WNK4 inhibits Ca2+-activated big-conductance potassium channels (BK) via mitogen-activated protein kinase-dependent pathway

Abstract

We used the perforated whole-cell recording technique to examine the effect of With-No-Lysine Kinase 4 (WNK4) on the Ca2+ activated big-conductance K channels (BK) in HEK293T cells transfected with BK–α subunit (BK-α). Expression of WNK4 inhibited BK channels and decreased the outward K currents. Coexpression of SGK1 abolished the inhibitory effect of WNK4 on BK channels and restored the outward K currents. Expression of WNK4S1169D//1196D, in which both SGK1-phosphorylation sites (serine 1169 and 1196) were mutated to aspartate, had no effect on BK channels. Moreover, coexpression of SGK1 had no additional effect on K currents in the cells transfected with BKα + WNK4 S1169D//1196D, suggesting that SGK1 reversed WNK4-induced inhibition of BK channels by stimulating WNK4 phosphorylation. Expression of WNK4 but not WNK4 S1169D//1196D increased the phosphorylation of ERK and p38 mitogen-activated protein kinase (MAPK); an effect was abolished by coexpression of SGK1. The role of ERK and p38 MAPK in mediating the effect of WNK4 on BK channels was further suggested by the finding that inhibition of ERK and P38 MAPK completely abolished the inhibitory effect of WNK4 on BK channels. In contrast, inhibition of MAPK failed to abolish the inhibitory effect of WNK4 on ROMK channels in both HEK cells and Xenopus oocytes. Expression of dominant negative dynaminK44A (DynK44A) or treatment of the cells with dynasore, a dynamin inhibitor, not only increased K currents but also largely abolished the inhibitory effect of WNK4 on BK channels. However, inhibition of MAPK still increased the outward K currents in the cells transfected with BKα+WNK4 and treated with dynasore. Similar results were obtained in experiments performed in the native tissue in which inhibition of ERK and p38 MAPK increased BK channel activity in the cortical collecting duct (CCD) treated with dynasore. We concluded that WNK4 inhibited BK channels by stimulating ERK and p38 MAPK and that activation of MAPK by WNK4 may inhibit BK channels partially via a mechanism other than stimulating endocytosis.

Cell-specific regulation of L-WNK1 by dietary K+

“…WNK4 reduces whole cell and surface expression of the BK ␣-subunit in HEK293 cells by a process that is kinase dependent and sensitive to inhibitors of lysosomal degradation, consistent with the notion that WNK4 enhances the routing of BK channels to lysosomes in a dynaminindependent manner (108) that is dependent on activation of MAP kinases (106). We have reported that WNK4 enhances ubiquitination of the BK ␣-subunit, consistent with a role for WNK4 in targeting the channel for internalization and/or degradation via a ubiquitin-dependent pathway (95).…”

“…Because FHHt is associated with hyperkalemia and impaired renal K ϩ secretion, it is not surprising that both L-WNK1 and WNK4 inhibit the expression of ROMK channels (11,28,37,42,69,91). While WNK4 also inhibits the expression of BK channels (95,106,108), the effects of L-WNK1 on BK channels have only recently begun to be addressed (48).…”

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.…”

“…8 We therefore determined whether WNK1 stimulates BK activity and protein expression by inhibiting ERK1/2 signaling pathway. In HEK BKa cells transfected with WNK1, WNK1 overexpression significantly decreased ERK1/2 phosphorylation in a dose-dependent manner (p-ERK1/2/t-ERK1/2 ratio of 1.060.07 with WNK1 at 1 mg, 0.8860.09 at 3 mg, 0.7160.08 at 6 mg, 0.5760.10 at 9 mg; n=4; P,0.05 compared with 1-mg WNK1 group) ( Figure 4, A and C), whereas knocking down WNK1 expression significantly increased ERK1/2 phosphorylation in a dose-dependent manner (p-ERK1/2/t-ERK1/2 ratio of 1.060.22 with siRNA control, 1.2260.36 with siRNA WNK1 15 nM, 1.5660.35 at 30 nM, 2.4060.58; n=4; P,0.05 compared with siRNA control group) as shown in Figure 4, B and D. These data suggested that WNK1 enhances BK channel activity and its protein expression, likely by inhibiting ERK1/2 signaling pathway.…”

“…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.…”