[with no lysine (k)] kinase is a serine/threonine kinase subfamily. Mutations in two of the WNK kinases result in pseudohypoaldosteronism type II (PHA II) characterized by hypertension, hyperkalemia, and metabolic acidosis. Recent studies showed that both WNK1 and WNK4 inhibit ROMK activity. However, little is known about the effect of WNK kinases on Maxi K, a large-conductance Ca 2ϩ and voltage-activated potassium (K) channel. Here, we report that WNK4 wild-type (WT) significantly inhibits Maxi K channel activity in HEK ␣BK stable cell lines compared with the control group. However, a WNK4 dead-kinase mutant, D321A, has no inhibitory effect on Maxi K activity. We further found that WNK4 inhibits total and cell surface protein expression of Maxi K equally compared with control groups. A dominant-negative dynamin mutant, K44A, did not alter the WNK4-mediated inhibitory effect on Maxi K surface expression. Treatment with bafilomycin A1 (a proton pump inhibitor) and leupeptin (a lysosomal inhibitor) reversed WNK4 WT-mediated inhibition of Maxi K total protein expression. These findings suggest that WNK4 WT inhibits Maxi K activity by reducing Maxi K protein at the membrane, but that the inhibition is not due to an increase in clathrin-mediated endocytosis of Maxi K, but likely due to enhancing its lysosomal degradation. Also, WNK4's inhibitory effect on Maxi K activity is dependent on its kinase activity. protein expression; lysosomal degradation WNK [WITH NO LYSINE (K)] KINASE belongs to a subfamily of serine/threonine kinases (55). Mutations in two members of this family, WNK1 and WNK4, result in pseudohypoaldosteronism type II (PHA II). PHA II, also referred to as Gordon's syndrome, is an autosomal dominant disorder, characterized by hypertension, hyperkalemia, and metabolic acidosis (51). This clinical phenotype suggests that WNK kinases might regulate renal potassium (K) channels, such as renal outer medullary potassium channel (ROMK) or Maxi K channels (BK channels) that are responsible for K handling by the distal nephron. A number of studies indicate that WNK kinases constitute a novel signaling pathway that is involved in the regulation of different ion transporters and channels controlling sodium and K homeostasis (23). In kidney tissue, there are two types of apical K channels identified in the distal nephron by patchclamp analysis (38). One type of K channel is a low-conductance secretory K (SK) channel that has high open probability at resting membrane potential and mediates K ϩ secretion under basal conditions. The properties of the SK channel are consistent with those of ROMK. The other type of K channel has a high single-channel conductance (Ͼ100 pS) and channel kinetics similar to Maxi K channels (34). Although it is generally accepted that ROMK is the K ϩ secretory channel in the mammalian distal nephron, recent in vitro and in vivo studies have provided evidence that Maxi K can also serves as a K ϩ secretory channel in renal tubules (37) and that it plays an important role in K ϩ secretion in ROM...
WNK kinase is a serine/threonine kinase that plays an important role in electrolyte homeostasis. WNK4 significantly inhibits the surface expression of the sodium chloride co-transporter (NCC) by enhancing the degradation of NCC through a lysosomal pathway, but the mechanisms underlying this trafficking are unknown. Here, we investigated the effect of the lysosomal targeting receptor sortilin on NCC expression and degradation. In Cos-7 cells, we observed that the presence of WNK4 reduced the steady-state amount of NCC by approximately half. Co-transfection with truncated sortilin (a dominant negative mutant) prevented this WNK4-induced reduction in NCC. NCC immunoprecipitated with both wild-type sortilin and, to a lesser extent, truncated sortilin. Immunostaining revealed that WNK4 increased the co-localization of NCC with the lysosomal marker cathepsin D, and NCC co-localized with wild-type sortilin, truncated sortilin, and WNK4 in the perinuclear region. These findings suggest that WNK4 promotes NCC targeting to the lysosome for degradation via a mechanism involving sortilin.
With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca 2+ -activated K + (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the a subunit of BK (HEK-BKa cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKa channel activity and open probability. Knockdown of WNK1 expression also significantly inhibited BKa protein expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKa expression and decreased ERK1/2 phosphorylation in a dose-dependent manner in HEK293 cells. Knockdown of ERK1/2 prevented WNK1 siRNA-mediated inhibition of BKa expression. Similarly, pretreatment of HEK-BKa cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of WNK1 siRNA on BKa expression in a dose-dependent manner. Knockdown of WNK1 expression also increased the ubiquitination of BKa channels. Notably, mice fed a high-K + diet for 10 days had significantly higher renal protein expression levels of BKa and WNK1 and lower levels of ERK1/2 phosphorylation compared with mice fed a normal-K + diet. These data suggest that WNK1 enhances BK channel function by reducing ERK1/2 signaling-mediated lysosomal degradation of the channel. With no lysine (WNK) kinase belongs to a family of serine/threonine kinases. Mutations of WNK1 and WNK4 are responsible for pseudohypoaldosteronism type II (PHAІІ), characterized by hypertension, hyperkalemia, and metabolic acidosis. 1,2 The disease mutation in WNK1 or WNK4 kinase resulting in hyperkalemia suggests a role of WNK in potassium handling in renal distal nephron, which contains two major potassium channels, renal outer medullary K + channels (ROMK) and Big K (BK) channels. 3,4 WNK4 inhibits ROMK channel activity and its surface expression, whereas WNK4 disease mutant enhances its inhibitory effect on ROMK. 5 WNK1 also inhibits ROMK activity; however, a kidney-specific form of WNK1 (KS-WNK1) reverses WNK1's effect on ROMK. 6 WNK4 inhibits BK channel activity and protein expression, 7-9 whereas WNK4 disease mutant also enhances its inhibitory effect on BK activity via a ubiquitin-dependent pathway. 9 BK channel (or Maxi K) is a large conductance Ca 2+ and voltage-activated K channel. 10 BK is encoded by the gene slo1 11 and is widely distributed in many different
WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.
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