Renal and Brain Isoforms of WNK3 Have Opposite Effects on NCCT Expression

BASIC RESEARCH www.jasn.org

Mark Glover, Annie Mercier Zuber, and Kevin M. O’Shaughnessy

Clinical Pharmacology Unit, Department of Medicine, University of Cambridge, Cambridge, United Kingdom

ABSTRACT Mutations in the WNK kinases WNK1 and WNK4 cause a rare familial form of hypertension (Gordon syndrome) by increasing expression of the thiazide-sensitive co-transporter NCCT in the kidney. Regu- lation of NCCT expression involves a scaffold of proteins composed of several kinases, including the third member of the WNK kinase family, WNK3. This protein, expressed in several tissues including kidney and brain, displays splice variation around exons 18 and 22. We expressed these proteins in Xenopus oocytes and found that the renal isoform of WNK3 increased but the brain isoform decreased NCCT expression and activity. Introduction of a kinase-inactivating mutation into renal WNK3 reversed its action on NCCT, and the same mutation in the brain isoforms led to loss of function. We also studied the effect of phosphorylation of a key NCCT threonine (T58) on the effects of WNK3/4 coexpression; NCCT mutants with a T58A or T58D substitution had the same surface expression as T58 but had significantly altered transporter activity; however, both isoforms of WNK3 as well as WNK4 still modulated expression of these NCCT mutants. Finally, experiments using kinase-dead STE20/SPS1-related proline/alanine-rich kinase (SPAK), a putative downstream target for WNKs, revealed that brain WNK3 acts in tandem with SPAK, whereas renal WNK3 seems to upregulate NCCT through a SPAK-independent pathway. Taken together, these results suggest that the C-terminal motifs contributed by exons 18 and 22 play an important role in the actions of WNK3 isoforms on NCCT.

J Am Soc Nephrol 20: 1314–1322, 2009. doi: 10.1681/ASN.2008050542

The Na-Cl transporter NCCT (SLC12A3) is ex- ing just four members and share an N-terminal cat- pressed in the distal convoluted tubule and targeted alytic domain and a regulatory C-terminal that in- by thiazide diuretics, one of the most widely used cludes a highly conserved acidic motif and two coil- classes of antihypertensive therapy.1,2 In the past coil domains6 (Figure 1). decade, the importance of NCCT in regulating BP Initially, it was thought that WNK4 inhibited has come from studying two rare familial BP syn- forward trafficking of NCCT and that WNK1 inter- dromes. The first of these, Gitelman syndrome, is acted with it to suppress WNK4 function and re- associated with low BP as a result of mutations in store NCCT expression at the cell surface5,7; how- NCCT itself that reduce either its function or its ever, it is now clear that SLC12A transporter expression in the distal convoluted tubule. In contrast, patients with the much rarer Gordon syn- Received May 27, 2008. Accepted January 29, 2009. drome (pseudohypoaldosteronism type II) have Published online ahead of print. Publication date available at high BP and overexpress NCCT. The mutations in www.jasn.org.

Gordon syndrome are not in NCCT itself but are M.G. and A.M.Z. contributed equally to this work. located in genes encoding two members of a novel Correspondence: Dr. Kevin M. O’Shaughnessy, Clinical Pharma- family of serine-threonine kinases called WNK ki- cology Unit, Box 110, Addenbrooke’s Hospital, Cambridge, CB2 nases (WNK13 and WNK43,4), which seem to regu- 2QQ, UK. Phone: ϩ44-1223-762578; Fax: ϩ44-1223-762576; E- late the trafficking of NCCT.4,5 The WNK kinases mail: [email protected] are a very small family within the kinome contain- Copyright ᮊ 2009 by the American Society of Nephrology

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er.16 Nevertheless, there is no evidence that WNK kinases actually phosphorylate NCCT directly. This is thought to involve WNK proteins phosphorylat- ing an intermediary kinase, STE20/ SPS1-related proline/alanine-rich kinase (SPAK), which is responsible for the actual phosphorylation of the T58 residue of NCCT.17 Indeed, SPAK is phosphorylated and activated by both WNK4 and WNK117,18 and can phosphorylate human NCCT at three conserved N-terminal residues, including T60.9 In HEK 293 cells, site mutation of T60 to alanine not only abrogates Figure 1. Structural differences between the brain and renal WNK3 isoforms. WNK3 in phosphorylation by SPAK at this resi- the brain exists as two isoforms. Isoform 1 contains a short version of exon 18 (18a), and due but also reduces phosphorylation isoform 2 contains a long version of exon 18 (18b) that has an additional 47 amino acids. at the nearby T46 and T55 sites and Both isoforms contain exon 22. Renal WNK3 (isoform 3) contains only the shorter exon 18 markedly attenuates NCCT activation (18a) and lacks exon 22. by a low-chloride medium.9 SPAK also seems necessary for (renal) WNK3 ac- regulation involves a complicated network of proteins that in- tivation of the related sodium co-transporter NKCC2.19 corporates diverse kinases, phosphatases, and scaffolding pro- Because splice variation within the C-terminal region of teins.8–11 One additional regulatory kinase is WNK3, the third WNK3 is likely to affect protein–protein interactions with the member of the WNK family and a protein of approximately complex scaffold of interacting proteins regulating NCCT, we 1800 amino acids.6 It shows significant homology with the hypothesized that the WNK3 splice variants may differentially other WNK kinases and is expressed widely in human and affect NCCT expression. In addition, we investigated how the mouse tissues.12,13 Human WNK3 has splice variation based WNK3–NCCT interaction might be affected by mutation of around exons 18 and 22 that affects tissue distribution.12 In the the key T residue in the N-terminal of NCCT (T58) to mimic brain, two isoforms of WNK3 exist. One contains a short ver- different phosphorylation states of the transporter. We have sion of exon 18 (exon 18a; isoform 1); the other contains a also looked at the involvement of SPAK in the effects of WNK3 longer exon 18 with an additional 47 amino acids (exon 18b; by coexpressing a dominant-negative KD mutant (SPAK KD). isoform 2), and both contain exon 22. WNK3 in the kidney We report here that renal and brain isoforms of WNK3 pro- contains exon 18a but not exon 22 (isoform 3; Figure 1). For duce opposite actions on NCCT expression and that SPAK KD the rest of this article, brain WNK3 refers to the brain-specific differentially blocks the actions of the WNK3 isoforms. Muta- isoform 2 and renal WNK3 refers to the renal-specific isoform tion of the T58 residue in NCCT also affects transporter activ- 3. In contrast to the inhibitory effects of WNK4 on NCCT ity but does not affect basal membrane expression or the ability expression, renal WNK3 has been shown to increase mem- of WNK3 isoforms or WNK4 to alter transporter density in brane expression of NCCT, NKCC1, and NKCC2 in oocytes oocyte membranes. and also to inhibit the basolateral K-Cl transporters KCC1 through 4.13,14 Kinase-dead (KD) renal WNK3 mutants produce opposite effects. The function of brain WNK3 is un- RESULTS known,15 but reports that a C-terminal fragment of renal WNK3 is able to stimulate NCCT expression points to key Alternative Splicing of WNK3 Exons 18 and 22 in motifs within it being responsible for the stimulatory actions of Mouse Kidney and Brain WNK3.8 PCR using exon 18–specific primers in the human renal and Although WNK kinases can affect the density of NCCT brain WNK3 clones used for the subsequent oocyte experi- transporters in the cell membrane through an effect on NCCT ments confirmed that a shorter product (291 bp) was present trafficking,6 it is clear that NCCT function can also be affected in the renal clone and a longer product in brain WNK3 (432 by the phosphorylation state of key serine/threonine residues bp) corresponding to exons 18a and 18b, respectively (Figure in the N-terminal of NCCT (especially T58 in rodent sequence 2A). PCR of mouse tissue cDNA showed that brain contains or T60 in human). For example, in Xenopus oocytes, increased both exon 18b and exon 18a, but the kidney contains only the NCCT phosphorylation in response to chloride depletion has shorter exon 18a. been observed to increase 22Naϩ flux through NCCT without Similarly, using primers to amplify exon 22, the human any change in surface membrane expression of the transport- clones showed a difference of 33 bp corresponding to the ex-

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Confocal microscopy of enhanced cyan flourescent protein (ECFP)-NCCT injected oocytes showed that WNK3 effects on NCCT flux activity were accompanied by parallel changes in

Figure 2. Agarose gel electrophoresis of WNK3 exons 18 and 22. (A) PCR of exon 18 of human WNK3 containing plasmid (renal and brain) showing exon 18a (291 bp) and exon 18b (432 bp), respectively. Reverse transcription–PCR (RT-PCR) using mouse tissue shows that the brain contains both exon 18a and exon 18b, whereas the mouse kidney contains only exon 18a. (B) PCR for exon 22 of the human WNK3 containing plasmid shows the shorter product attributable to exon 22 deletion from the renal isoform. RT-PCR of mouse tissue confirms that only the brain expresses exon 22. W and M are water and 100-bp ladders respectively. K, kidney; B, brain. pected additional 11 amino acids in the brain form of WNK3 contributed by exon 22 (Figure 2B). Using the mouse cDNAs, only the brain contains a transcript corresponding to exon 22. Thus, as in humans, murine brain expresses both exon 18a and exon 18b and also contains exon 22, whereas WNK3 in the kidney contains only exon 18a and lacks exon 22.

Brain WNK3 Produces Opposite Effects to Renal WNK3 For comparison of the effects of the two isoforms of WNK3, the clones were first coexpressed with wild-type (wt) NCCT by injection of the respective cRNAs into Xenopus oocytes. The 22Naϩ flux measurable in NCCT-injected oocytes was com- pletely inhibited by hydrochlorothiazide (100 ␮M; Figure 3A). Renal WNK3 co-injection with NCCT increased 22Naϩ uptake by 2.5-fold (6.96 Ϯ 1.29 to 16.88 Ϯ 1.60 nmol/oocyte per h; P Ͻ 10Ϫ4), in keeping with previous reports; however, brain WNK3 produced the opposite effect, reducing 22Naϩ uptake through NCCT by almost one half (6.96 Ϯ 1.29 to 3.51 Ϯ 0.21 nmol/oocyte per h; P Ͻ 10Ϫ4). The effects of both isoforms were kinase dependent, but the loss of kinase activity affected ϩ Figure 3. Effect of WNK3 isoforms versus WNK4. (A) 22Na flux in the isoforms differently. Hence, KD renal WNK3 (D294A) re- 22 ϩ oocytes injected with NCCT and either WNK3 (R, renal; B, brain) or duced Na flux compared with NCCT injected alone, and WNK4 as indicated. Significant differences from NCCT alone are KD brain WNK3 (D294A) lost its ability to inhibit NCCT. The indicated. *P Ͻ 0.001. (B) ECFP-NCCT fluorescence signal from action of brain WNK3 was reminiscent of WNK4 in this system oocytes injected as in A. Significant differences are indicated. *P Ͻ in that both were able to suppress NCCT expression and the 0.001; **P Ͻ 0.05. (C) Representative confocal microscopy images KD mutant was not functional (Figure 3A). for each injection condition in B. HCTZ, hydrochlorothiazide.

1316 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1314–1322, 2009 www.jasn.org BASIC RESEARCH surface membrane expression of the transporter (Figure 3). Figure 3B shows that renal WNK3 increased membrane expression of ECFP-NCCT approximately two-fold (31.2 Ϯ 4.1 to 64.2 Ϯ 5.8 arbitrary fluorescence units [AFU]; P Ͻ 10Ϫ3), whereas brain WNK3 reduced it by approximately 50% (31.2 Ϯ 4.1 to 15.2 Ϯ 1.6 AFU; P Ͻ 10Ϫ3). The KD forms of WNK3 also produced changes in expression that paralleled their effects on 22Naϩ flux.

NCCT T58 Mutants Display Altered Activity but not Surface Expression Xenopus oocytes were injected with cDNA for wt ECFP-NCCT or the T58A or T58D mutants of ECFP-NCCT. Figure 4A shows that 22Naϩ uptake by NCCT T58A, which mimics the nonphos- phorylated form of the transporter, was reduced by one half compared with wt. Conversely, 22Naϩ flux through NCCT T58D, a mimic of the phosphorylated transporter, was increased two-fold over wt. Despite these changes in activity, the membrane expression of NCCT was not altered in either of the T58 mutants on the basis of their membrane fluorescence (Figure 4B).

Effect of WNK3 Isoforms on NCCT Are not Altered by NCCT T58 Mutation The brain isoform of WNK3 inhibited 22Naϩ flux through the T58D and T58A mutants of NCCT in the same qualitative

ϩ Figure 5. Effect of brain WNK3 on T58 NCCT mutants. (A) 22Na flux in oocytes injected with NCCT wt or T58 mutants and brain WNK3 isoform as indicated. Significant difference from NCCT clone used is indicated. *P Ͻ 0.001; ***P Ͻ 0.01. (B) ECFP-NCCT fluorescence signal from oocytes injected as in A. Significant difference from NCCT is indicated. *P Ͻ 0.001; ***P Ͻ 0.01; **P Ͻ 0.05.

manner as wt NCCT, and the converse held for coexpression of the renal isoform of WNK3 that stimulated 22Naϩ flux through NCCT T58D mutant (Figures 5A and 6A). The KD forms of brain and renal WNK3 (D294A) affected 22Naϩ flux through the NCCT T58D mutant in the same way as wt NCCT: The brain isoform was nonfunctional and the renal isoform reversed its function, inhibiting flux. Confocal microscopy of the same oocytes showed that the changes in activity when WNK3 isoforms were coexpressed with the NCCT T58 mutants was accompanied by exactly parallel changes in surface expression (Figures 5B and 6B).

WNK4 Effect on NCCT Is not Influenced by NCCT Threonine 58 Mutation For determination of whether WNK4 behaved in a similar manner to WNK3 when coexpressed with the T58 mutants, wt WNK4 cRNA was injected into Xenopus oocytes along with ϩ ϩ 22 Figure 4. Effect of T58 substitution on NCCT. (A) 22Na flux in either wt T58D or T58A NCCT. The Na flux was reduced by oocytes injected with NCCT wt, T58A NCCT, or T58D NCCT mu- WNK4 for all three NCCT transporters (Figure 7A), and con- tants. Significant difference from NCCT wt is indicated. *P Ͻ 0.001. focal microscopy showed that this reflected a parallel reduction (B) ECFP-NCCT fluorescence signal from oocytes injected as in A. in surface membrane expression of ECFP-NCCT (Figure 7B).

J Am Soc Nephrol 20: 1314–1322, 2009 Regulation of NCCT Function 1317 BASIC RESEARCH www.jasn.org

DISCUSSION

The control of distal tubular sodium handling through NCCT is regulated by diverse kinases, phosphatases, and scaffolding proteins.8–11 In addition to WNK1 and WNK4, WNK3 is now recognized as an important regulator of NCCT activity,15 although there are no reports of mutations within this protein among families with Gordon syndrome. WNK4 seems to reduce NCCT expression by reducing forward trafficking, but WNK1 does not affect NCCT expression in Xenopus oocytes unless coexpressed with WNK4.5 This is in marked contrast to the interaction of WNK proteins with the ROMK ion channel, where WNK1 and WNK4 both independently regulate inter- nalization.20 Our findings with WNK3 confirm previous reports that renal WNK3 increases NCCT Naϩ flux by increasing surface membrane expression.10,13 This effect requires intact WNK3 kinase activity; however, the KD mutation does not simply lose its activity but actually reverses it. In fact, this behavior is seen in other members of the WNK kinase family in which inter- molecular protein–protein interactions are as important as the catalytically active kinase domain to the functionality of the WNK protein. Wang et al.21 also highlighted the importance of

ϩ Figure 6. Effect of renal WNK3 on T58 NCCT mutants. (A) 22Na flux in oocytes injected with NCCT wt or T58 mutants and renal WNK3 isoform as indicated. Significant difference from NCCT clone is indicated. *P Ͻ 0.001; ***P Ͻ 0.01. (B) eCFP-NCCT fluorescence signal from oocytes injected as in A. Significant difference from NCCT is indicated. *P Ͻ 0.001.

Role of SPAK in the Regulation of NCCT by WNK3 Isoforms Coexpression of SPAK wt with NCCT did not affect 22Naϩ flux through NCCT; however, coexpression of the dominant-negative SPAK KD (D212A) reduced 22Naϩ flux through NCCT by at least 55% (7.99 Ϯ 1.79 versus 3.55 Ϯ 0.46 nmol/oocyte per h; P Ͻ 10Ϫ4; Figure 8A). Similarly, SPAK wt did not affect regulation of NCCT by either WNK3 isoform (Figure 8, B and C). The coexpression of SPAK KD did not prevent NCCT regulation by renal WNK3 or brain WNK3 wt, but it did abolish the NCCT flux with brain WNK3 KD (13.60 Ϯ 2.44 versus 3.13 Ϯ 0.72 nmol/oocyte per h; P Ͻ 10Ϫ4; Figure 8C).

NCCT, WNK3, and SPAK Do not Directly Interact Immunoprecipitation of 35S-Methionine–labeled proteins with anti-NCCT antibody failed to show an interaction be- ϩ Figure 7. Effect of WNK4 on T58 NCCT mutants. (A) 22Na flux tween NCCT and either WNK3 isoform (expected approxi- in oocytes injected with NCCT wt or T58 mutants and WNK4 as mately 190 kD; Supplemental Figures S1 and S2). Using an- indicated. Significant difference from NCCT clone is indicated. ti-HA antibody to precipitate SPAK, there was also no *P Ͻ 0.001; ***P Ͻ 0.01. (B) ECFP-NCCT fluorescence signal from demonstrable interaction between SPAK and either WNK3 oocytes injected as in A. Significant difference from NCCT is isoform or with NCCT (Supplemental Figures S1 and S2). indicated. *P Ͻ 0.001; ***P Ͻ 0.01; **P Ͻ 0.05.

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reported to be involved in the regulation of the related CC co-transporter NKCC2.19 Expression of SPAK wt does not significantly alter NCCT function in our hands; neither does it affect NCCT regulation by either wt or KD WNK3 isoforms. In contrast, the dominant- negative SPAK KD (D212A) reduces basal NCCT activity but does not affect either the upregulation of NCCT caused by renal WNK3 wt or downregulation by renal WNK3 KD. This result differs from NKCC2, for which SPAK KD reduced basal NKCC2 activity but also antago- nized the upregulation of NKCC2 by renal WNK319; however, the interaction of SPAK KD and brain WNK3 is strikingly different, with SPAK KD suppressing NCCT function when coexpressed with brain WNK3 KD. This suggests that brain WNK3 acts in tandem with SPAK, whereas the renal isoform seems to upregulate NCCT through a pathway that is not dependent on SPAK. This could explain the divergent effects of the two WNK3 isoforms with the brain form op- erating purely in tandem with SPAK; however, the different effects of the two isoforms do not seem to be explained by differences in the physical interaction of SPAK with either isoform and/or NCCT. In fact, under the conditions we used, we cannot show any evidence that either WNK3 isoform interacts with NCCT or SPAK. Figure 8. Effect of SPAK wt and SPAK KD on NCCT and its regulation by WNK3 isoforms. It has been suggested that WNK3 ϩ (A) SPAK KD but not SPAK wt inhibits 22Na flux through wt NCCT. Significance from might have a pivotal role in regulating Ϫ NCCT is indicated. *P Ͻ 0.001. (B) Neither SPAK wt nor SPAK KD affects regulation by neuronal excitability by stimulating Cl Ϫ renal WNK3. (C) SPAK wt does not affect regulation of NCCT by brain WNK3 wt or KD; entry through NKCC1 and inhibit Cl however, SPAK KD inhibits NCCT when coexpressed with brain WNK3 KD but not brain exit through the K/Cl co-transporter WNK3 wt. Significance from NCCT ϩ WNK3 KD is indicated. *P Ͻ 0.001. KCC2.13 The resulting increase in intracellular [ClϪ] would affect the neuronal intramolecular salt bridge formation in the functioning of response to GABAA/chloride ionophore activation by pushing WNK1. Our novel finding is that the splice variant of WNK3 its response in a depolarizing direction; however, this work was found in the brain behaves substantially differently from the based on expression of the renal isoform of WNK3 in oocytes renal isoform. The brain isoform behaves, in fact, like WNK4 and not the brain isoform. Because the brain isoform clearly in that it inhibits NCCT by reducing its surface expression, and behaves very differently against NCCT, its effects on KCC2 and it does this in a kinase-dependent manner,5 so the KD mutants NKCC1 need to be formally explored before a clear role for of WNK4 and brain WNK3 are nonfunctional and do not show WNK3 in membrane excitability within the central nervous the reversal of function seen with renal WNK3. This splice system can be made. variation in WNK3 is not confined to the human genome; we The difference between the two WNK3 isoforms amounts have demonstrated that tissue-specific WNK3 splice variation to two small peptide insertions into the C-terminal encoded by also occurs in the mouse. exon 18b and exon 22. The C-terminal is thought to be impor- We investigated the role of the putative downstream kinase tant for the renal isoform of WNK3 because C-terminal frag- SPAK in regulating NCCT function, because it was previously ments but not an N-terminal fragment encompassing the ki-

J Am Soc Nephrol 20: 1314–1322, 2009 Regulation of NCCT Function 1319 BASIC RESEARCH www.jasn.org nase domain are sufficient to reproduce the activating effect on nal phosphorylation affecting the intrinsic activity of individ- NCCT in oocytes.8 Coil–coil domains are important for pro- ual transporters within the membrane. This model reflects the tein interaction, and deletion of the second coil–coil domain mutations seen in patients with Gitelman syndrome, which of WNK4 disrupts its interaction with WNK3.8 Because the either block trafficking of NCCT (through impaired synthesis peptides encoded by exons 18 and 22 in WNK3 occur imme- or glycosylation) or reduce its intrinsic transporter activity. diately distal to C-terminal coil–coil domains, it is conceivable The T60M mutation in particular24 (homologous to the mouse that the difference between the two isoforms reflects alteration T58) emphasizes that the inability to phosphorylate this key in protein–protein interactions through the coil–coil regions, threonine has pathophysiologic consequences. Nevertheless, for example, in the recruitment of a phosphatase that may the ability of WNK3/4 to alter NCCT expression regardless of occur in the interaction of renal WNK3 with KCC1 through the phosphorylation state of T58 suggests the two mechanisms 4.10 The exon 18b sequence also contains two predicted myris- operate relatively independently. toylation sites and three protein kinase C phosphorylation sites, but their role in regulating membrane targeting or response to signal transduction is uncertain. CONCISE METHODS Phosphorylation of three conserved N-terminal residues in members of the SLC12A transporter family was previously re- Cloning and cRNA Synthesis ported to alter transporter activity without affecting expression Wt DNA for human full-length renal WNK3 in pcDNA3 and brain of the transporter at the cell surface. This has been reported for WNK3 in pT7TS plasmid were gifts of Dr. Shmuel Muallem (Univer- NKCC1 phosphorylation by SPAK/OSR117,22 and for NCCT sity of Texas, Dallas, TX) and Dr. Lucy Raymond (University of Cam- after hypotonic low-chloride stimulation.16 Here we focused bridge, Cambridge, United Kingdom), respectively. Human HA- on the T58 residue because it seemed in the Xenopus oocyte SPAK wt and HA-SPAK KD (212D3A) in pCMV5 plasmid from Dr. system to be the most important of the three residues in Hilary McLauchlan (University of Dundee, Dundee, United King- NCCT.16 The Alessi group9 recently confirmed this in HEK dom) were subcloned into a modified pTNT vector. Wt mouse NCCT cells and showed that alanine substitution of this residue in ECFP-TNT with cyan fluorescence protein at its N-terminal and wt blocked phosphorylation of the adjacent threonine residues WNK4 in pcDNA3 were as described previously.5 (T46 and T55). This suggests that T58 phosphorylation may Both WNK3 isoforms were mutated to produce KD (294D3A) pro- force a conformational change in the NCCT protein that facil- teins, and wt NCCT was mutated to 58T3D or 58T3A, respectively, itates further phosphorylation. In our hands, the nonphos- using site-directed mutagenesis (Stratagene, Amsterdam, The Nether- phorylatable T58A NCCT mutant also shows reduced trans- lands). All sequences were verified using an ABI 377 and Big Dye fluores- porter activity, and the constitutively phosphorylated T58D cence chemistry (Applied Biosystems, Foster City, CA). Copy RNA was NCCT has substantially increased activity. Interestingly, the transcribed in vitro from linearized plasmids using T7 and SP6 mMES- same aspartate substitution in NKCC1 produces an inactivat- SAGE mMACHINE kits (Ambion, Austin, TX) and quantified using ul- ing effect like T58A in NCCT.23 The reason for this difference is traviolet absorption spectroscopy (Nanodrop, Wilmington, DE). not clear; however, despite high sequence homology within the phosphorylation motif, the N-terminal of NKCC1 is approxi- Expression in Xenopus Oocytes mately 130 residues longer. Differences in three dimensional Xenopus laevis oocytes were harvested and defolliculated as detailed structure of their N-terminal domains may explain the diver- previously.4,5,20 Briefly, 10 ng of NCCT cRNA was injected in a total gent impact of T58 mutation in the two CC co-transporter volume of 50 nl per oocyte, and for co-injections involving WNK3, proteins. For both T58 mutants, the level of expression of WNK4, SPAK, or one of the mutants, an additional 10 ng of cRNA NCCT at the cell surface is the same, suggesting that this phos- was added to the injectate. RNAase and DNAase-free water-injected pho-threonine activates intrinsic transporter kinetics. More oocytes were used as controls throughout. Oocytes were then incu- important, neither NCCT mutation affects the ability of bated in ND96 containing 2 mM sodium pyruvate and 0.1 mg/ml WNK4 or either isoform of WNK3 to affect transporter traf- gentamicin at 18°C for 5 d before use. ficking as measured by expression at the cell surface. This sug- For 22Naϩ flux studies, oocytes were placed for 24 h in ClϪ-free gests that in oocytes at least, the effect of WNK3/4 on NCCT ND96 solution containing 96 mM sodium isethionate, 2 mM potas- trafficking is not dependent on phosphorylation of T58. In sium gluconate, 1.8 mM calcium gluconate, 1 mM magnesium glu- HEK cells, it has been suggested that the WNK1–SPAK path- conate, 5 mM HEPES, 2.5 mM sodium pyruvate, and 50 ␮g/ml gen- way regulates NCCT activity by T58 phosphorylation.9 There tamicin. Thirty minutes before the addition of uptake medium, are no reports that either WNK3 or WNK4 uses the same sig- oocytes were added to ClϪ-free ND96 with inhibitors (1 mM ouabain, naling pathway in HEK cells. In fact, our results suggest they do 100 ␮M amiloride, and 100 ␮M bumetanide) according to the proto- not, and it is worth remembering that WNK1 is able to affect col of Gamba et al.25 Oocytes were then transferred to isotonic uptake NCCT expression in oocytes only when coexpressed with WNK4. medium (58 mM NaCl, 38 mM M-methyl-D-glucamine, 2 mM KCl,

Our in vitro findings confirm that there are two distinct 1.8 mM CaCl2, and 5 mM HEPES with inhibitors [pH 7.4]) contain- methods for regulating NCCT function: One by modulating ing 22Naϩ (2.5 ␮Ci/ml) and incubated in shaking incubator at 30°C NCCT trafficking to the cell surface and the other by N-termi- for 1 h. Oocytes were then washed five times with 5 ml of ice-cold

1320 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1314–1322, 2009 www.jasn.org BASIC RESEARCH aliquots of isotonic medium and counted individually in a gamma DISCLOSURES counter (Perkin-Elmer Cobra 5003, Perkin-Elmer, Waltham, MA). None. Thiazide sensitivity was shown using 100 ␮M hydrochlorothiazide (data not shown). Membrane expression measurements were performed 5 d after injection by laser-scanning confocal microscopy with a Leica DMRXA confocal microscope as described previously5,20 and REFERENCES presented as mean total fluorescence intensity in AFUs. 1. ALLHAT Officers and Coordinators for the ALLHAT Collaborative Re- Reverse Transcription–PCR of Alternatively Spliced search Group. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial: Major outcomes in high-risk hypertensive Murine WNK3 patients randomized to angiotensin-converting enzyme inhibitor or RNA was extracted from mouse renal and brain tissue using Triazol calcium channel blocker vs diuretic: The Antihypertensive and Lipid- (Invitrogen) reagent and reverse-transcribed using Superscript III Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) [published (Invitrogen). PCR amplification of the splice variants of exon 18 (pro- erratum appears in JAMA 289: 178, 2003 and JAMA 291: 2196, 2004]. ducing 291- or 432-bp products) used a forward primer 5Ј-ATTCAA- JAMA 288: 2981–2997, 2002 GATAGCCCTGCACAAT-3Ј in exon 17 and reverse primer 5Ј-GT- 2. Williams B, Poulter NR, Brown MJ, Davis M, McInnes GT, Potter JF, Ј Sever PS, McInnes GT, Thom S: Guidelines for management of hyper- CAGAGGAATGGATCAGAAG-3 in exon 19. Similarly, alternatively tension: Report of the fourth working party of the British Hypertension spliced transcripts of exon 22 were amplified with a forward primer in Society, 2004-BHS IV. J Hum Hypertens 18: 139–185, 2004 exon 21 (5Ј-GGTGGTCAGTCTTCAAACACAA-3Ј) and reverse 3. Wilson FH, Disse-Nicodeme S, Chate KA, Ishikawa K, Nelson-Williams primer in exon 23 (5Ј-GTCAACATCCCCTTCTTACTGG-3Ј). Exon C, Desitter I, Gunal M, Milford DV, Lipkin GW, Achard JM, Feeley MP, 22 deletion reduced the product size by 33 bp. As a control, the same Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeun- emaitre X, Lifton RP: Human hypertension caused by mutations in primers were used to amplify human WNK3 renal and brain clones WNK kinases. Science 293: 1107–1112, 2001 used in the oocyte experiments. 4. Golbang AP, Murthy M, Hamad A, Liu C-H, Cope G, Van’t Hoff W, Cuthbert AW, O’Shaughnessy KM: A new kindred with pseudohypoal- Immunoprecipitation Experiments dosteronism type II and a novel mutation (564DϾH) in the acidic motif Xenopus oocytes were injected with cRNA as already described and of the WNK4 gene. Hypertension 46: 295–300, 2005 incubated at 18°C for4din1mCi/ml 35S-Methionine containing 5. Golbang AP, Cope G, Hamad A, Murthy M, Liu C-H, Cuthbert AW, O’Shaughnessy KM: Regulation of the expression of the Na/Cl co- MBS medium (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO , 0.82 mM 3 transporter by WNK4 and WNK1: Evidence that accelerated dynamin- MgSO4, 0.33 mM CaCl2, 10 mM HEPES-NaOH, and 5 mg/dl genta- dependent endocytosis is not involved. Am J Physiol Renal Physiol micin). Proteins were extracted using Digitonin homogenization 291: F1369–F1376, 2006 buffer (100 mM NaCl, 0.5% Digitonin, 20 mM Tris-HCl [pH 7.6], 1 6. Cope G, Golbang AP, O’Shaughnessy KM: WNK kinases and the mM PMSF, and protease inhibitor cocktail). Protein extract was then control of blood pressure. Pharmacol Ther 106: 221–231, 2006 7. Subramanya AR, Wade JB, Ellison DH, Welling PA: The carboxyl incubated overnight at 4°C with primary antibody (polyclonal anti- terminus of WNK4 suppresses forward trafficking of the thiazide- NCCT antibody [Chemicon, Billerica, MA] or monoclonal anti-HA sensitive cotransporter. FASEB J 21: 938.14–2007. www.fasebj.org/ antibody [Sigma, St. Louis, MI]) and then incubated with Protein A cgi/content/meeting/abstract/21/6/A1337-a. Accessed April 9, 2009. Sepharose (Amersham) and Protein G agarose (Calbiochem) beads 8. Yang CL, Zhu X, Ellison DH: The thiazide-sensitive Na-Cl cotransporter for 1 h. The beads were washed, and proteins were eluted with loading is regulated by a WNK kinase signaling complex. J Clin Invest 117: 3403–3411, 2007 buffer. Samples were separated on an 8% SDS-PAGE. The gel was 9. Richardson C, Rafiq FH, Karlsson HKR, Molelek N, Vandewalle A, dried after fixation, and imaging was performed using x-ray film Campbell DG, Morrice NA, Alessi DR: Activation of the thiazide- (Fuji, Du¨sseldorf, Germany). sensitive Na-Cl cotransporter by the WNK-regulated kinases SPAK and OSR1. J Cell Sci 121: 675–684, 2008 Statistical Analysis 10. De los Heros P, Kahle KT, Rinehart J, Bobadilla NA, Vazquez N, San For all oocyte experiments, 10 to 15 oocytes were injected for each Cristobal P, Mount DB, Lifton RP, Hebert SC, Gamba G: WNK3 by- cRNA used, and the results are presented as means Ϯ SEM. Differ- passes the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway. Proc Natl Acad Sci U S A 103: ences between groups were compared by one-way ANOVA with post 1976–1981, 2006 hoc analyses using Tukey or Scheffe test. Figures show representative 11. Gagnon KB, England R, Diehl L, Delpire E: Apoptosis-associated experiments that were replicated using at least four different batches tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals of oocytes from different donor animals and cRNA batches. SPSS 12 a novel pathway for Na-K-Cl cotransporter regulation. Am J Physiol (SPSS, Chicago, IL) was used throughout with significance defined as Cell Physiol 292: C1809–C1815, 2007 Ͻ 12. Holden S, Cox J, Raymond FL: Cloning, genomic organization, alter- P 0.05. native splicing and expression analysis of the human gene WNK3 (PRKWNK3). Gene 335: 109–119, 2004 13. Kahle KT, Rinehart J, De los Heros P, Louvi A, Meade P, Vazquez N, ACKNOWLEDGMENTS Hebert SC, Gamba G, Gimenez I, Lifton RP: WNK3 modulates transport of ClϪ in and out of cells: Implications for control of cell volume and neuronal excitability. Proc Natl Acad Sci U S A 102: 16783–16788, M.G. is supported by a British Heart Foundation Clinical PhD stu- 2005 dentship award and the Sackler Foundation. A.M.Z. is supported by 14. Rinehart J, Kahle KT, De los Heros P, Vazquez N, Meade P, Wilson FH, the Swiss National Science Foundation. Hebert SC, Gimenez I, Gamba G, Lifton RP: WNK3 kinase is a positive

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regulator of NKCC2 and NCC, renal cation-ClϪ cotransporters re- potassium channel independent of WNK4. J Am Soc Nephrol 17: quired for normal blood pressure homeostasis. Proc Natl Acad Sci 1867–1874, 2006 USA102: 16777–16782, 2005 21. Wang HR, Liu Z, Huang CL: Domains of WNK1 kinase in the 15. Kahle KT, Ring AM, Lifton RP: Molecular physiology of the WNK regulation of ROMK1. Am J Physiol Renal Physiol 295: F438–F445, kinases. Annu Rev Physiol 70: 329–355, 2008 2008 16. Pacheco-Alvarez D, Cristobal PS, Meade P, Moreno E, Vazquez N, 22. Gagnon KB, England R, Delpire E: Volume sensitivity of cation ClϪ Munoz E, Diaz A, Juarez ME, Gimenez I, Gamba G: The Naϩ:ClϪ cotransporters is modulated by the interaction of two kinases: Ste- cotransporter is activated and phosphorylated at the amino-terminal related proline-alanine-rich kinase and WNK4. Am J Cell Physiol 290: domain upon intracellular chloride depletion. J Biol Chem 281: C134–C142, 2006 28755–28763, 2006 23. Darman RB, Forbush B: A regulatory locus of phosphorylation in the N 17. Moriguchi T, Urushiyama S, Hisamoto N, Iemura S, Uchida S, Natsume terminus of the Na-K-Cl cotransporter, NKCC1. J Biol Chem 277: T, Matsumoto K, Shibuya H: WNK-1 regulates phosphorylation of 37542–37550, 2002 cation-chloride-coupled cotransporters via the STE related kinases, 24. Lin S-H, Shiang J-C, Huang C-C, Yang S-S, Hsu Y-J, Cheng C-J: SPAK and OSR-1. J Biol Chem 280: 42685–42693, 2005 Phenotype and genotype analysis in Chinese Patients with Gitelman’s 18. Vitari AC, Deak M, Morrice NA, Alessi DR: The WNK1 and WNK4 syndrome. J Clin Endocrinol Metab 90: 2500–2507, 2005 protein kinases that are mutated in Gordon’s hypertension syndrome 25. Gamba G, Miyanoshita A, Lombardi M, Lytton J, Lee WS, Hediger MA, phosphorylate and activate SPAK and OSR1 protein kinases. Biochem Hebert SC: Molecular cloning, primary structure and characterization J 391: 17–24, 2005 of two members of the mammalian electroneutral sodium-(potassium)- chloride cotransporter family expressed in kidney. J Biol Chem 269: 19. Ponce-Coria J, San Cristobal P, Kahle KT, Vasquez N, Pacheco-Alvarez 17713–17722, 1994 D, De los Heros P, Juarez P Munoz E, Michel G, Bobadilla NA, Gimenez I, Lifton RP, Hebert SC, Gamba G: Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases. See related editorial, “Splicing a Kinase and the Regulation of Salt Transport,” Proc Natl Acad Sci U S A 105: 8458–8463, 2008 on pages 1166–1168. 20. Cope G, Murthy M, Golbang AP, Hamad A, Liu CH, Cuthbert AW, Supplemental information for this article is available online at http://www. O’Shaughnessy KM: WNK1 affects surface expression of the ROMK jasn.org/.

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