Antagonistic Regulation of ROMK by Long and Kidney-Specific WNK1 Isoforms

Antagonistic regulation of ROMK by long and kidney-specific WNK1 isoforms

Ahmed Lazrak*, Zhen Liu*, and Chou-Long Huang†

Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8856

Communicated by Steven C. Hebert, Yale University School of Medicine, New Haven, CT, December 8, 2005 (received for review November 8, 2005) WNK kinases are serine-threonine kinases with an atypical place- of hypertension (7, 8). Others have reported that WNK4 phos- ment of the catalytic lysine. Intronic deletions with increased phorylates claudins 1–4, the tight-junction proteins involved in expression of a ubiquitous long WNK1 transcript cause pseudohy- the regulation of paracellular ion permeability (9, 10). The poaldosteronism type 2 (PHA II), characterized by hypertension and paracellular chloride permeability is greater in cells expressing hyperkalemia. Here, we report that long WNK1 inhibited ROMK1 WNK4 mutants than in cells expressing wild-type proteins. Thus, by stimulating its endocytosis. Inhibition of ROMK by long WNK1 hypertension in patients with WNK4 mutations may be caused by was synergistic with, but not dependent on, WNK4. A smaller an increase in NaCl reabsorption through the Na-Cl cotrans- transcript of WNK1 lacking the N-terminal 1–437 amino acids is porter and the paracellular pathway. Wild-type WNK4 inhibits expressed highly in the kidney. Whether expression of the KS- the ROMK1 channel and WNK4 mutants that cause disease WNK1 (kidney-specific, KS) is altered in PHA II is not known. We exhibit increased inhibition of ROMK (11), suggesting that found that KS-WNK1 did not inhibit ROMK1 but reversed the WNK4 mutations cause hyperkalemia by inhibiting ROMK. inhibition of ROMK1 caused by long WNK1. Consistent with the Expression of WNK1 abolishes inhibition of the sodium lack of inhibition by KS-WNK1, we found that amino acids 1–491 chloride cotransporter caused by WNK4 in Xenopus oocytes (7), ؉ of the long WNK1 were sufficient for inhibiting ROMK. Dietary K suggesting that WNK1 mutations cause hypertension by releasing restriction decreases ROMK abundance in the renal cortical-collect- WNK4-mediated inhibition of the cotransporter in the distal ing ducts by stimulating endocytosis, an adaptative response convoluted tubule. However, PHA II patients with WNK1 ؉ ؉ important for conservation of K during K deficiency. We found mutations are not particularly sensitive to thiazide diuretics (12). ؉ that K restriction in rats increased whole-kidney transcript of long Moreover, patients with WNK1 mutations do not have hyper- WNK1 while decreasing that of KS-WNK1. Thus, KS-WNK1 is a calciuria, whereas patients with WNK4 mutations have hyper- physiological antagonist of long WNK1. Hyperkalemia in PHA II calciuria that is Ϸ6-fold more sensitive to thiazide treatment than patients with PHA II mutations may be caused, at least partially, by normal individuals (13, 14). A recent study by Xu et al. (15) increased expression of long WNK1 with or without decreased shows that WNK1 activates SGK leading to activation of ENaC. expression of KS-WNK1. Thus, hypertension in PHA II patents with WNK1 mutations may be caused by increased activity of Na-Cl cotransporter and dietary potassium intake ͉ endocytosis ͉ clathrin-coated vesicle ͉ dynamin ENaC. The mechanism for hyperkalemia in patients with WNK1 mutations is unknown. NK (with no lysine [K]) kinases are a new family of large Although WNK4 is expressed predominantly in kidney and Wserine-threonine protein kinases conserved in multicellu- several extrarenal epithelial tissues, WNK1 is widely expressed lar organisms with an atypical placement of the catalytic lysine. in multiple spliced forms (2, 16). A long WNK1 transcript There are four mammalian WNK family members (1). WNK1, (produced from 28 exons) encoding a polypeptide of Ͼ2,100 the first member identified, is Ͼ2,100 amino acids long (2). It amino acids in length is expressed in all cell lines and tissues contains an Ϸ270-aa kinase domain located near the amino examined (2, 17–19). A shorter WNK1 transcript encoding a terminus (e.g., amino acids 218–491 of the rat WNK1). WNK2, polypeptide (Ϸ1,700 amino acids in length) lacking the amino 3, and 4 are products of different genes and Ϸ1,200 to 1,600 terminal 1–437 amino acids of the long WNK1 is expressed amino acids in length (1, 2). The kinase domain of the four highly in the kidney but not in other tissues (18, 19). The WNKs that share 85–90% sequence identity are unique in having KS-WNK1 (KS, kidney-specific) is produced by replacing the the catalytic lysine located in the subdomain I instead of the first 4 exons with an alternative 5Ј exon (exon 4A). The remain- conserved subdomain II of most protein kinases (1–3). Other ing exons 5–28 are the same as the long transcript. Quantitative conserved domains of WNK kinases include an autoinhibitory analysis of WNK1 transcripts reveals that KS-WNK1 is expressed

domain, 1–2 coiled-coil domains, and multiple PXXP proline- in kidney more abundantly than long WNK1 (Ϸ85% vs. Ϸ15%) PHYSIOLOGY rich motifs for potential protein–protein interaction (1–4). Be- (18, 19). A large deletion of intron 1 causes increased expression yond the aforementioned conserved domains͞motifs, sequence of the long WNK1 isoform (6). Whether the expression of identity among the four WNKs is much lower and few homol- KS-WNK1 is altered in PHA II and the physiological role of ogous regions exist. KS-WNK1 are currently unknown. Pseudohypoaldosteronism type II (PHA II) is an autosomal- Kϩ secretion by kidney is critical for controlling serum Kϩ dominant disease characterized by hypertension and hyperkale- levels and overall Kϩ homeostasis (20, 21). ROMK Kϩ channels mia (5). Recently, Wilson et al. (6) reported that mutations of present on the apical membrane of the distal renal tubules are WNK1 and WNK4 cause PHA II. Mutations in the WNK1 gene important for baseline renal Kϩ secretion (20–23). Another type are large deletions of the first intron leading to increased expression. Mutations in the WNK4 gene are missense mutations in the coding sequence outside the protein kinase domain. Conflict of interest statement: No conflicts declared. Several recent studies have examined the mechanisms for hy- Abbreviations: CCV, clathrin-coated vesicle; HEK, human embryonic kidney; KS, kidney- pertension and hyperkalemia in PHA II patients. WNK4 inhibits specific; PHA II, pseudohypoaldosteronism type II; siRNA, small interference RNA. the activity of the sodium chloride cotransporter. WNK4 mu- *A.L. and Z.L. contributed equally to this work. tants that cause disease fail to inhibit the sodium chloride †To whom correspondence should be addressed. E-mail: chou-long.huang@ cotransporter, suggesting that an increase in sodium chloride utsouthwestern.edu. cotransporter activity in the distal convoluted tubule is a cause © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510609103 PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1615–1620 Downloaded by guest on October 1, 2021 Fig. 2. Effect of WNK1 or WNK4 siRNA on ROMK1 expression in HEK cells. (A) Cells were cotransfected with ROMK1 (0.5 ␮g) plus WNK1 siRNA (200 nM in transfection mixture) or control oligonucleotide. (B) Cells were cotransfected with ROMK1 (0.5 ␮g) plus WNK4 siRNA (200 nM) or control oligonucleotide. (B Inset) mRNA expression of endogenous WNK4 analyzed by reverse- transcription PCR. Cells were mock-transfected (Mock) or transfected with control oligos (Control) or siRNA for WNK4 (WNK4-si). Experiments above were repeated 2–3 times with similar results.

Fig. 1. Effect of WNK1 on ROMK1 expressed in HEK cells. (A) Whole-cell recording, voltage-clamp protocol, and current-voltage (I-V) relationships of currents. (B) Dose-dependent inhibition of ROMK1 by WNK1. Cells were ROMK1 undergoes clathrin-coated vesicle (CCV)-mediated transfected with ROMK1 plus WNK1 (0–2.5 ␮g of plasmid DNA). In each endocytosis, a process believed to be an important mechanism ϩ experiment, the total amount of DNA for transfection was balanced by using for regulating K secretion in physiological and͞or pathophys- empty vector. Ba2ϩ-sensitive (after subtraction of residual currents in the iological states (27, 28). To determine whether WNK1 inhibits ϩ presence of 10 mM Ba2 ) inward current density is shown. *, P Ͻ 0.05 vs. ROMK1 by stimulating CCV-mediated endocytosis of the chan- ROMK1 alone. (C) Effect of wild-type (WT-DII) or dominant-negative (K44A) nel, we examined the effect of WNK1 on ROMK1 by coexpres- dynamin II (DN-DII) on WNK1 inhibition of ROMK1. (D) Effect of WNK1 on wild-type ROMK1 (WT-RK) vs. N375I ROMK1 mutant. Experiments above were sion with wild-type or a dominant-negative dynamin II. As repeated 3–5 times with similar results. NS, not signiﬁcant. reported previously, coexpression with dominant-negative dynamin II increased basal ROMK1 current density (Fig. 1C; 124 Ϯ 10 pA͞pF vs. 364 Ϯ 20 pA͞pF for coexpression with ϩ of K channels, maxi-K, are also present in the distal renal wild-type vs. dominant-negative dynamin II, respectively; P Ͻ ϩ tubules and important for K secretion in response to increase 0.01), indicating that endocytosis of ROMK1 occurs in the basal ϩ in tubular fluid flow (23, 24). To maintain K homeostasis, the state. Coexpression with dominant-negative dynamin II (lysine- abundance of ROMK in the distal nephron decreases or in- 44 to alanine; K44A) prevented the inhibition of ROMK1 by ϩ creases during low or high dietary K intake, respectively (25, long WNK1. For comparison, coexpression with wild-type dy- 26). Alteration of abundance of ROMK during changes of ϩ namin II had no effect on long WNK1-induced inhibition. dietary K intake involves endocytosis and subsequent degra- Dynamin II may also be involved in internalization via caveolae dation of the channel protein (27, 28). (29). ROMK1 contains a canonical NPXY motif for internal- In the present study, we show that long WNK1 inhibits ROMK1, ization via CCV (30), and mutation of the critical asparagine-375 whereas KS-WNK1 reverses inhibition of ROMK1 caused by long ϩ in the motif abolished endocytosis (28). We found that WNK1 WNK1. Dietary K restriction in rats increases the abundance of failed to inhibit N375I mutant while inhibiting wild-type transcript for long WNK1 and decreases the abundance for KS- ROMK1 (Fig. 1D). Thus, WNK1 inhibits ROMK1 by stimulating WNK1 in the kidney. These results suggest that the physiological endocytosis of the channel via CCV. The incomplete inhibition role of KS-WNK1 as an antagonist of long WNK1. Furthermore, of ROMK by WNK1 (Fig. 1B) is probably due to limitation of hyperkalemia in PHA II patients with WNK1 mutations may be endogenous endocytic machinery. caused, at least partially, by the inhibition of ROMK channels resulting from increased expression of long WNK1 with or without Endogenous WNK1 and WNK4 Are Important for Regulating ROMK1 decreased expression of KS-WNK1. Channels. WNK1 and WNK4 are widely expressed. In the next Results series of experiments, we examined whether endogenous WNK1 Long WNK1 Inhibits ROMK1 by Stimulating Endocytosis of the Channel. or WNK4 in HEK cells contributes to the basal endocytosis of ROMK1. We found that cells transfected by small interference We examined regulation of ROMK1 by long WNK1 coexpressed Ϸ in the human embryonic kidney (HEK) cells by using whole-cell RNA (siRNA) for WNK1 expressed 2.2 fold higher ROMK1 patch-clamp recording. Cells transfected with ROMK1 exhibited current density than cells transduced by control RNA oligonu- Ϯ ͞ Ϯ ͞ the characteristic weak inward-rectifying Ba2ϩ-sensitive Kϩ cur- cleotide (current density: 380 24 pA pF vs. 170 13 pA pF ϩ Ͻ rents (Fig. 1A). K currents were not detected in mock- for WNK1 siRNA vs. control RNA, respectively, P 0.01; Fig. transfected cells (Fig. 1A). Coexpression with long WNK1 2A). Knockdown of endogenous WNK1 by siRNA was con- inhibited Kϩ currents through ROMK1 (Fig. 1A). Fig. 1B shows firmed by Western blot analysis of endogenous WNK1 in ref. 31. dose-dependent inhibition of ROMK1 current by long WNK1. Similarly, cells transfected with WNK4 siRNA expressed higher The mean inward ROMK current density (at Ϫ100 mV) were ROMK1 currents (current density: 341 Ϯ 36 pA͞pF vs. 192 Ϯ 15 135 Ϯ 14 pA͞pF and 37 Ϯ 13 pA͞pF for without WNK1 and pA͞pF for WNK4 siRNA vs. control RNA, respectively, P Ͻ coexpression with 2.5-␮g long WNK1 DNA, respectively (Fig. 0.01; Fig. 2B). Knockdown of endogenous WNK4 messenger 1B). No further inhibition of ROMK1 was observed by coex- RNA by siRNA was confirmed by reverse-transcription PCR pression with Ͼ2.5-␮g WNK1 DNA (not shown). It has been analysis (Fig. 2B Inset). We could not consistently detect en- reported that WNK4 regulates membrane trafficking of ion dogenous WNK4 protein by using the antibodies against WNK4 channels and transporters (7, 8, 11). available to us.

1616 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510609103 Lazrak et al. Downloaded by guest on October 1, 2021 Fig. 3. Relationships between WNK1 and WNK4 regulation of ROMK1. (A) Dose-dependent inhibition of ROMK1 by WNK4. Cells were transfected with ROMK1 plus WNK4 (0–2.5 ␮g plasmid DNA). (B) Cells were cotransfected with ROMK1 plus 0.5 ␮g WNK1 and͞or 0.5 ␮g WNK4. (C) Cells were cotransfected with ROMK1 plus 1 ␮g WNK1 and͞or 0.5 ␮g WNK4. (D) Cells were cotransfected with ROMK1 (0.5 ␮g), WNK1 (2.5 ␮g), and WNK4 siRNA (200 nM) or control oligonucleotide. *, P Ͻ 0.05 vs. ROMK1 alone. Experiments above were repeated 3–5 times with similar results.

Fig. 4. Domain of WNK1 involved in regulation of ROMK1. (A) Domain Regulation of ROMK1 by WNK1 Is Synergistic with but not Dependent structure of full-length long WNK1 and location of the catalytic lysine-233. on WNK4. It has been reported that WNK4 also inhibits ROMK by Autoinhibitory domain (AID), kinase domain (KD), and N terminus preceding stimulating endocytosis (11). As shown in Fig. 3A, WNK4 inhibited kinase domain (N-ter) are shown, but not drawn in scale. Fragments of WNK1 ROMK1 dose-dependently. WNK4 did not inhibit N375I mutant used are shown. (B) Cells were transfected with ROMK1 alone or cotransfected ␮ (data not shown), confirming that endocytosis is involved. WNK1 with indicated Myc-tagged WNK1 construct (each at 0.5 g). Experiments above were repeated three times with similar results. *, P Ͻ 0.05 vs. ROMK1 interacts with WNK4 (7). We examined the relationships between alone. (C) Western blot analysis of each construct blotted by anti-Myc anti- WNK1 and WNK4 regulation of ROMK1. Although transfecting body. Arrowhead on the left indicates molecular mass in kilodaltons (kDa). cells with 0.5 ␮g WNK1 plasmid DNA or WNK4 DNA alone did Experiments above were repeated three times with similar results. not cause inhibition of ROMK1, transfection with both WNK1 and 4 each at 0.5 ␮g DNA caused a significant inhibition of ROMK1 (Fig. 3B). Also, coexpression with 0.5 ␮g of WNK4 potentiated an autoinhibitory domain to the kinase core (4) (Fig. 4A). Accord- WNK1 inhibition of ROMK1 (Fig. 3C). These results indicate that ingly, WNK1 (1–555) construct does not exhibit kinase activity (4). WNK1 inhibits ROMK1 synergistically with WNK4. We further Lysine-233 of WNK1 is the catalytic active lysine and WNK1 examined whether WNK1 inhibition of ROMK1 depends on (1–491͞K233M) is kinase-dead (2). We found that both WNK1 WNK4 by knocking down endogenous WNK4. We found that (1–555) and WNK1 (1–491͞K233M) failed to inhibit ROMK1 (Fig. WNK1 inhibited ROMK1 similarly in cells transfected with WNK4 4B). Efficient expression of each individual protein in cells was siRNA and cells transfected with control oligonucleotide (Fig. 3D; confirmed by Western blot analysis (Fig. 4C). Together, these 80% vs. 82% for siRNA vs. control, respectively), indicating that results indicate that region of WNK1, including the kinase domain inhibition of ROMK1 by WNK1 does not depend on endogenous and preceding N terminus, contains amino acids sufficient for WNK4 in HEK cells. regulation of ROMK1.

The Kinase Domain of WNK1 Is Required for Inhibition of ROMK1. KS-WNK1 Does Not Inhibit ROMK1 but Reverses the Inhibition Caused

We PHYSIOLOGY have recently shown that WNK1 activates serum- and glucocorti- by Long WNK1. The KS-WNK1 lacks the N-terminal 1–437 amino coid-inducible protein kinase SGK, leading to activation of the acids of the long WNK1, including most of the kinase domain epithelial channel ENaC (15). The N-terminal 220 amino acids of (Fig. 5A) and is therefore kinase-defective. To examine whether WNK1 preceding the kinase domain are necessary and sufficient the region of WNK1 beyond the kinase domain can inhibit for activating SGK and ENaC. To determine whether the WNK1 ROMK1, we compared full-length WNK1, WNK1 (1–491), and kinase domain is necessary for regulating ROMK1, we compared KS-WNK1. We found that KS-WNK1 had no effect on ROMK1, the effects of amino acids 1–491 and 1–220 (Fig. 4A). We found that whereas the full-length long WNK1 and WNK1 (1–491) both WNK1 (1–491) inhibited ROMK1 and the full-length WNK1, inhibited ROMK1 (Fig. 5B). These results support the idea that whereas WNK1 (1–220) had no effect (Fig. 4B), indicating that region of amino acids 1–491 of WNK1 is the only region kinase domain is required for inhibition of ROMK1. Efficient necessary (and sufficient) for inhibition of ROMK1. The level of expression of WNK1 (1–220) in HEK cells is shown in Fig. 4C.A expression of KS-WNK1 in kidney is severalfold higher than that construct containing kinase domain but no amino acids preceding of long WNK1 (18, 19). We examined the hypothesis that it, WNK1 (218–491) did not cause inhibition of ROMK1 (data not KS-WNK1 may modulate long WNK1-induced inhibition of shown). The level of expression of WNK1 (218–491) was compa- ROMK1 by coexpressing KS-WNK1 with ROMK1 plus full- rable to that of WNK1 (1–491) (data not shown). Thus, both the length WNK1 or with ROMK1 plus WNK1 (1–491). Interest- WNK1 kinase domain and the preceding N terminus are required ingly, KS-WNK1 reversed inhibition of ROMK1 caused by for regulation of ROMK1. Amino acids 486–555 of WNK1 contain full-length WNK1 (Fig. 5B), suggesting that it functions as an

Lazrak et al. PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1617 Downloaded by guest on October 1, 2021 Fig. 6. Effect of dietary Kϩ intake on long and KS-WNK1 expression in kidney. (A) Relative abundance of transcript (normalized to the control Kϩ diet) for long WNK1 in rat kidney fed low (LK), control (CK) or high Kϩ diet (HK). (B) Relative abundance for KS-WNK1. (C) Ratio of abundance of transcript for L-WNK1 vs. for KS-WNK1. Ratio relative to the control Kϩ diet is shown. *, P Ͻ 0.05 vs. CK.

WNK1 (1–491) did not coimmunoprecipitate Flag-pod1 [see lack of protein in 22 kDa position (predicted molecular mass of pod1); Fig. 5C]. Conversely, anti-Flag antibody immunoprecipitated Flag-KS-WNK1 (1–253), which coimmunoprecipitated Myc-WNK1 (1–491). Also, Flag-pod1 did not coimmunoprecipitate Myc-WNK1 (1–491) (Fig. 5C). These results suggest that KS-WNK1 antagonizes inhibition of ROMK1 by long WNK1 by Fig. 5. Role of KS-WNK1 in regulation of ROMK1. (A) Comparison of domain binding to amino acids 1–491 of long WNK1. structure of long WNK1 (L-WNK1) with KS-WNK1. KS-WNK1 lacks the ﬁrst 437 amino acids of L-WNK1 but contains unique 30 amino acids coded by an Effects of Changes of Dietary K؉ Intake on Expression of Long and alternatively spliced exon 4A (the ﬁrst exon of KS-WNK1). The vertical dotted KS-WNK1. ROMK channel in the apical membrane of the distal line indicates position of amino acid in L-WNK1 equivalent to amino acid 31 of nephron is an exit pathway for baseline renal Kϩ secretion KS-WNK1. Amino acids of L-WNK1 and KS-WNK1 distal to the dotted line are ϩ identical. Amino acid 660 of L-WNK1 is equal to amino acid 253 of KS-WNK1. (20–22). To maintain K homeostasis, the abundance of ROMK (B) Cells with transfected with ROMK alone or cotransfected with ROMK1 plus in the apical membranes of distal nephron increases and de- L-WNK1, WNK1 (1–491), and͞or KS-WNK1 or KS-WNK1 (1–253) as indicated. creases with high and low dietary Kϩ intake, respectively (25, (C) Lysates from mock-transfected cells or cells cotransfected with Myc-tagged 26). Dietary Kϩ restriction decreases the abundance of ROMK ͞ WNK1 (1–491), Flag-tagged KS-WNK1 (1–253), and or an unrelated Flag- of cortical collecting ducts by stimulating endocytosis (27). To tagged protein pod1 were immunoprecipitated by either anti-Myc or anti- Flag antibody and probed for Western blot analysis by the respective antibody see whether the opposite regulation of ROMK1 by long and as indicated. The molecular mass of Myc-WNK1 (1–491), Flag-KS-WNK1 (1– KS-WNK1 is physiologically important, we examined the ex- ϩ 253), and Flag-pod1 are 60, 32, and 22 kDa, respectively (as indicated by ar- pression of long and KS-WNK1 in rats fed K -deficient (LK), rowhead). Experiments above were repeated three times with similar results. control (CK) or high Kϩ (HK) diet. The abundance of transcript for Kϩ-deficient and high Kϩ diet relative to the control Kϩ diet was determined by using quantitative real-time PCR. As shown antagonist of long WNK1. Consistent with the finding that in Fig. 6, the transcript for long WNK1 was apparently increased WNK1 (1–491) inhibits ROMK1 equally to the full-length ϩ by feeding a K -restriction diet (Fig. 6A; 115 Ϯ 7% of the WNK1, KS-WNK1 also reversed inhibition of ROMK1 caused control, P ϭ 0.05; mean Ϯ SEM, n ϭ 11 for each) but not by WNK1 (1–491) (Fig. 5B). significantly altered by a high Kϩ diet (88 Ϯ 11% of the control, It has been reported that the region of amino acids 480–660 of long WNK1 interacts with the region of amino acids 1–491 not significant). As reported in refs. 18 and 19, we found that KS-WNK1 is much more abundant than long WNK1 in rat (32). Amino acids 1–253 of KS-WNK1 contain the region of Ϯ Ϯ amino acids 480–660 of long WNK1 (Fig. 5A). We found that kidney (91 8% vs. 9 4%). In contrast to the apparent KS-WNK1 (1–253), like full-length KS-WNK1, was capable of increase for the long WNK1, the abundance of transcript for ϩ Ϯ reversing long WNK1-mediated inhibition of ROMK1 (Fig. 5B). KS-WNK1 was decreased by K -deficient diet (Fig. 6B,50 6% of the control). Conversely, the transcript for KS-WNK1 was KS-WNK1 (1–253) by itself had no effect on ROMK. We next ϩ examined the interaction between WNK1 (1–491) and KS- increased by high K diet (142 Ϯ 10% of control). Besides the WNK1 (1–253) by coexpressing the respective Myc- and Flag- opposite direction of changes, the magnitude of changes of ϩ tagged proteins. As shown in Fig. 5C, anti-Myc antibody immu- KS-WNK1 by variations of dietary K intake was much greater noprecipitated Myc-WNK1 (1–491) (indicated by a 60-kDa than that in long WNK1. The ratio (relative to control) of protein band), which coimmunoprecipitated Flag-KS-WNK1 abundance of transcript for long over KS-WNK1 for Kϩ- (1–253) (indicated by a 32-kDa protein band). Pod1 is an deficient and high Kϩ diet were calculated at 230% of control unrelated transcription factor protein (33). As a control, Myc- and 62% of control, respectively (Fig. 6C).

1618 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510609103 Lazrak et al. Downloaded by guest on October 1, 2021 Discussion by WNK1 requires the N-terminal amino acids 1–220 (15). Also, PHA II is an autosomal-dominant disease featured by hypertension release of WNK4-mediated inhibition of Na-Cl cotransporter by and hyperkalemia (5, 6). A recent genetic study reported that WNK1 requires the kinase domain of WNK1 (35). Thus, it appears mutations of WNK1 and WNK4 cause PHA II (6). Mutations in the that hypertension in PHA II patients with WNK1 mutation is likely WNK1 gene are large deletions of the first intron resulting in also caused by an increase in long WNK1, not KS-WNK1. Activa- increased expression of the gene. How increased expression of tion of ENaC and release of WNK4 inhibition of Na-Cl cotrans- WNK1 causes hyperkalemia is unknown. Schambelan et al. (34) porter likely contribute to pathogenesis of hypertension for patients found that infusion of sodium sulfate in substitution for NaCl with WNK1 mutations. These results of WNK1 on ENaC and Na-Cl ameliorates hyperkalemia in PHA II patients and proposed that cotransporter support our results on ROMK1. How deletion of hyperkalemia was due to reduced NaCl delivery to the cortical intron 1 caused an increase in the expression of long WNK1 is not collecting ducts consequent to increased ClϪ reabsorption in tu- known. Interestingly, in contrast to the increased expression by bules proximal to the cortical collecting ducts. However, although large deletions of intron 1, insertion of a large piece of DNA (a all affected individuals develop hyperkalemia, only 50% of those Ϸ8-kb gene trap DNA) into intron 1 caused a decrease in the develop hypertension (12–14). Also, hyperkalemia in PHA II of expression of long WNK1 in mice (36). Future experiments should WNK1 and WNK4 mutations typically occurs Ϸ10 to 20 years before examine the expression of KS-WNK1 in these mice. development of hypertension (12–14), suggesting that other mech- The KS-WNK1 is expressed in the kidney much more abundantly anisms may also be involved. To support this observation, Kahle et than long WNK1 (18, 19). We found that KS-WNK1 does not al. (11) recently found that WNK4 inhibits ROMK. Here, we report inhibit ROMK1 but rather reverses the inhibition caused by long that WNK1 inhibits ROMK1 by stimulating its endocytosis via WNK1. Variations of dietary Kϩ intake in rats cause reciprocal CCV. These results suggest that inhibition of ROMK by WNK1 changes of the long vs. KS-WNK1. These results suggest that may contribute to hyperkalemia in PHA II with WNK1 mutations. KS-WNK1 functions as a physiological antagonist of the long ϩ How does the finding of direct inhibition of K secretion WNK1. A recent report that KS-WNK1 inhibits long WNK1 through ROMK reconcile with the observation by Schambelan regulation of Na-Cl cotransporter supports this idea (37). Dual et al. (34)? Recent studies have found that maxi-K channels, control by both positive and negative regulators allows biological although relatively quiescent at the basal state, are activated by systems to respond to variations of external and͞or internal milieu an increase in the luminal fluid flow and predominantly respon- ϩ with much greater flexibility. Our findings that greater changes in sible for flow-stimulated K secretion (23, 24). Thus, despite of ϩ ϩ the ratio of long vs. KS-WNK1 caused by variations of dietary K inhibition of ROMK, increase in the distal Na and fluid intake compared to changes in long or KS-WNK1 alone exemplifies delivery by infusion of nonreabsorbable sodium sulfate would this idea. Differential expression of WNK1 isoforms suggests that stimulate Kϩ secretion through maxi-K channels to correct the ϩ WNK1 will display widely variable tissue-specific regulatory prop- hyperkalemia. Flow-stimulated K secretion through maxi-K erties depending on the forms that are present. can also explain the effectiveness of thiazide diuretics in ame- As a critical exit pathway for Kϩ secretion, ROMK channels liorating the hyperkalemia manifested by these patients (12, 13). are regulated by acute (such as arginine vasopressin) and long- How does WNK1 inhibit ROMK1? Long WNK1 physically term (such as dietary Kϩ intake) factors that affect Kϩ secretion interacts with WNK4 (35), which also stimulates endocytosis of ϩ (20–22). To maintain K homeostasis, the density of ROMK in ROMK (11). Thus, it is interesting to know whether WNK1 cortical collecting ducts increases and decreases during high and regulates ROMK1 synergistically with WNK4. Our results indicate ϩ ϩ low dietary K intake, respectively (25, 26). Low dietary K that WNK1 and WNK4 inhibit ROMK1 synergistically. However, intake decreases ROMK, likely by stimulating endocytosis via knockdown of endogenous WNK4 by using siRNA does not affect CCVs followed by degradation via lysosomes (27, 28). Our inhibition of ROMK1 by WNK1, suggesting that regulation by results suggest that changes in the ratio of long vs. KS-WNK1 in WNK1 does not depend on WNK4. A presumed kinase-dead WNK4 mutant remains capable of inhibiting ROMK1, suggesting kidney may, at least partly, mediate the regulation of ROMK by dietary Kϩ intake. Changes in the ratio of long vs. KS-WNK1 by that kinase activity is not required (11). Our results indicate that ϩ inhibition of ROMK1 requires the WNK1 kinase domain. The dietary K intake occurred predominantly through changes of KS-WNK1. Thus, KS-WNK1 may be the primary mediator WNK1 kinase domain, however, is not sufficient for regulating ϩ ROMK1; the N terminus before kinase domain is also required. regulating renal K secretion in some settings. The N terminus of WNK1 preceding the kinase domain shows little Materials and Methods homology with WNK4. Thus, the mechanism by which WNKs stimulate endocytosis of ROMK1 cannot be deduced from se- DNA Constructs and siRNA for WNK1 and WNK4. GFP-ROMK1 was created by subcloning coding sequence of ROMK in-frame and

quence comparison alone. WNK1 and 4 may interact with different PHYSIOLOGY sets of proteins leading to stimulation of endocytosis of ROMK. downstream of GFP by using pEGFP-N3 vector. Full-length rat Future experiments should focus on identification of these potential WNK1 and WNK4 (gifts of Melanie Cobb and Bing-e Xu, Uni- proteins. versity of Texas Southwestern Medical Center) are in pCMV5-Myc WNK1 is expressed in multiple splice variants (2, 18, 19). At least vector (2, 15, 31, 32). WNK1 fragments were amplified by PCR with two transcripts have been detected in kidney (18, 19). A longer full-length rat WNK1 cDNA as the template and subcloned into transcript (Ϸ12 kb in size) is ubiquitously expressed and encodes pCMV5-Myc. KS-WNK1 (gift of Aniko Naray-Fejes-Toth, Dart- amino acids 1–491 necessary and sufficient for regulating ROMK1. mouth Medical School, Hanover, NH; ref. 38) was amplified by A smaller kidney-specific transcript lacking the N-terminal 437 PCR and subcloned into pCMV5-myc vector. Fragments of KS- residues is highly expressed in kidney (18, 19). Leukocytes, in which WNK1 were amplified by PCR and subcloned into a C-terminal increased WNK1 expression in PHA II were demonstrated (6), Flag vector (pIRES-hrGFP-1a) (Stratagene). Point mutation was contain the ubiquitous long WNK1 but not the KS-WNK1 (18, 19). generated by site-directed mutagenesis (QuikChange kit, Strat- Limited by the availability of kidney tissue from patients with PHA agene) and confirmed by sequencing. Sense and antisense oligo- II, the expression of KS-WNK1 has not been examined. Neverthe- nucleotide for WNK1 siRNA were 5Ј-UGU CUA ACG AUG GCC less, our results suggest that increased expression of the long WNK1 GCUUdTdTand5Ј-AAG CGG CCA UCG UUA GAC A dT contributes to hyperkalemia in patients with WNK1 mutations. dT, respectively. Sense and antisense oligonucleotide for WNK4 Expression of KS-WNK1 in PHA II, if altered, is likely reduced siRNA were CGG GCA CGC UCA AGA CGU AUU and 5Ј-P compared to the control. It has been shown that activation of ENaC UAC GUC UUG AGC GUG CCC GUU, respectively.

Lazrak et al. PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1619 Downloaded by guest on October 1, 2021 Tissue Culture, Immunoprecipitation, and Western Blots. HEK293 the effects of low dietary Kϩ intake, rats were pair-fed either a cells were maintained in Dulbecco’s modified Eagle’s medium with Kϩ-deficient diet (no added Kϩ; TD95006, Harlan TEKLAD, 10% FBS͞2mML-glutamine͞100 units/ml penicillin/steptomycin Madison, WI), a control Kϩ diet (Kϩ content 6.7 g͞kg in KCl; and transfected as described in ref. 15. Transfected cells were TD88238) or a high Kϩ diet (Kϩ content 49.5 g͞kg; TD94121) for harvested in isotonic lysis buffer containing 1% Triton X-100 and 48–72 h as described in ref. 27. All animals were allowed to drink phosphatase and protease inhibitors as described in ref. 15. For distilled water freely. Food intake and body weight were measured coimmunoprecipitation experiments, cells were lysed with a daily. Kidneys from control and Kϩ-deficient rats were dissected Dounce homogenizer in buffer lacking Triton X-100. Proteins were immediately after killing. After homogenization, RNA was ex- immunoprecipitated from cell lysates by using monoclonal anti-Myc tracted from kidney by using RNAzol (Invitrogen) according to the or anti-Flag (2, 15, 31, 32) antibodies at 1:100 dilution. For Western manufacturer’s instructions. Two hundred nanograms was used for blots, total lysates or immunoprecipitates were resolved by SDS͞ PAGE and proteins were transferred onto nitrocellulose mem- RT with the TaqMan RT kit (Applied Biosystems). Real-time branes. The membranes were incubated with the indicated anti- quantitative PCR was performed on ABI 7000 as described in bodies and developed by using enhanced chemiluminescence. Applied Biosystems User Bulletin no. 2 by using the TaqMan assay. Forward and reverse primers and TaqMan probe from the 5Ј Ј Whole-Cell Patch-Clamp Recording of ROMK Channels. HEK cells sequence of exon1 (WNK1 P1 forward, 5 -GGC ACT CCT GGC were transfected with cDNAs (0.5 ␮g each) for GFP-ROMK. As TTC CTT TC-3Ј; P1 reverse, 5Ј-ATC GGA GCT TGA GCC ATT indicated, cells were cotransfected with long WNK1, WNK4, KS- CTT-3Ј; P1 TaqMan probe, 5Ј-CCT CCG GCT CCA GTC-3Ј) were WNK1, wild-type, and͞or dominant-negative rat dynamin II (gifts used to amplify the full-length WNK1 isoform under P1 promoter of Joseph Albanesi, University of Texas Southwestern Medical control (18, 19). Primer from exon 4a (RP forward, 5Ј-GCT GCT Center). In each experiment, the total amount of DNA for trans- GTT CTC AAA AGG ATT GTA T-3Ј) and from exon 5 (RP fection was balanced by using empty vectors. Approximately 36–48 reverse, 5Ј-CAG GAA TTG CTA CTT TGT CAA AAC TG-3Ј) h after transfection, whole-cell currents were recorded by using an and TaqMan probe (5Ј-TGA GGG AGT GAA GCC A-3Ј) were Axopatch 200B amplifier as described in refs. 15 and 39. Trans- used to amplify the kidney-specific isoform (18, 19). Relative fected cells were identified by using epifluorescent microscopy. The WNK1 mRNA levels were calculated with 18s rRNA as the internal bath and pipette solution contained 145 mM KCl, 2 mM MgCl2,2 control. mM CaCl2, 10 mM Hepes (pH 7.4) and 145 mM KCl, 2 mM EDTA, 10 mM Hepes (pH 7.4), respectively. Capacitance and access We thank Drs. M. Cobb and B. Xu for WNK1 and WNK4 constructs and resistance were monitored and 75% compensated. The voltage for discussion; Dr. M. Baum for reading of the manuscript; Dr. G. He protocol consists of 0 mV holding potential and 400 ms steps from (University of Texas Southwestern Medical Center) for the N375I point Ϫ 100 to 100 mV in 20-mV increments. Statistical comparison was mutant of ROMK1; Drs. P. Igarashi and T. Hiesberger (University of made by using the unpaired Student t test. Texas Southwestern Medical Center) for the Flag-pod1 construct; and members of the Huang laboratory for discussion. This work was sup- Experimental Animals, Diets, and Real-Time PCR. Male or female ported, in part, by National Institute of Health Grants DK-54368, Sprague–Dawley rats (150–200 g) were used for the study. To study DK-59530, and DK-59530S1 (to C.-L.H.).

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