WNK4 kinase inhibits Maxi K channel activity by a kinase-dependent mechanism

Abstract

[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...

WNK1 Activates Large-Conductance Ca2+-Activated K+ Channels through Modulation of ERK1/2 Signaling

“…This finding suggests that WNK1 activates BK channel activity by reducing its degradation through a lysosomal pathway, likely by inhibiting an ERK1/2 signaling pathway, a mechanism similar to WNK4-mediated modulation of BK but in the opposite direction. 7,8 WNK1 was found to inhibit ROMK activity and its protein expression and KS-WNK1 reversed wild-type (WT)-WNK1's inhibitory effects on ROMK. 6,45 Several studies have shown that the ratio of WT-WNK1 to KS-WNK1 is relevant to K secretion.…”

“…HEK293 cells stably expressing the a subunit of BK (HEK BKa cells) were previously generated in our laboratory and maintained in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), G418 200 mg/10 ml, and 10% FBS. 7 Lipofectamine 2000 (Life Technologies) was used for transfection according to the manufacturer's instructions. Forty-eight hours after transfection of cells with a series of doses of WNK1 plasmid, cells were lysed.…”

“…7,51 Electrodes were fabricated from Corning 7052 glass (Garner Glass, Fullerton, CA) in two steps on a Narishige PP-83 electrode puller (Narishige, Tokyo, Japan). The bath solution used in the cell-attached mode contained (in mM) 140 NaCl, 1 CaCl 2 , 5 KCl, 1 EGTA, and 10 HEPES, pH adjusted to 7.4.…”

“…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][8][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.…”

Collecting Duct Intercalated Cell Function and Regulation

“…These channels, which are less abundant in principal cells, are activated by depolarization, stretch/luminal flow, intracellular calcium increases, or hypoosmotic stress (reviewed in reference 67). Moreover, the BK channel is inhibited by the with-no-lysine kinase 4 (WNK4) in a phosphorylationdependent manner (68,69). In a later study performed in a cell line of intercalated cell characteristics, WNK4 inhibited BK channel activity in part by increasing channel ubiquitination and degradation (70).…”