thus L-WNK1 kinase, which is ubiquitously expressed, is expected to be a modulator in the SLC12 family of cotransporters. Although previous studies have shown that other WNKs mediate the activity of all members of the SLC12 family (10,12,19,44,52), the precise effect of L-WNK1 on these cotransporters remains unknown. Rinehart et al (51) observed that knocking down L-WNK1 with two distinct siRNAs reduced the phosphorylation of the key residues T991 and T1048 of the KCC3a isoform, a condition that is associated with its activation, suggesting that by promoting KCC3 phosphorylation, L-WNK1 could also be an inhibitor of this cotransporter.…”