BASIC RESEARCH www.jasn.org 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) kinases are members of the serine/threonine kinase 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 protein 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. Mutations of WNK1 and activity via a ubiquitin-dependent pathway.9 WNK4are responsible for pseudohypoaldosteronism 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 gene slo111 and is widely distributed in many different mutation 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 cell-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.