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

† ‡ | Yingli Liu,* Xiang Song, Yanling Shi,* Zhen Shi,§ Weihui Niu,§ Xiuyan Feng,* Dingying Gu,§ | Hui-Fang Bao,¶ He-Ping Ma,¶ Douglas C. Eaton,¶ Jieqiu Zhuang,§ and Hui Cai* ¶

*Renal Division, Department of Medicine, and ¶Department of Physiology, Emory University School of Medicine, Atlanta, Georgia; §Department of Nephrology, The Second Affiliated Hospital, Wenzhou Medical University, Zhejiang, China; †Department of Nephrology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; ‡Department of Cardiology, The Fourth Affiliated Hospital, Harbin Medical University, Heilongjiang, China; and |Renal Section, Atlanta Veterans Affairs Medical Center, Decatur, Georgia

ABSTRACT With no lysine (WNK) are members of the serine/threonine family. We previously showed that WNK4 inhibits renal large-conductance Ca2+-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 expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKa expression and decreased ERK1/2 phosphor- ylation 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. Knock- down 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.

J Am Soc Nephrol 26: ccc–ccc, 2014. doi: 10.1681/ASN.2014020186

With no lysine (WNK) kinase belongs to a family of mutant also enhances its inhibitory effect on BK serine/threonine kinases. of WNK1 and activity via a ubiquitin-dependent pathway.9 WNK4are responsible for BK channel (or Maxi K) is a large conductance Ca2+ type II (PHAІІ), characterized by hypertension, and voltage-activated K channel.10 BK is encoded by the hyperkalemia, and metabolic acidosis.1,2 The disease slo111 and is widely distributed in many different in WNK1 or WNK4 kinase resulting in hyperkalemia suggests a role of WNK in potassium Received February 17, 2014. Accepted June 16, 2014. handling in renal distal nephron, which contains two Y.L. and X.S. contributed equally to this work. major potassium channels, renal outer medullary K+ channels (ROMK) and Big K (BK) channels.3,4 Published online ahead of print. Publication date available at WNK4 inhibits ROMK channel activity and its sur- www.jasn.org. face expression, whereas WNK4 disease mutant en- Correspondence: Dr.HuiCai,RenalDivision,EmoryUniversity hances its inhibitory effect on ROMK.5 WNK1 also School of Medicine, 1639 Pierce Drive, WMB Room 338, Atlanta, fi GA 30322, or Jieqiu Zhuang, Department of Nephrology, The inhibits ROMK activity; however, a kidney-speci c Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, form of WNK1 (KS-WNK1) reverses WNK1’s effect Zhejiang 325000, China. Email: [email protected] or jieqiuzhuang@ on ROMK.6 WNK4 inhibits BK channel activity and hotmail.com – protein expression,7 9 whereas WNK4 disease Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 26: ccc–ccc, 2014 ISSN : 1046-6673/2604-ccc 1 BASIC RESEARCH www.jasn.org tissues.12 BK channels are composed of two subunits: a pore- using a -attached technique was performed in human forming a subunit and a modulatory b subunit.13 The a subunit embryonic kidney 293 (HEK293) BKa stably expressing cells of BK (BKa) channels functions as a channel by itself and is mod- (HEK BKa cells) transfected with siRNA WNK1 (45 nM) or ulated by cAMP-dependent protein kinase A,14–16 protein kinase scramble siRNA control. As shown in Figure 1, knocking down C,14,16–18 cGMP-dependent PKG,17,19 and cSrc.20 It is also regu- WNK1 expression significantly inhibited both BKa NPo lated extensively by alternative splicing,21 phosphorylation and de- (0.019260.0074, n=18 versus 0.131660.0311 in control group, phosphorylation,22 andassociatedregulatoryb subunits.23 The b n=36; P=0.006) and open probability (Po; 0.019460.0023 ver- subunits alter the apparent Ca2+ and voltage sensitivity of the a sus 0.051260.0096 in control group; P,0.001). These data sug- subunit, modify channel kinetics, and alter the pharmacologic gest that WNK1 activates BK channel activity. properties of the channel.24 The b1 subunit rapidly traffics to surfacemembranetoassociatewithBKa and control functional Effect of WNK1 on BK Protein Expression BK activity in response to nitric oxide stimulation.25 BKa is ex- Because WNK1 activates BK channel activity, likely by in- pressed in various renal tubular segments, including medullary and creasing the channel number, we investigated whether WNK1 cortical thick ascending limbs,26 distal convoluted tubule,27 con- affects BK protein expression, which could be responsible for necting tubule,28 principal cells and intercalated cells of the cortical increased BK activity. First, we determined the effect of siRNA collecting duct (CCD).29 BK channel is responsible for the flow- WNK1 on BK protein expression in HEK BKa cells. As shown dependent K+ secretion in CCD.30 Inhibition of mitogen-activated in Figure 2, WNK1 expression was markedly decreased by protein kinase (MAPK) stimulates BK activity in CCD cells.31 Our siRNA WNK1, and knockdown of WNK1 expression signifi- previous study showed that WNK4 inhibits BK channel activity by cantly reduced BK protein expression in a dose-dependent enhancing its degradation via a lysosomal pathway.7 WNK4 inhib- manner (100%60% with siRNA WNK1 at 0 nM, 87.9% its BK activity by activating extracellular signal-regulated kinase 1/2 61.6% at 15 nM, 73.2%68.5% at 30 nM, 57.1%629.4% at (ERK1/2) and p-38 signaling pathways.8 WNK1 phosphorylates 45 nM; n=5; P,0.05 compared with WNK1 at 0 nM group), as ERK5 via MEKK2/3.32 However, little is known about the effect of shown in Figure 2B. We repeated the above knockdown ex- WNK1 kinase on BK activity and ERK1/2 signaling pathway. Here, periment with second siRNA WNK1 (45 nM), knockdown of we report that the knockdown of WNK1 expression significantly WNK1 expression also significantly reduced BK protein ex- inhibited both BKa NPo and its open probability. Knockdown of pression (Supplemental Figure 1). WNK1 expression significantly inhibited BKa protein expression, which was reversed by bafilomycin A1, a lysosomal inhibitor, and overexpressing WNK1 significantly en- hanced BKa protein expression. Overex- pressing WNK1 also decreased ERK1/2 phosphorylation, whereas WNK1 knock- down increased ERK1/2 phosphorylation. Knocking down ERK1/2 abolished the small interfering RNA (siRNA) WNK1-mediated inhibitory effect on BKa expression. The knockdown of WNK1 expression reduced BKa while increasing BKa ubiquitination. In vivo mice study showed that protein abun- dances of BKa and WNK1 were significantly higher in mice fed with a high-K diet while reducing ERK1/2 phosphorylation. These data suggest that WNK1 activates BK chan- nel activity by reducing BK degradation through a lysosomal pathway via ERK1/2- dependent signaling. Figure 1. Knockdown of WNK1 expression inhibits BKa channel activity in HEK293 stably expressing cells (HEK BKa cells). HEK BKa cells were transfected with siRNA WNK1 (45 nM) or scramble control siRNA. Single cell recordings using a cell-attached configuration were performed 48 hours after transfection. (A) The representative recording RESULTS traces for both siRNA WNK1 and scramble control siRNA groups at a hold potential of 240 mV. (B) Bar graph for BKa channel NPo. (C) Bar graph for BK Po. Knockdown of Effect of WNK1 on BK Channel WNK1 expression significantly inhibits both BKa channel NPo (0.019260.0074 in Activity siRNA WNK1, n=18, compared with the siRNA control group, 0.131660.0311, n=36; To determine whether WNK1 affects the ac- *P=0.006) and Po (0.019460.0023, compared with the siRNA control group, 0.051260.0096; tivityof BK channel, a single-channel recording #P,0.001).

2 Journal of the American Society of Nephrology J Am Soc Nephrol 26: ccc–ccc,2014 www.jasn.org BASIC RESEARCH

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 phosphoryla- tion 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.

Effect of siRNA ERK1/2 on BK Protein Expression To confirm that WNK1 modulates BK protein expression through the ERK1/2 signaling pathway, we performed ERK1/2 knockdown experiments. In HEK BKa cells transfected with siRNA WNK1 (45 nM) with or without siRNA ERK 1/2, WNK1 expression was significantly reduced by .85% (Figure 5C). BK protein expression was significantly reduced in the siRNA WNK1 transfected group (50.3%610.2%) compared Figure 2. Knockdown of WNK1 expression decreases BKa pro- with siRNA control group (100%611.7%; n=8; P,0.01), as a a tein expression in HEK BK cells. HEK BK cells were transfected shown in Figure 5B, while increasing EKR1/2 phosphorylation with scramble control siRNA or a series of doses of siRNA WNK1. (p-ERK1/2/t-ERK1/2 ratio of 1.4860.35 versus 1.060.08; Forty-eight hours after transfection, cells were lysed and sub- n=8; P,0.01) (Figure 5D). However, when in HEK BKa cells jected to SDS-PAGE and Western blot analysis. (A) Representa- tive immunoblots for BK, WNK1, and actin. (B) Bar graph shows cotransfected with both siRNA WNK1 (45 nM) and siRNA mean6SEM band densities of BK from five independent experi- ERK1/2 (15 nM), ERK1/2 protein expression and ERK1/2 ments. Knockdown of WNK1 expression significantly decreases phosphorylation were remarkably reduced, as expected (Fig- total BK protein expressions in a dose-dependent manner. n=5; ure 5A). BK protein expression was back to the control level *P,0.05 versus control. (100.1%622.8%) (Figure 5B), suggesting that knockdown of ERK1/2 abolished the effect of WNK1 on BK. These data fur- ther supported that WNK1 modulates BK through an ERK1/2 Wethen performed WNK1-overexpressing experiments. As signaling-dependent pathway. shown in Figure 3, WNK1 overexpression significantly in- creased BK protein expressions in a dose-dependent manner Effect of WNK1 on BK Ubiquitination either in HEK293 cells transfected with myc-BK or in HEK Activation of ERK 1/2 signaling increases ubiquitination of the stably expressing BKa cells. WNK1 significantly increased BK sodium chloride cotransporter, ultimately leading to NCC protein expression (100%60% with WNK1 at 0 mg, 172.3% degradation.33 To determine whether WNK1 affects BK ubiq- 613.6% at 1 mg, 221.0%629.2% at 2 mg, and 272.9%638.5% uitination after inhibiting ERK1/2 phosphorylation, we as- at 4mg; n=4; P,0.05 compared with WNK1 0-mg group) in sessed the effect of siRNA WNK1 on BK ubiquitination. As both HEK293 cells transiently expressing BK and WNK1 (Fig- shown in Figure 6, in HEK BKa cells transfected with siRNA ure 3, A and C) and HEK BKa stable cells expressing WNK1 WNK1 (45 nM), knockdown of WNK1 markedly reduced (100%60% with WNK1 at 1 mg, 164.1%644.3% at 3 mg, total BK protein expression (Figure 6B), whereas knockdown 203.1%669.6% at 6 mg, 409.0%6121.9% at 9 mg; n=5; of WNK1 expression significantly increased BKubiquitination P,0.05 compared with WNK1 1-mg group) (Figure 3, B (302.7%641.0% versus 100%627.7% in siRNA control and D). These data suggested that WNK1 significantly in- group; n=4; P,0.05) (Figure 6C), suggesting that knockdown creases BK protein expressions, ultimately leading to activat- of WNK1 reduces BK protein levels by increasing BK ubiqui- ing BK activity. tination, ultimately leading to BK degradation.

Effect of WNK1 on ERK1/2 Phosphorylation WNK1 Stabilizes BK Protein Expression by Reducing Its WNK4 inhibits BK activity by activating ERK1/2 and p-38 signal- Degradation through a Lysosomal Pathway ing pathways.8 We therefore determined whether WNK1 stimu- Given the inhibitory effect of WNK1 knockdown on BK lates BK activity and protein expression by inhibiting ERK1/2 sig- protein expression, we further investigated whether knock- naling pathway. In HEK BKa cells transfected with WNK1, WNK1 down of WNK1 enhances the degradation of BK protein. Thus, overexpression significantly decreased ERK1/2 phosphorylation we determined the effect of bafilomycin A1 (Baf A1), a

J Am Soc Nephrol 26: ccc–ccc, 2014 WNK1 Stimulates BK Channel Activity via ERK1/2 3 BASIC RESEARCH www.jasn.org

Figure 3. Overexpression of WNK1 increases BK protein expressions in HEK293 cells with transiently expressing BKa or stably ex- pressing BKa. HEK293 cells were transfected with both pCMV-myc-BKa and a series of doses of pcDNA4.0-his-myc-WNK1 (A) in four independent experiments. HEK BKa cells were transfected with a series of doses of WNK1 in five independent experiments (B). Forty- eight hours after transfection, cells were lysed and subjected to SDS-PAGE and Western blot analysis. (A and B) Representative im- munoblots for WNK1, BK, and actin. (C and D) Bar graphs of summarized data from respective immunoblots. Anti-myc antibody was used for detecting WNK1 and BK protein expressions in A. Anti-WNK1 and anti-BK antibodies were used to detect WNK1 and BK protein ex- pressions, respectively, in B. Overexpression of WNK1 both in transiently transfected HEK293 cells and HEK stably expressing BKa cells significantly increased total BK protein expressions in a dose-dependent manner. *P,0.01 compared with the control. lysosomal inhibitor, on BK protein expression. Baf A1 specif- in mice. After 10 days of high dietary K loading (10%), urinary ically inhibits the vacuolar-type H+-ATPase and thereby affects K excretions were significantly increased in the HK group acidic proteases by disturbing the pH of endocytic organelles, compared with those in the normal K (NK) diet group including lysosomes, ultimately interfering with the lysosomal (2.4860.50 mmol/d versus 0.5960.19 mmol/d; n=8; degradation pathway. As shown in Figure 7, BK protein ex- P,0.001). As shown in Figure 8, protein expressions of BK pression was significantly reduced in HEK BKa cells transfec- and WNK1 were significantly increased in HK groups ted with siRNA WNK1 (45 nM) (48.9%613.2% versus 100% (208.6%624.4% versus 100%618.7%; n=8; P,0.01) and 611.2% in siRNA control; n=4; P,0.05). However, in HEK (188.6%630.0% versus 100%614.3%; n=8; P,0.01), respec- BKa cells pretreated with Baf A1, BK protein level was signif- tively, compared with those in NK groups. ERK1/2 phosphor- icantly increased in a dose-dependent manner (189.8% ylations were significantly reduced in HK groups compared 650.2% at Baf A1 0.5 mM and 294.6%663.3% at Baf A1 with those in NK groups (p-ERK1/2/t-ERK1/2 ratio of 1.0 mM) compared with siRNA WNK1 group (48.9% 0.4960.15 versus 1.060.44; n=8; P,0.05). These data sugges- 613.2%; n=4; P,0.05). These data suggested that WNK1 en- ted that dietary K challenge enhanced BK-mediated K excre- hances BK protein expression by reducing BK degradation tion through WNK1-dependent ERK 1/2 signaling pathway. through a lysosomal pathway.

Effects of Dietary K on Protein Expressions of WNK1 DISCUSSION and BKa and ERK 1/2 Phosphorylation A high-K (HK) diet increases BK protein expression and flow- In this study we have demonstrated for the first time that mediated K excretion.34 To explore the physiologic relevance WNK1 activates BK channel activity by inhibiting the MAPK to preceding findings, we performed the metabolic cage study ERK1/2 signaling pathway. We have shown that knockdown of

4 Journal of the American Society of Nephrology J Am Soc Nephrol 26: ccc–ccc,2014 www.jasn.org BASIC RESEARCH

Figure 4. WNK1 reduces ERK1/2 phosphorylations in HEK BKa cells. HEK BKa cells were transfected with a series of doses of WNK1 plasmids (A) or a series of doses of siRNA WNK1 (B). Forty-eight hours after transfection, cells were lysed and subjected to SDS-PAGE and Western blot analysis. (A and B) Representative immunoblots for WNK1, p-ERK 1/2, t-ERK1/2, and actin. (C and D) Bar graphs show the ratios of p-ERK1/2 to total ERK1/2 from four independent experiments for the respective immunoblot (mean6SD). Overexpression of WNK1 significantly reduces ERK1/2 phosphorylation, whereas knockdown of WNK1 expression significantly enhances ERK1/2 phosphorylation in a dose-dependent manner. n=4; *P,0.05.

WNK1 expression reduced BK activity and BK protein WNK1 acts upstream of the MAPK-ERK1/2 signaling pathway expression while increasing ERK1/2 phosphorylation. Knock- and that WNK family members closely intermingle with down ERK1/2 abolished siRNA WNK1-mediated inhibitory MAPK signaling cascades. effect on BK. Wehavefurther shown that knockdown of WNK1 The BK channel is a large-conductance . enhanced BK ubiquitination while reducing total BK protein It is unique among potassium channels in that it is activated, or expression as a result of enhanced BK degradation via alyso- gated, both by membrane depolarization and intracellular somal pathway. Dietary potassium challenge in mice increased calcium (Ca2+), being particularly subjected to intracellular BK and WNK1 protein abundances while reducing ERK1/2 Ca2+ regulation.38 BKa is expressed in nearly every segment phosphorylation. Our data suggest that WNK1 modulates BK of the nephron.39–41 The BK channel is localized on the apical function through an ERK1/2 signaling pathway, indicating membrane of these cells, including both principal and inter- that WNK1 plays an important role in the regulation of BK calated cells, along with the ROMK that were exclusively ex- activity and its protein expression. pressed in the principal cells, which underlie baseline luminal WNK kinase is structurally categorized into MAPK kinase K+ secretion.34 Studies from the BKa null mouse also showed kinase.35 As a member of MAPKs, ERK1/2 is usually phos- that K+ secretion is disrupted in response to the increased flow, phorylated and activated as part of Ras/Raf/MEK1/2/ERK1/2 as well as to high K+ load.34 A functional BK channel is com- signaling cascade upon stimulation of cells by various cyto- posed of a tetramer of a-subunits that form the pore of the kines, growth factors, and extracellular stresses. WNK1 was channel. BKa functions as a channel by itself. BKa is exten- found to activate ERK5 by an MEKK2/3-dependent mecha- sively regulated by alternative splicing, phosphorylation and nism.32 WNK2 reduces cell proliferation by inhibiting the dephosphorylation, and regulatory b subunits. The b1sub- MEK1/ERK1/2 signaling pathway.36 We previously showed unit of BK was found to facilitate the forward trafficking of that WNK4 overexpression enhances ERK1/2 phosphoryla- BKa.42 The b1 subunit itself can also rapidly traffictothe tion.37 In the present study, we found that overexpression of surface in response to nitric oxide stimulation and can activate WNK1 decreased ERK1/2 phosphorylation, whereas knock- the BKa channel.25 However, it is not entirely clear whether down of WNK1 expression using an siRNA technique en- the b subunit of the BK channel affects the internalization and hanced ERK1/2 phosphorylation. These data suggested that degradation of BKa.BKa itself can traffictotheplasma

J Am Soc Nephrol 26: ccc–ccc, 2014 WNK1 Stimulates BK Channel Activity via ERK1/2 5 BASIC RESEARCH www.jasn.org

Figure 5. Knockdown of ERK1/2 expression abolished WNK1-mediated effects on BK protein expression. HEK BKa cells were transfected with scramble control siRNA or 45 nM of siRNA WNK1 with or without siRNA ERK1/2 (15 nM). Forty-eight hours after transfection, cells were lysed and subjected to SDS-PAGE and Western blot analysis. (A) Representative immunoblots for WNK1, BK, phospho-ERK1/2, total-ERK1/2, and actin. Bar graphs show mean6SD band densities of BK (B) and WNK1 (C) and ratio of p-ERK1/2 over t-ERK1/2 (D). The knockdown of WNK1 expression significantly decreases total BK protein expressions, whereas knocking down ERK1/2 abolished siRNA WNK1-mediated inhibitory effect on BK protein expressions. n=8; *P,0.05 versus control.

membrane in the absence of its regulatory BK b subunit,43,44 likely due to both decreases in channel number on the cell and the motif in the C-terminus of BKa also plays an impor- surface and its channel activity, suggesting that WNK1 not tant role in BKa forward trafficking.43,44 Whether WNK1 af- only affects BKa protein expression but also affects BKa chan- fects the expression of BK b subunit and subsequently alters nel intrinsic kinetics. Whether WNK1 phosphorylates BKa, BKa forward trafficking remains to be further explored. ultimately leading to the change of BKa channel kinetics, BKa is modulated by many protein kinases.14,16–20 In ad- remains to be determined. dition, MAPK was reported to inhibit BK channel activity in In this study we also found that reduced WNK1 expression rat principal cells.31 We have previously shown that WNK4 increases BK ubiquitination while reducing total BK protein inhibits BK channel activity by enhancing its degradation expression. Pretreatment with bafilomycin A1, a lysosomal through a lysosomal pathway. In this study, we found that inhibitor, reversed the siRNA WNK1-mediated inhibitory overexpression of WNK1 increased BKa protein expressions effect on BK expression. This finding suggests that WNK1 while reducing ERK1/2 phosphorylation, whereas knockdown activates BK channel activity by reducing its degradation of WNK1 expression decreased BKa protein expression while through a lysosomal pathway, likely by inhibiting an ERK1/2 increasing ERK1/2 phosphorylation. We further found that signaling pathway, a mechanism similar to WNK4-mediated knockdown of ERK1/2 expression abolished the WNK1- modulation of BK but in the opposite direction.7,8 mediated effects on BKa protein expression. These data WNK1 was found to inhibit ROMK activity and its protein suggested that WNK1 affects BKa protein expression by mod- expression and KS-WNK1 reversed wild-type (WT)-WNK1’s ulating ERK1/2 signaling pathway. Our cell-attached patch inhibitory effects on ROMK.6,45 Several studies have shown experiments also showed that knockdown of WNK1 expres- that the ratio of WT-WNK1 to KS-WNK1 is relevant to K sion significantly inhibited both BKa NPo and Po, which is secretion.6,46 TheKS-WNK1levelappearstobemore

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abundance, remains to be further eluci- dated.49,50 Since WNK1 expression is upregulated in patients with PHA-II,1 in- hibition of ROMK by WNK1 could explain the clinical phenotype of hyperkalemia.45 However, it is still to be determined whether the ratio of WNK1 over KS- WNK1 is crucial in directly modulating BK channel activity. How the interaction between WNK1 and KS-WNK1 affects BK activity needs to be further explored. Whether upregulation of WNK1 expres- sion in patients with PHA II enhances BK channel activity to alleviate hyperkalemia as a result of inhibitory effect of ROMK Figure 6. Effect of knockdown of WNK1 expression on BK ubiquitination in HEK BKa by WNK1 remains to be further investi- cells. HEK BKa cells were transfected with scramble control siRNA or siRNA WNK1. gated. The above-mentioned questions Forty-eight hours after transfection, cells were lysed and subjected to SDS-PAGE, need to be addressed in future studies. Immunoprecipitation, and Western blot analysis. A representative immunoblot of four In summary, our data demonstrated for independent experiments is shown here. (A) Sample was immunoprecipitated with the first time that WNK1 directly activates a anti-BK antibody and immunoblotted (IB) with anti-ubiquitin antibody. B. Sample was BK channel activity and its protein expres- a immunoprecipitated with anti-BK antibody, and immunoblotting was performed with sion by inhibiting the ERK1/2 signaling the same anti-BKa antibody. (C). Bar graph shows summary data of the ratio of pathway. This stimulatory effect of WNK1 ubiquitinated-BK over total BK protein expressions from four independent experi- ments (mean6SD). Knockdown of WNK1 expression markedly reduces total BK pro- on BK activity is a result of WNK1-mediated tein expression (B) while significantly increasing BK ubiquitination (A). reduction of BK degradation through a lyso- somal pathway. These data suggest that WNK1 directly modulates BK activity by altering sensitive to dietary potassium modulation.47 In this study, we ERK1/2 signaling pathway. WNK kinase playsan important role found that 10 days of HK K (10% KCl) diet feeding in mice in regulation of BK activity that is contributing to maintaining significantly enhanced BK protein expression, which is con- potassium homeostasis. sistent with previous findings.34 An HK diet also reduced ERK1/2 phosphorylation, suggesting that dietary K modu- lates BK expression through an ERK1/2 signaling pathway. MATERIAL AND METHODS We also found that an HK diet enhanced WNK1 protein ex- pression, which is not consistent with previous findings that Animals, Dietary Manipulation, and Metabolic Cage WNK1 expression was not significantly changed after 2 days Studies of HK diet (10% KCl) feeding in rats.6 Different durations of The Institutional Animal Care and Use Committees from an HK diet challenge between two studies and different spe- Emory University and the Atlanta Veterans Affairs Medical cies of animals that were used in the experiments might con- Center approved all animal-related procedures. The C57BL/6 tribute to the discrepancy of the two studies. However, mice (8 weeks old) were purchased from The Jackson another study showed that 10 days of HK (3.3% K) feeding Laboratory (Bar Harbor, ME). The mice were assigned to in mice appears to have increased the WNK1 mRNA level, but different dietary groups: (1) control K group: eight mice were this finding did not reach statistically significance; high K did fed with control potassium diet (control K; 1% KCl, TD induce more dramatic increase in KS-WNK mRNA level.48 #88238) and (2) HK group: eight mice were fed with high Whether a lower amount of dietary HK feeding could explain potassium diet (HK; 10% KCl, TD #09075). All diets were an insignificant increase in WNK1 expression needs to be purchased from Harlan Laboratories (Indianapolis, IN). The further studied. animals were fed with these diets for 10 days and then studied. Hadchouel et al. reported that BKa protein abundance At the end of the animal study, all mice were perfused with along with ROMK is upregulated in KS-WNK1 KO mice,49 0.9% saline before kidneys were harvested. Mice were fully suggesting that KS-WNK1 regulates BK channel activity. anesthetized with isoflurane, then moved to a Styrofoam board However,Cheng et al. also reported that BK channel-mediated and kept on continuous isoflurane inhalation. The four paws K secretion is not significantly affected during HK diet feeding were pinned. An abdominal incision was made up to the chest in KS-WNK1 knockout mice.50 Whether knockout of KS- cavity and the diaphragm was cut. A 21-gauge needle was WNK1 affects WNK1 kinase activity or WNK1 expression inserted into the left ventricular chamber and a small hole was level, which could contribute to the increased BK protein snipped in the right atrium simultaneously to release 13PBS.

J Am Soc Nephrol 26: ccc–ccc, 2014 WNK1 Stimulates BK Channel Activity via ERK1/2 7 BASIC RESEARCH www.jasn.org

matched the human WNK1 sequence (GenBank accession no.: NM_018979). The N-terminal myc-taggedandHis-tagged WNK1 WT constructs were generated by subcloning the WNK1 cDNA into pcDNAä4/myc-His vector (Life Technol- ogies, Grand Island, NY). Myc-tagged wild-type rabbit Maxi K plasmid was a gift from Dr. William Guggino (Johns Hopkins University, Baltimore, MD). All constructs were confirmed by DNA sequencing. These constructs have been successfully ex- pressed in the HEK293 cells, as confirmed by Western blot analysis.

Cell Culture and Transfection HEK293 cells were maintained in DMEM medium containing 10% FBS, 1% penicillin (100 U/ml), and 1% streptomycin (100 U/ml). HEK293 cells stably expressing the a subunit of BK (HEK BKa cells) were previously generated in our labo- ratoryandmaintainedinDMEMsupplementedwith penicillin (100 U/ml), streptomycin (100 U/ml), G418 200 mg/10 ml, and 10% FBS.7 Lipofectamine 2000 (Life Technolo- gies) was used for transfection according to the manufacturer’s instructions. Forty-eight hours after transfection of cells with a Figure 7. Effect of bafilomycin A1, a lysosomal inhibitor, on BK series of doses of WNK1 plasmid, cells were lysed. Cell lysates a a protein expressions in HEK BK cells. HEK BK cells were were subjected to SDS-PAGE and Western blot analysis. transfected with control siRNA or siRNA WNK1. Sixteen hours before Western blot analysis, two doses of bafilomycinA1(Baf WNK1 Knockdown Experiments with siRNA A1) were added to culture medium. Forty-eight hours after a transfection, cells were lysed and subjected to SDS-PAGE and WNK1 expression was knocked down in HEK BK cells using Western blot analysis. (A) Representative immunoblots for WNK1, siRNA. WNK1 siRNA and scramble control siRNA were pur- BK, and actin. (B) Bar graphs of mean band densities (mean6SD) chased from Santa Cruz Biotechnology. Cells were grown in from four independent experiments. Lanes 1–3indicatethe six-well plates with 2 ml antibiotic-free normal culture me- control siRNA group treated with Baf A1 (0.5 and 1.0 mM). Lanes dium with 10% FBS 1–2 days before transfection day. WNK1 4–6 indicate siRNA WNK1 group treated with Baf A1 (0.5 and siRNA ([h], Sc-39526), WNK1 siRNA ([h2], Sc-96133), or 1.0 mM). Knockdown of WNK1 expression (lane 4) significantly scramble control siRNA was transfected into HEK-293 BKa reduces total BK protein expression compared with the control cells with siRNA reagent (Santa Cruz Biotechnology). ERK 1/2 , group (lane 1). n=4; *P 0.05. Baf A1 treatment reverses the in- siRNA (Sc-29308) was also purchased from Santa Cruz Bio- hibitory effects by knocking down WNK1 expression (lanes 5 and 6). technology. Forty-eight hours after transfection, cell lysates n=4; #P,0.05 compared with the untreated siRNA WNK1 group were collected and analyzed by Western blot analysis. (lane 4). These results suggest that Baf A1 reverses the inhibitory effects of WNK1 siRNA on BKa protein expression in a dose- dependent manner. Single-Channel Recording All experiments in this study were carried out using the cell- attached configuration of the patch-clamp technique in HEK PBS was continuously infused for 1 minute to cleanse all blood BKa cells transfected with siRNA WNK1 or siRNA scramble until the color of the kidney tissue changed from red to pale control, as previously described.7,51 Electrodes were fabricated pink. One kidney was harvested for Western blot analysis and from Corning 7052 glass (Garner Glass, Fullerton, CA) in two the other kidney tissue was stored at 280°C. steps on a Narishige PP-83 electrode puller (Narishige, Tokyo, Metabolic cages were used to collect 24-hour urine before Japan). The bath solution used in the cell-attached mode con- and after dietary K manipulations. Two days prior to being fed tained (in mM) 140 NaCl, 1 CaCl2, 5 KCl, 1 EGTA, and 10 with special diets and 2 days prior to euthanasia, the animals HEPES, pH adjusted to 7.4. The pipette solution used con- were placed in metabolic cages, and 24-hour urine samples tained (in mM) 140 KCl, 4 MgCl2,1CaCl2, 1 EGTA, and 5 were collected for the measurement of urine volume and HEPES (pH 7.2). Recordings were performed at room tem- electrolytes. The electrolyte concentrations were measured perature. After formation of a high-resistance (5 GV) seal, the using an Na/K/Cl/Li analyzer (Medica, MA). channel currents were filtered at 1 kHz, recorded with an Axopatch 1-D amplifier (Molecular Devices) and sampled at Plasmids and Constructs 5 kHz. Channel activity (NPo) was calculated from pClampfit Human WT WNK1 was amplified by PCR using a human 9.2 data analysis software (Molecular Devices). Channel num- kidney cDNA library as the template. The PCR product ber (N) was determined from the maximal number of

8 Journal of the American Society of Nephrology J Am Soc Nephrol 26: ccc–ccc,2014 www.jasn.org BASIC RESEARCH

Figure 8. The effects of high dietary K on protein expressions of WNK1 and BKa, and ERK1/2 phosphorylation in normal mice. Mice were fed with NK (1%) diet (8 mice) or HK K (10%) diet (8 mice) for 10 days. After 10 days of dietary K challenges, kidneys were harvested for Western blot analysis. (A) Representative immunoblots for WNK1, BK, phospho-ERK1/2, total-ERK1/2, and actin. Bar graphs show mean6SD band densities of BK (B), WNK1 (C), and ratio of p-ERK1/2 over t-ERK1/2 (D). BK and WNK1 protein expressions were significantly increased in the HK group compared with those in the NK group, whereas ERK1/2 phosphorylations were signifi- cantly reduced in HK group compared with those in NK group. n=8; *P,0.05.

transitions during 10–20 min of recording, and channel Po were performed according to standard procedures as described was calculated as the ratio of NPo to N. previously.37 Antibodies were used including p-ERK1/2 and t-ERK1/2 (Cell Signaling Technology, Boston, MA), anti-Actin Western Blotting Analysis (EMD Millipore, Billerica, MA), anti-WNK1 (Catalog #GTX Cells were harvested and processed as described previously.52 106197, from GeneTex, Irvine, CA), and anti-BKa (Alomone Briefly, cells transiently transfected with various DNA con- Labs, Jerusalem, Israel). structs or siRNA as indicated were lysed in lysis buffer con- taining 20 mM Hepes, pH 7.5, 120 mM NaCl, 5.0 mM EDTA, Immunoprecipitation 1.0% Triton X-100, 0.5 mM dithiothreitol, 1.0 mM phenyl- Immunoprecipitation was performed as previously de- methylsulfonyl fluoride, and complete protease inhibitor scribed.53 Briefly, HEK293 cells were cotransfected with (Roche Diagnostics, Mannheim, Germany; 1 tablet per 50-ml siRNA WNK1 or siRNA control as indicated. Forty-eight solution). The lysates were centrifuged at 6000 g for 5 minutes to hours after transfection, the cells were lysed. The cell lysates pellet the insoluble material, and the from supernatant were incubated with primary antibody (anti-BKa antibody) were quantified using a Pierce BCA Protein Assay kit (Pierce, for 2 hours, then Protein G/A Sepharose beads were added, Rockford, IL). After mixing in Laemmli buffer (Bio-Rad, Her- and the lysates were further mixed and incubated overnight at cules, CA) and incubating at 37°C for 30 minutes, the protein 4°C. After washing with lysis buffer three times, beads were sample was separated by SDS-PAGE and electrophoretically then eluted with Laemmli buffer (Bio-Rad). The eluted pro- transferred to polyvinylidene difluoride (PVDF) membranes teins were separated by SDS-PAGE, transferred onto PVDF (Amersham Biosciences, Piscataway, NJ) for Western blot. Prob- membrane, and probed with antiubiquitin antibody (Thermo ing with specific antibodies and subsequent detection with ECL Fisher Scientific, Rockford, IL). The PVDF membranes were plus system (Amersham Biosciences) or Super signal (Pierce) then stripped and reprobed with anti-BKa antibody.

J Am Soc Nephrol 26: ccc–ccc, 2014 WNK1 Stimulates BK Channel Activity via ERK1/2 9 BASIC RESEARCH www.jasn.org

Statistical Analyses 9. Wang Z, Subramanya AR, Satlin LM, Pastor-Soler NM, Carattino MD, The data are presented as the mean6SD. Statistical significance Kleyman TR: Regulation of large-conductance Ca2+-activated K+ chan- – was determined by t test when two groups were compared or nels by WNK4 kinase. Am J Physiol Cell Physiol 305: C846 C853, 2013 10. Wang WH, Giebisch G: Regulation of potassium (K) handling in the by one-way ANOVA, followed by Bonferroni post hoc tests renal collecting duct. Pflugers Arch 458: 157–168, 2009 when multiple groups were compared. We assigned signifi- 11. Butler A, Tsunoda S, McCobb DP, Wei A, Salkoff L: mSlo, a complex cance at P,0.05. mouse gene encoding “maxi” calcium-activated potassium channels. Science 261: 221–224, 1993 12. Kaczorowski GJ, Knaus HG, Leonard RJ, McManus OB, Garcia ML: High-conductance calcium-activated potassium channels; structure, pharmacology, and function. J Bioenerg Biomembr 28: 255–267, 1996 ACKNOWLEDGMENTS 13. Toro L, Wallner M, Meera P, Tanaka Y: Maxi-K(Ca), a Unique Member of the Voltage-Gated K Channel Superfamily. News Physiol Sci 13: 112– We thank Dr. Janet Klein for her suggestions and critical reading of the 117, 1998 manuscript. 14. Liu W, Wei Y, Sun P, Wang WH, Kleyman TR, Satlin LM: Mechano- regulation of BK channel activity in the mammalian cortical collecting This work is supported by Norman S. Coplon Satellite Grant duct: Role of protein kinases A and C. Am J Physiol Renal Physiol 297: (H.C.),the DepartmentofVeteranAffairsMERITAward 5I01BX000994 F904–F915, 2009 (H.C.), National Institutes of Health grants R37-DK037963 (D.C.E.), 15. Tian L, Duncan RR, Hammond MS, Coghill LS, Wen H, Rusinova R, Clark 5R01-DK067110(H.M.),NationalNaturalScienceFoundationofChina AG, Levitan IB, Shipston MJ: Alternative splicing switches potassium – #81170709 (J.Z.) and #31300811 (Y.L.), Zhejiang Provincial Natural channel sensitivity to protein phosphorylation. J Biol Chem 276: 7717 7720, 2001 Science Foundation of China #Y2080291 (J.Z.) and Wenzhou City 16. Zhou XB, Arntz C, Kamm S, Motejlek K, Sausbier U, Wang GX, Ruth P, Science and Technology Cooperation ProgramH20090071 (D.G.). Parts Korth M: A molecular switch for specific stimulation of the BKCa of data were presented at the annual American Society of Nephrology channel by cGMP and cAMP kinase. JBiolChem276: 43239–43245, meeting in 2013. 2001 17. Barman SA, Zhu S, White RE: PKC activates BKCa channels in rat pul- monary arterial smooth muscle via cGMP-dependent protein kinase. Am J Physiol Lung Cell Mol Physiol 286: L1275–L1281, 2004 DISCLOSURES 18. Wu SN, Wang YJ, Lin MW: Potent stimulation of large-conductance None. Ca2+-activated K+ channels by rottlerin, an inhibitor of protein kinase C-delta, in pituitary tumor (GH3) cells and in cortical neuronal (HCN-1A) cells. JCellPhysiol210: 655–666, 2007 19. 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J Am Soc Nephrol 26: ccc–ccc, 2014 WNK1 Stimulates BK Channel Activity via ERK1/2 11 Supplemental File

Figure S1. Knock-down of WNK1 expression using siRNA WNK1 (h2) decreases BKα protein expression in HEK BKα cells. HEK BKα cells were transfected with either scramble control siRNA or siRNA WNK1(h2) at 45 nM. Forty-eight hours after transfection, cells were lysed and subjected to SDS-PAGE and western blot analysis. A shows representative immunoblots for WNK1, BK and actin. B indicates a bar graph showing mean ± SD band densities of BK protein expression from four independent experiments. The knock-down of

WNK1 expression significantly decreases total BK protein expressions (32.7 ± 18.9 % in siRNA

WNK1 (h2) vs 100 ± 15.3 % in control siRNA, n=4, p < 0.001).

Figure S1