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WNK1 Affects Surface Expression of the ROMK Potassium Channel Independent of WNK4

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WNK1 Affects Surface Expression of the ROMK Potassium Channel Independent of WNK4 WNK1 Affects Surface Expression of the ROMK Potassium Channel Independent of WNK4 Georgina Cope, Meena Murthy, Amir P. Golbang, Abbas Hamad, Che-Hsiung Liu, Alan W. Cuthbert, and Kevin M. O’Shaughnessy Department of Medicine, University of Cambridge, Cambridge, United Kingdom The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D>A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression. J Am Soc Nephrol 17: 1867–1874, 2006. doi: 10.1681/ASN.2005111224 he WNK (with no lysine kinase) kinases WNK1 and interaction extends to the effects of WNK on other transporters WNK4 are widely expressed in mammalian transport- or ion channels. Lifton’s laboratory has shown, for example, T ing epithelia (1,2), and expression studies in Xenopus that WNK4 also inhibits expression of the Na-K-Cl cotrans- oocytes suggest that they are able to modify the expression of porter SLC12A2 (NKCCl) and Cl/base exchanger SLC26A6 several co-transporters and ion channels (3,4). The details of the (CFEX) transporters and the renal outer-medullary K channel interaction are best understood for WNK4, which reduces sur- KCNJ1 (ROMK) (1,11). The effect on ROMK is intriguing be- face expression of the thiazide-sensitive NaCl co-transporter cause of the part that it plays in K secretion in the distal (NCCT; gene symbol SLC12A3) in Xenopus oocytes (5–8). This nephron and a reduction in its expression in the collecting duct effect of WNK4 depends on its serine-threonine (S/T) kinase could explain the hyperkalemia that is seen in patients with activity as well as a highly conserved downstream acidic motif Gordon syndrome. We recently confirmed this effect of WNK4 (EPEEPEADQH). Mutations that cause charge-changing amino (8), but, to date, there have been no reports on WNK1 effects on acid substitutions within this motif abolish the inhibitory effect ROMK expression. To address this, we studied their coexpres- of wild-type WNK4 and cause the phenotype of hypertension sion using Xenopus oocytes. We show that in this system, and hyperkalemia that characterizes Gordon syndrome WNK1 directly affects ROMK trafficking independent of (pseudohypoaldosteronism type 2 [PHA2]; OMIM #145260) (9). WNK4 and that this effect is not kinase dependent. Using WNK1 mutations also can cause this phenotype, but published truncations or fragments of WNK1, we further show that the C data suggest that WNK1 protein is effective only in regulating terminal of WNK1 is not necessary for this effect and that the NCCT trafficking when coexpressed with WNK4 (5,10). This acidic motif and coiled-coil regions alone may be sufficient. suggests that the WNK may form a multimeric complex with This has important implications for the physiology of WNK1 NCCT and that protein–protein interactions between WNK1 because its predominant isoform in the kidney is an N-terminal and WNK4 are key to the functionality of WNK1. truncation that lacks a functional kinase S/T domain. It is not known whether this paradigm of WNK1–WNK4 Materials and Methods Received January 6, 2006. Accepted April 20, 2006. Cloning and cRNA Synthesis Full-length cDNA for ROMK2 was identified in an IMAGE clone Published online ahead of print. Publication date available at www.jasn.org. (clone no. 4611308) and then subcloned in-frame into pEGFP-C1 to G.C. and M.M. contributed equally to this work. produce a GFP fusion protein with the N-terminal of ROMK2. A full-length clone for rat WNK1 was a gift of Dr. Melanie Cobb (12). The Address correspondence to: Dr. Kevin M. O’Shaughnessy, Clinical Pharmacology Ͼ Unit, Level 6, ACCI, Box 110, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK. WNK1 sequence was mutated to produce 637Q E (to reproduce the Phone: ϩ44-1223-762578; Fax: ϩ44-1223-762576; E-mail: [email protected] WNK4 565QϾE disease mutation) and 368DϾA (kinase-dead) mutants Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1707-1867 1868 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1867–1874, 2006 using site-directed mutagenesis. The WNK1 protein fragments were microscope. All images were captured using an equatorial section produced by PCR amplification off the full-length WNK1 template to through each oocyte. Images were collected using a ϫ10 objective lens produce a product with a 5Ј EcoRI site and a 3Ј XhoI site for direct with brightness and contrast settings kept constant for all oocytes in cloning into pET29b (Novagen, EMDBiosciences, Damstadt, Germany). each injection series. The fluorescence signal in the membrane was This created an in-frame N-terminal (His)6 fusion and a C-terminal quantified using Leica confocal software (version 2.61 of LCS Lite, Leica S-Tag. Details of the primers that were used to generate them are Microsystems, Heidelberg, Germany) with sampling made at 16 equi- shown in Table 1. Full-length sequence for WNK4 was PCR-amplified spaced points on the circumference and averaged to give mean total from mouse kidney cDNA and cloned into pcDNA3. Wild-type and fluorescence intensity in arbitrary fluorescence units. K44A dynamin clones were gifts of Dr. Peter Friedman (13). All clones were verified by sequencing before being used to run off cRNA. Copy S-Tag–Agarose Pulldown Method RNA was transcribed in vitro from linearized plasmids using either the Recombinant fusion proteins of truncated WNK1 proteins with N- T7 or the SP6 mMESSAGE mMACHINE kit (Ambion, Austin, TX) and terminal (His)6-Tag and C-terminal S-Tag were generated using Esche- quantified by ultraviolet absorption spectroscopy. richia coli protein expression system, cells were lysed using lysozyme (10 mg/ml), and proteins were extracted and purified using IMAC Expression in Xenopus oocytes (Akta Prime, GE Healthcare, Uppsala, Sweden). Purified proteins were Xenopus laevis eggs were harvested and de-folliculated as detailed dialyzed on a Slide-a-lyser dialysis cassette (Pierce, Rockford, IL) with previously (8). Briefly, the cRNA (10 ng of ROMK) was injected in a a molecular weight cutoff of 10 kD and concentrated using Centricon total volume of 100 nl per oocyte, and for co-injections involving WNK1 filters (Millipore Corp., Billerica, MA). Protein concentration was esti- constructs, an additional 10 ng of cRNA was added to the injectate mated using the BCA assay (Pierce). again in a total volume of 100 nl. Water-injected oocytes were used as For pulldown assays, 20 ␮g of fusion protein was adsorbed onto controls throughout. Oocytes then were incubated in ND96 that con- S-Tag agarose (Novagen) and washed once in wash buffer (20 mM tained 2 mM sodium pyruvate and 0.1 mg/ml gentamicin at 18°C for Tris-HCl [pH 7.5], 1.5 M NaCl, and 1% Triton X-100). In preliminary 2 d unless stated otherwise. experiments, binding of fusion protein to the beads was confirmed by both Coomassie staining and Western blotting with an anti-polyhis Two-Electrode Voltage Clamp Recording antibody (Novagen). Bead-bound protein was exposed to 200 ␮lofa Two-electrode voltage clamp used microelectrodes that were filled lysate from oocytes that were injected with ROMK cRNA and pro- with 3 M KCl (1 to 2 M⍀) for voltage sensing and current passing. cessed using a lysis buffer that contained 50 mM Tris-HCl (pH 7.5), 150 External voltage and current electrodes consisted of fine, chloride- mM NaCl, 2 mM EDTA, 1% Triton X-100, and 10 ␮l of Protease coated silver wires. Oocytes were held in a small chamber and perfused Inhibitor Cocktail Set III (Calbiochem, La Jolla, CA). Beads were incu- continuously with ND96 (2 ml/min) at room temperature (18 to 20°C). bated at 4°C for4honarotating stage. They then were pelleted and After impaling, oocytes were held at a holding potential of Ϫ60 mV. washed twice with affinity wash buffer before SDS-PAGE separation Current-voltage (I-V) plots were obtained from voltage step protocols and Western blotting. that ranged from Ϫ140 mV to 40 mV in 20-mV increments. The oocytes were held at each voltage step for 500 ms with 100-ms intervals be- Western Blotting tween the voltage steps. For the I-V plots, the steady-state current at Oocytes were placed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 each voltage step was used.
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