Regulation of ROMK Channel and K+ Homeostasis by Kidney-specific WNK1 Kinase
2009; Elsevier BV; Volume: 284; Issue: 18 Linguagem: Inglês
10.1074/jbc.m806551200
ISSN1083-351X
AutoresZhen Liu, Hao-Ran Wang, Chou-Long Huang,
Tópico(s)Pancreatic function and diabetes
ResumoWNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. WNK1, the first member discovered, has multiple alternatively spliced isoforms, including a ubiquitously expressed full-length long form (L-WNK1) and a kidney-specific form (KS-WNK1) predominantly expressed in the kidney. Intronic deletions of WNK1 that increase WNK1 transcript cause pseudohypoaldosteronism type 2, an autosomal-dominant disease characterized by hypertension and hyperkalemia. L-WNK1 inhibits renal K+ channel ROMK, likely contributing to hyperkalemia in PHAII. Previously, we reported that KS-WNK1 by itself has no effect on ROMK1 but antagonizes L-WNK1-mediated inhibition of ROMK1. Amino acids 1–253 of KS-WNK1 (KS-WNK1(1–253)) are sufficient for reversing the inhibition of ROMK1 caused by L-WNK1(1–491). Here, we further investigated the mechanisms by which KS-WNK1 counteracts L-WNK1 regulation of ROMK1. We reported that two regions of KS-WNK1(1–253) are involved in the antagonism of L-WNK1; one includes the first 30 amino acids unique for KS-WNK1 encoded by the alternatively spliced initiating exon 4A, and the other is equivalent to the autoinhibitory domain (AID) of L-WNK1. Mutations of two phenylalanine residues known to be critical for autoinhibitory function of AID abolish the ability of the AID region of KS-WNK1 to antagonize L-WNK1. To examine the physiological role of KS-WNK1 in the regulation of renal K+ secretion, we generated transgenic mice that overexpress amino acids 1–253 of KS-WNK1 under the control of a kidney-specific promoter. Transgenic mice have lower serum K+ levels and higher urinary fractional excretion of K+ compared with wild type littermates despite the same amount of daily urinary K+ excretion. Moreover, transgenic mice (compared with wild type littermates) displayed a higher abundance of ROMK on the apical membrane of distal nephron. Thus, KS-WNK1 is an important physiological regulator of renal K+ excretion, likely through its effects on the ROMK1 channel. WNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. WNK1, the first member discovered, has multiple alternatively spliced isoforms, including a ubiquitously expressed full-length long form (L-WNK1) and a kidney-specific form (KS-WNK1) predominantly expressed in the kidney. Intronic deletions of WNK1 that increase WNK1 transcript cause pseudohypoaldosteronism type 2, an autosomal-dominant disease characterized by hypertension and hyperkalemia. L-WNK1 inhibits renal K+ channel ROMK, likely contributing to hyperkalemia in PHAII. Previously, we reported that KS-WNK1 by itself has no effect on ROMK1 but antagonizes L-WNK1-mediated inhibition of ROMK1. Amino acids 1–253 of KS-WNK1 (KS-WNK1(1–253)) are sufficient for reversing the inhibition of ROMK1 caused by L-WNK1(1–491). Here, we further investigated the mechanisms by which KS-WNK1 counteracts L-WNK1 regulation of ROMK1. We reported that two regions of KS-WNK1(1–253) are involved in the antagonism of L-WNK1; one includes the first 30 amino acids unique for KS-WNK1 encoded by the alternatively spliced initiating exon 4A, and the other is equivalent to the autoinhibitory domain (AID) of L-WNK1. Mutations of two phenylalanine residues known to be critical for autoinhibitory function of AID abolish the ability of the AID region of KS-WNK1 to antagonize L-WNK1. To examine the physiological role of KS-WNK1 in the regulation of renal K+ secretion, we generated transgenic mice that overexpress amino acids 1–253 of KS-WNK1 under the control of a kidney-specific promoter. Transgenic mice have lower serum K+ levels and higher urinary fractional excretion of K+ compared with wild type littermates despite the same amount of daily urinary K+ excretion. Moreover, transgenic mice (compared with wild type littermates) displayed a higher abundance of ROMK on the apical membrane of distal nephron. Thus, KS-WNK1 is an important physiological regulator of renal K+ excretion, likely through its effects on the ROMK1 channel. WMK (with no lysine (K)) kinases are a recently discovered family of large serine-threonine protein kinases characterized by an atypical placement of the catalytic lysine (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). There are four family members, WNK1–4 (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 2Veríssimo F. Jordan P. Oncogene. 2001; 39: 5562-5569Crossref Scopus (226) Google Scholar, 3Wilson F.H. Disse-Nicodème S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. Simon D.B. Farfel Z. Jeunemaitre X. Lifton R.P. Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1233) Google Scholar), each encoded by a separate gene. WNK1 protein is over 2,100 amino acids long and contains an ∼270-amino acid kinase domain located near the amino terminus (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). WNK2, WNK3, and WNK4 are between 1,200 and 1,600 amino acids in length (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 2Veríssimo F. Jordan P. Oncogene. 2001; 39: 5562-5569Crossref Scopus (226) Google Scholar, 3Wilson F.H. Disse-Nicodème S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. Simon D.B. Farfel Z. Jeunemaitre X. Lifton R.P. Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1233) Google Scholar). The four WNK kinases share a conserved kinase domain with 85–90% sequence identity, an autoinhibitory domain (AID), 2The abbreviations used are: AID, autoinhibitory domain; L-WNK1, long form of WNK1; KS-WNK1, kidney-specific form of WNK1; HEK, human embryonic kidney; PHAII, pseudohypoaldosteronism type II; TG, transgenic; PBS, phosphate-buffered saline; GFP, green fluorescent protein; ERK, extracellular signal-regulated kinase. one or two coiled-coiled domains, and multiple PXXP motifs for potential protein-protein interaction (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 2Veríssimo F. Jordan P. Oncogene. 2001; 39: 5562-5569Crossref Scopus (226) Google Scholar, 3Wilson F.H. Disse-Nicodème S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. Simon D.B. Farfel Z. Jeunemaitre X. Lifton R.P. Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1233) Google Scholar). Beyond these conserved domains and motifs, amino acid sequences of WNK1–4 are divergent. The human WNK1 gene spans more than 150 kb in chromosome 12 and consists of 28 exons (3Wilson F.H. Disse-Nicodème S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. Simon D.B. Farfel Z. Jeunemaitre X. Lifton R.P. Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1233) Google Scholar). A WNK1 transcript produced from all 28 exons (encodes a peptide referred to herein as long WNK1 (L-WNK1)) is ubiquitously expressed (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). A shorter WNK1 transcript produced by an alternative 5′ exon (exon 4A) and exon 5–28 is expressed exclusively in the kidney, encoding a peptide known as kidney-specific WNK1 (KS-WNK1) (4Delaloy C. Lu J. Houot A.M. Disse-Nicodeme S. Gasc J.M. Corvol P. Jeunemaitre X. Mol. Cell. Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 5O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar). KS-WNK1 is ∼1,700 amino acids in length and lacks amino acids 1–437 of the long WNK1 that are encoded by exon1–4. The first 30 amino acids of KS-WNK1 are encoded by exon 4A (4Delaloy C. Lu J. Houot A.M. Disse-Nicodeme S. Gasc J.M. Corvol P. Jeunemaitre X. Mol. Cell. Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 5O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar) and unique to KS-WNK1. In the kidney, KS-WNK1 is predominantly in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (6O'Reilly M. Marshall E. Macgillivray T. Mittal M. Xue W. Kenyon C.J. Brown R.W. J. Am. Soc. Nephrol. 2006; 17: 2402-2413Crossref PubMed Scopus (111) Google Scholar), suggesting a role in these segments. Analysis by real time PCR reveals that the transcript for KS-WNK1 in kidney is more abundant than that for L-WNK1 (4Delaloy C. Lu J. Houot A.M. Disse-Nicodeme S. Gasc J.M. Corvol P. Jeunemaitre X. Mol. Cell. Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 5O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar). The relative protein abundance of KS-WNK1 versus L-WNK1 has not been determined. Alternative splicing of exon 11 and 12 also occurs and produces isoforms with peptide length between that of L-WNK1 and KS-WNK1 and with differential tissue distributions (4Delaloy C. Lu J. Houot A.M. Disse-Nicodeme S. Gasc J.M. Corvol P. Jeunemaitre X. Mol. Cell. Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 5O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar). Large deletions within the first intron of WNK1 increase the abundance of WNK1 transcript and cause pseudohypoaldosteronism type 2 (PHAII; also known as familial hyperkalemic hypertension or Gordon syndrome), an autosomal-dominant disorder featured by hypertension and hyperkalemia (3Wilson F.H. Disse-Nicodème S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. Simon D.B. Farfel Z. Jeunemaitre X. Lifton R.P. Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1233) Google Scholar, 7Schambelan M. Sebastian A. Rector Jr., F.C. Kid. Int. 1981; 19: 716-727Abstract Full Text PDF PubMed Scopus (194) Google Scholar). Many studies have examined the role of WNK kinases in the regulation of renal ion transport (8Yang C.L. Angell J. Mitchell R. Ellison D.H. J. Clin. Investig. 2003; 111: 1039-1045Crossref PubMed Scopus (401) Google Scholar, 9Wilson F.H. Kahle K.T. Sabath E. Lalioti M.D. Rapson A.K. Hoover R.S. Hebert S.C. Gamba G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 680-684Crossref PubMed Scopus (361) Google Scholar, 10Yamauchi K. Rai T. Kobayashi K. Sohara E. Suzuki T. Itoh T. Suda S. Hayama A. Sasaki S. Uchida S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4690-4694Crossref PubMed Scopus (228) Google Scholar, 11Xu B. Stippec S. Chu P.-Y. Li X.-J. Lazrak A. Ortega B. Lee B.H. English J.M. Huang C.-L. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10315-10320Crossref PubMed Scopus (166) Google Scholar). With respect to WNK1 on K+ transport, it was reported that L-WNK1 decreases cell surface abundance of renal K+ channel ROMK1 (renal outer medullary potassium (K) channel 1) by increasing clathrin-coated vesicle-mediated endocytosis of the channel (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar, 13He G. Wang H.R. Huang S.K. Huang C.L. J. Clin. Investig. 2007; 117: 1078-1087Crossref PubMed Scopus (129) Google Scholar). ROMK K+ channels are expressed in the connecting tubule and the cortical collecting duct and are important for base-line (non-flow-stimulated) renal K+ secretion (14Hebert S.C. Desir G. Giebisch G. Wang W. Physiol. Rev. 2005; 85: 319-371Crossref PubMed Scopus (273) Google Scholar). Thus, a decrease in K+ secretion by the kidney resulting from the inhibition of ROMK by L-WNK1 may contribute to hyperkalemia in patients of PHAII with WNK1 mutations. However, there are multiple alternatively spliced WNK1 isoforms differentially expressed in tissues (4Delaloy C. Lu J. Houot A.M. Disse-Nicodeme S. Gasc J.M. Corvol P. Jeunemaitre X. Mol. Cell. Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 5O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar). The effects of deletions of the first intron on splice variants of WNK1 and the effects of individual isoforms on K+ transport remain largely unknown. Recently, we and others reported that kidney-specific WNK1, by itself, has no effect on ROMK1 but antagonizes the inhibition of ROMK1 caused by L-WNK1 (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar, 15Wade J.B. Fang L. Liu J. Li D. Yang C.L. Subramanya A.R. Maouyo D. Mason A. Ellison D.H. Welling P.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8558-8563Crossref PubMed Scopus (114) Google Scholar). K+ secretion by kidney is critical for controlling serum K+ levels and overall K+ homeostasis (14Hebert S.C. Desir G. Giebisch G. Wang W. Physiol. Rev. 2005; 85: 319-371Crossref PubMed Scopus (273) Google Scholar). As an important pathway for K+ secretion in kidney, the abundance of ROMK on the apical membrane of distal nephron is regulated by dietary K+ intake (14Hebert S.C. Desir G. Giebisch G. Wang W. Physiol. Rev. 2005; 85: 319-371Crossref PubMed Scopus (273) Google Scholar). The apical ROMK abundance decreases or increases during low or high dietary K+ intake, respectively (16Palmer L.G. Frindt G. Am. J. Physiol. 1999; 277: F805-F812PubMed Google Scholar, 17Wang W.-H. Lerea K.M. Chan M. Giebisch G. Am. J. Physiol. 2000; 278: F165-F172Crossref PubMed Google Scholar). The decrease in the apical abundance of ROMK in response to dietary K+ restriction involves an increase in the clathrin-mediated endocytosis and subsequent degradation of the channel protein (18Zeng W.Z. Babich V. Ortega B. Quigley R. White S.J. Welling P.A. Huang C.L. Am. J. Physiol. 2002; 283: F630-F639Crossref PubMed Scopus (66) Google Scholar, 19Chu P.Y. Quigley R. Babich V. Huang C.L. Am. J. Physiol. 2003; 285: F1179-F1187Crossref PubMed Scopus (44) Google Scholar). We reported that dietary K+ restriction in rats increases the expression of L-WNK1 and decreases that of KS-WNK1 (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). The increase in the L-WNK1 to KS-WNK1 ratio would be expected to cause inhibition of ROMK1. These results suggest that KS-WNK1 is an important physiological antagonist of L-WNK1, and the ratio of L-WNK1 to KS-WNK1 regulates surface abundance of ROMK1 and renal K+ secretion during changes in dietary K+ intake. In the present study, we further examined the mechanism by which KS-WNK1 antagonizes L-WNK1 regulation of ROMK1. We identified two regions within amino acids 1–253 of KS-WNK1 that are involved in binding to and antagonism of L-WNK1. Furthermore, to examine the physiological role of KS-WNK1 in the regulation of K+ secretion in vivo, we generated transgenic mice overexpressing amino acids 1–253 of KS-WNK1 and found that they display lower serum K+ levels and increased tubular excretion of K+ relative to wild type littermates despite a similar K+ intake. These results further support the important physiological role of KS-WNK1 in the regulation ROMK1 activity and renal K+ excretion. Plasmid DNA Constructs—pEGFP-ROMK1, pCMV-Myc-WNK1(1–491), and pIRES-FLAG-KS-WNK1(1–253) were described previously (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). WNK1 fragments were amplified by PCR using rat WNK1 cDNA as the template and subcloned into a pCMV5-Myc vector. Fragments of rat KS-WNK1 were amplified by PCR and subcloned into a carboxyl-terminal FLAG vector (pIRES-hrGFP-1a) (Stratagene). Point mutations were generated by site-directed mutagenesis (QuikChange kit; Stratagene) and confirmed by sequencing. Cell Culture, Immunoprecipitation, and Western Blot Analysis—HEK 293 cells were cultured, transfected, and harvested as described previously (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). For coimmunoprecipitation, the proteins were immunoprecipitated from cell lysates by using monoclonal anti-FLAG antibody (1:100 dilution; Sigma) and followed by protein A-Sepharose beads. The precipitates were washed three time with 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.5% Triton X-100. For Western blot analysis, total lysates, immunoprecipitates, or kidney homogenate were resolved by SDS-PAGE gel electrophoresis, and proteins were transferred onto nitrocellulose membranes. The membranes were incubated with the indicated antibodies and developed using enhanced chemiluminescence. Whole Cell Patch-Clamp Recording of ROMK1 Channels—HEK 293 cells were cotransfected with cDNAs encoding GFP-ROMK1 and a fragment of L-WNK1 and/or KS-WNK1. In each experiment, the total amount of DNA for transfection was balanced by using empty vectors. Approximately 36–48 h after transfection, whole cell currents were recorded by using an Axopatch 200B amplifier as previous described (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). Transfected cells were identified by using epifluorescent microscopy. The bath and pipette solution contained 145 mm KCl, 2 mm MgCl2, 2 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 resistance were monitored and 75% compensated. The voltage protocol consists of 0-mV holding potential and 400-ms steps from -100 to 100 mV in 20-mV increments. Generation of Transgenic Mice—The FLAG-KS-WNK1(1–253) fusion fragment was generated by PCR using plasmid pIRES-hrGFP-KS-WNK1(1–253) as template. The ∼0.8-kb restriction fragment was isolated and cloned into unique SbfI and SmaI sites downstream to the Ksp-cadherin promoter of pKsp-BGH plasmid (provided by Dr. Peter Igarashi, University of Texas Southwestern Medical Center at Dallas). The plasmid insert was verified by DNA sequencing. The ∼2.6-kb transgene fragment Ksp-FLAG-KS-WNK1(1–253) was isolated by digestion with NdeI and KpnI followed by agarose gel electrophoresis, electroelution, and purification by anion exchange chromatography (Elutip-d, NH). Purified DNA was concentrated in Microcon 30 filters (Millipore, MA), resuspended at a concentration of 80 ng/μl in microinjection buffer (10 mm Tris-Cl, pH 7.4, 0.25 mm EDTA), and sterilized by filtration through 0.2-μm filters. Transgene DNA was microinjected into the pronuclei of fertilized oocytes by standard pronuclear injection. Fertilized oocytes were from C57BL/6 crosses. Microinjection was performed by the University of Texas Southwestern Transgenic Mouse Core Facility. The microinjected embryos were transferred into the oviducts of pseudopregnant foster mothers and were permitted to develop to term. Genotyping of Transgenic Mice—Founder (G0) mice were identified by PCR analysis. Genomic DNA was isolated from tails of transgenic mice using a standard method. Two pairs of primers were used for genotyping by PCR. One is specific for endogenous mouse WNK1 (forward, 5′-AAA ATA CTC TGT CAG GCT TAA GTG T-3′, and reverse, 5′-TGA AGC CAG GCA TTA AGC ACT C-3′), which would produce a 266-bp fragment in both wild type and transgenic mice. The other is specific for transgenic fragment (forward, 5′-GCA GAT CAG CAT CAA CAG CTG-3′, and reverse, 5′-CAA TGC GAT GCA ATT TCC TC-3′), which would produce a 320-bp fragment only in transgenic mice. The condition for PCR includes 35 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 45 s. PCR products were detected by electrophoresis on 2.0% agarose gels. Quantitative Real Time PCR Analysis—Total RNA was isolated from whole kidney with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Gene expression by quantitative real time PCR was carried out on ABI 7000 as described in Applied Biosystems User Bulletin no. 2 using the TagMan assay. Primer from exon 4A (RP forward, 5′-GCT GCT GTT CTC AAA AGG ATT GTA T-3′), from exon 5 (RP reverse, 5′-CAG GAA TTG CTA CTT TGT CAA AAC TG-3′), and from TagMan probe (5′-TGA GGG AGT GAA GCC A-3′) were used to amplify the kidney-specific isoform (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). Relative KS-WNK1 mRNA levels were calculated with 18 S rRNA as the internal control. Kidney cDNA was prepared from three to five animals as described above, and each sample was assayed in triplicate. Immunofluorescent Staining—The mice were anesthetized by Avertin and perfused via the heart with 15 ml of PBS followed by 15 ml of 4% paraformaldehyde in PBS. The kidneys were harvested and postfixed for 4 h in 4% paraformaldehyde in PBS at 4 °C, dehydrated by immersion in 30% sucrose in PBS overnight at 4 °C, and mounted in OTC (Tissue-Tek) for sectioning. The sections (4–5-μm thickness) were stained with primary antibodies: rabbit polyclonal anti-FLAG antibody (1:300, Sigma) or anti-ROMK (1:1000), followed by secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (1:500) or Alexa Fluor 568 goat anti-rabbit IgG (1:500). The fluorescent images were obtained using Zeiss LSM510 confocal microscope as described (19Chu P.Y. Quigley R. Babich V. Huang C.L. Am. J. Physiol. 2003; 285: F1179-F1187Crossref PubMed Scopus (44) Google Scholar). Blood and Urine Measurements—Under anesthesia by Avertin, blood was drawn from mice by retro-orbital bleeding into heparinized tubes. Electrolytes were measured using a flame photometer. Creatinine was measured by capillary electrophoresis (P/ACE MDQ; Beckman Coulter). Spot urine samples were collected by catching spontaneous voids. 24-h urine samples were collected using metabolic cages (Hatteras Instruments). All of the experiments involving animals were performed in compliance with relevant laws and institutional guidelines and were approved by the University of Texas Southwestern Medical Center at Dallas Institutional Animal Care and Use Committee. Statistical Analysis—Statistical comparisons between two groups of data were made using two-tailed unpaired Student's t tests. Multiple comparison were made using one-way analysis of variance followed by two-tailed Student's t tests adjusted for multiple comparisons. p values less than 0.05 and 0.01 were considered significant for single and multiple comparisons, respectively. Experiments shown in each panel of the figures were repeated at least three times with similar results. Two Regions of KS-WNK1(1–253) Are Involved in the Antagonism of L-WNK1—We have reported that KS-WNK1(1–253) antagonizes inhibition of ROMK1 by L-WNK1 by binding to amino acids 1–491 of L-WNK1 (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). To further define the molecular determinant(s) of KS-WNK1 involved in antagonism of L-WNK1 regulation of ROMK1, we generated several overlapping and nonoverlapping smaller fragments of KS-WNK1(1–253) and examined their effects on WNK1(1–491)-mediated inhibition of ROMK1. These fragments of KS-WNK1 include amino acids 1–196, 1–137, 1–77, and 31–253, respectively (Fig. 1A). To examine the effects of these fragments on antagonism of L-WNK1 regulation of ROMK1, HEK cells were cotransfected with plasmids expressing green fluorescent protein (GFP)-tagged ROMK1, WNK1(1–491) and one each of KS-WNK1 fragment and recorded for ROMK1 current density using whole cell patch-clamp recording. We have shown that WNK1(1–491) fully recapitulates the effect of full-length L-WNK1 on ROMK1 (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar, 13He G. Wang H.R. Huang S.K. Huang C.L. J. Clin. Investig. 2007; 117: 1078-1087Crossref PubMed Scopus (129) Google Scholar). As shown in Fig. 1B, we confirmed that WNK1(1–491) inhibits ROMK1 (compare bars 1 and 2) and that KS-WNK1(1–253) reverses WNK1(1–491)-mediated inhibition of ROMK1 (bar 3) as reported previously by us (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). Expression of KS-WNK1(1–253) exerts no effect on ROMK1 in the absence of WNK1(1–491) (not shown in Fig. 1B; see Ref. 12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar). Here, we found that each of the smaller KS-WNK1 constructs generated could reverse the inhibition of ROMK1 caused by WNK1(1–491) (Fig. 1B, bars 4–7). WNK1 contains an AID just carboxyl-terminal to the catalytic domain that is conserved in WNKs across species (1Xu B. English J.M. Wilsbacher J.L. Stippec S. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: 16795-16801Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 20Xu B.E. Min X. Stippec S. Lee B.H. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2002; 277: 48456-48462Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The AID domain binds with and suppresses the activity of the kinase domain (20Xu B.E. Min X. Stippec S. Lee B.H. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2002; 277: 48456-48462Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). We have recently shown that AID domain of WNK1 can antagonize WNK1(1–491)-mediated inhibition of ROMK1 (12Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar, 21Wang H.R. Liu Z. Huang C.L. Am. J. Physiol. 2008; 295: F438-F445Crossref PubMed Scopus (21) Google Scholar). Because KS-WNK1(1–253), KS-WNK1(1–196), KS-WNK1(1–137), and KS-WNK1(31–253) each contain the AID domain, it is not surprising that they each can reverse the effect of WNK1(1–491). The ability of KS-WNK1(1–77) to reverse the inhibition of ROMK1 by WNK1(1–491), however, is unexpected and suggests that additional region is involved in interacting with and reversing the effect of WNK1(1–491). KS-WNK1(1–77) contains the unique 30 amino acids encoded by exon 4A. We hypothesized that this region encoded by exon 4A is responsible for reversal of WNK1(1–491)-mediated inhibition of ROMK1 by KS-WNK1(1–77). In this hypothesis, the two regions within KS-WNK1(1–253) (i.e. AID and region encoded by exon 4A) can interact with WNK1(1–491) independently of one another to reverse the effect of WNK1(1–491). To test this hypothesis, we examined the interaction between WNK(1–491) and KS-WNK1(1–253), KS-WNK1(1–77) or KS-WNK1(31–253) by coimmunoprecipitation. HEK cells were cotransfected with Myc-tagged WNK1(1–491) and each of FLAG-tagged KS-WNK1 fragments as indicated. As shown in Fig. 1C (bottom panel), anti-FLAG antibody immunoprecipitated FLAG-KS-WNK1(1–77), KS-WNK1(31–253), and KS-WNK1(1–253) (lanes 2–4 indicated by 10-, 29-, and 32-kDa protein bands, respectively). The higher molecular size band (∼160-kDa protein band) in lysates of cells expressing KS-WNK1(1–77) (Fig. 1C, lane 2) suggests that KS-WNK1(1–77) may form oligomers. However, we did not observe large molecular size bands for KS-WNK1(1–253) expressed in HEK cells (not shown) nor in transgenic mice (see "Results" below), suggesting that oligomerization of KS-WNK1 does not occur in vivo and thus is not physiologically important. As shown, Myc-WNK1(1–491) (indicated by the 60-kDa protein band in the middle panel of Fig. 1C) coimmunoprecipitated strongly with KS-WNK1(1–253) (Fig. 1C, lane 4) and to a lesser degree with KS-WNK1(1–77) and with KS-WNK1(31–253) (Fig. 1C, lanes 2 and 3). As a control, Myc-WNK1(1–491) did not coimmunoprecipitate with FLAG-tagged pod (Fig. 1C, lane 5), an unrelated transcription factor protein (22Quaggin S.E. Schwartz L. Cui S. Igarashi P. Deimling J. Post M. Rossant J. Development. 1999; 126: 5771-5783PubMed Google Scholar). The abundance of input Myc-WNK1(1–491) was not different among experimental groups (Fig. 1C, top panel). These results support the hypothesis that the region encoded by exon 4A (as in "KS-WNK1(1–77)") and the AID domain (as in "KS-WNK1(31–253)") can interact with WNK1(1–491), independently. Consistently, the interaction with WNK1(1–491) is stronger for KS-WNK1(1–253), which contains both regions. It should be noted that the relatively weaker interaction between WNK1(1–491) and KS-WNK1(1–77) or KS-WNK1(31–253) was sufficient for reversing the inhibitory effect of WNK1(1–491) (Fig. 1B). Role of AID Domain of KS-WNK1 in the Interaction with L-WNK1 and Regulation ROMK1 Channel—In our recent study reporting that AID reverses WNK1(1–491) inhibition of ROMK1 (21Wang H.R. Liu Z. Huang C.L. Am. J. Physiol. 2008; 295: F438-F445Crossref PubMed Scopus (21) Google Scholar), we did not examine whether the effect was mediated by binding of AID to WNK1(1–491). Here, we examined the binding interaction between WNK1(1–491) and AID domain (as in "FLAG-WNK1(491–555)"). Two phenylalanine residues withi
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