WNK4 Kinase Stimulates Caveola-mediated Endocytosis of TRPV5 Amplifying the Dynamic Range of Regulation of the Channel by Protein Kinase C
2010; Elsevier BV; Volume: 285; Issue: 9 Linguagem: Inglês
10.1074/jbc.m109.056044
ISSN1083-351X
AutoresSeung‐Kuy Cha, Chou-Long Huang,
Tópico(s)Ion channel regulation and function
ResumoWNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca2+ channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca2+ reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (∼215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca2+ reabsorption by parathyroid hormones or the tissue kallikrein in vivo. WNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca2+ channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca2+ reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (∼215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca2+ reabsorption by parathyroid hormones or the tissue kallikrein in vivo. IntroductionWNK (With-no-lysine (K)) kinases are large serine-threonine protein kinases characterized by an atypical placement of the catalytic lysine (1.Xu 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 (407) Google Scholar). Mammalian WNK kinases consist of four members, named WNK1–4 (1.Xu 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 (407) Google Scholar, 2.Veríssimo F. Jordan P. Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (222) Google Scholar, 3.Wilson 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 (1206) Google Scholar). Each WNK kinase is encoded by a separate gene. WNK1–4 are between 1,200- and 2,100-amino acids long. Each contains a ∼270-amino acid conserved kinase domain located near the amino terminus (1.Xu 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 (407) Google Scholar). In addition, they share an autoinhibitory domain, one to two coiled-coil domains, and multiple PXXP proline-rich motifs for potential protein-protein interaction (1.Xu 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 (407) Google Scholar, 2.Veríssimo F. Jordan P. Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (222) Google Scholar, 3.Wilson 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 (1206) Google Scholar). The remaining amino acid sequences of WNK1–4 are not conserved.Mutations in WNK1 and WNK4 cause the autosomal-dominant disease pseudohypoaldosteronism type 2 (PHA2) 2The abbreviations used are: PHA2pseudohypoaldosteronism type 2PKCprotein kinase CFLfull-lengthOAG1-oleoyl-acetyl-sn-glycerolPTHparathyroid hormoneGFPgreen fluorescent proteinNCCthiazide-sensitive NaCl co-transporterROMKrenal outer medullary K+ channelHEKhuman embryonic kidneyCCVclathrin-coated vesiclesiRNAsmall interference RNAPTH1Rtype 1 PTH receptorSH3Src homology-3WTwild type. (3.Wilson 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 (1206) Google Scholar). PHA2-causing mutations in WNK1 are large deletions within the first intron resulting in an increase in the abundance of transcript. Mutations in WNK4 are missense mutations in the coding sequence leading to altered protein function. PHA2 is characterized by hypertension and hyperkalemia (3.Wilson 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 (1206) Google Scholar, 4.Schambelan M. Sebastian A. Rector Jr., F.C. Kidney Int. 1981; 19: 716-727Abstract Full Text PDF PubMed Scopus (192) Google Scholar). Interestingly, patients with PHA2 caused by WNK4 mutations, but not patients with WNK1 mutations, also have abnormally high urinary calcium excretion (hypercalciuria) (5.Mayan H. Munter G. Shaharabany M. Mouallem M. Pauzner R. Holtzman E.J. Farfel Z. J. Clin. Endocrinol. Metab. 2004; 89: 4025-4030Crossref PubMed Scopus (85) Google Scholar, 6.Achard J.M. Warnock D.G. Disse-Nicodème S. Fiquet-Kempf B. Corvol P. Fournier A. Jeunemaitre X. Am. J. Med. 2003; 114: 495-498Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).WNK kinases are broadly distributed. WNK1 has multiple alternatively spliced isoforms, including an ubiquitous long isoform and a kidney-specific isoform lacking the kinase domain and predominantly expressed in distal tubules of the kidney (7.Delaloy 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 (138) Google Scholar, 8.O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (142) Google Scholar). WNK2 is predominantly expressed in heart, brain, and colon (7.Delaloy 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 (138) Google Scholar); WNK3 and WNK4 are mostly expressed in kidney, heart, and brain (7.Delaloy 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 (138) Google Scholar, 9.Holden S. Cox J. Raymond F.L. Gene. 2004; 335: 109-119Crossref PubMed Scopus (50) Google Scholar, 10.Kahle K.T. Gimenez I. Hassan H. Wilson F.H. Wong R.D. Forbush B. Aronson P.S. Lifton R.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2064-2069Crossref PubMed Scopus (149) Google Scholar). In addition, WNK4 is expressed in many other epithelial tissues (10.Kahle K.T. Gimenez I. Hassan H. Wilson F.H. Wong R.D. Forbush B. Aronson P.S. Lifton R.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2064-2069Crossref PubMed Scopus (149) Google Scholar). The distribution of WNK1 and WNK4 in the renal tubules, and that mutations in them cause hypertension and hyperkalemia, suggest that they play important roles in regulating renal ion transport. Indeed, WNK1 and WNK4 have been shown to regulate renal ion transport proteins, including paracellular tight junction proteins claudins; cation-chloride co-transporters NCC, KCl cotransporters, and Na+-K+-2Cl− cotransporters; epithelial Na+ channel; and renal K+ channel ROMK (11.Kahle K.T. Ring A.M. Lifton R.P. Annu. Rev. Physiol. 2008; 70: 329-355Crossref PubMed Scopus (195) Google Scholar, 12.Huang C.L. Yang S.S. Lin S.H. Curr. Opin. Nephrol. Hypertens. 2008; 17: 519-525Crossref PubMed Scopus (78) Google Scholar).TRPV5 (transient receptor potential vanilloid 5) channel is localized to the apical membrane of distal convoluted tubules and connecting tubules and functions as a gatekeeper for transcellular Ca2+ reabsorption in the kidney. Many hormones, including parathyroid hormone (PTH), regulate renal Ca2+ reabsorption at least partly via TRPV5 (13.Hoenderop J.G. Nilius B. Bindels R.J. Annu. Rev. Physiol. 2002; 64: 529-549Crossref PubMed Scopus (205) Google Scholar, 14.Lambers T.T. Bindels R.J. Hoenderop J.G. Kidney Int. 2006; 69: 650-654Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). One mechanism for PTH regulation of TRPV5 is by increasing the protein expression of TRPV5, providing a mechanism for long term regulation of renal Ca2+ reabsorption (15.van Abel M. Hoenderop J.G. van der Kemp A.W. Friedlaender M.M. van Leeuwen J.P. Bindels R.J. Kidney Int. 2005; 68: 1708-1721Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In addition, PTH can enhance renal Ca2+ reabsorption within 10–15 min of its administration. The mechanism of acute regulation of TRPV5 by PTH involves activation of protein kinase A and PKC (16.Hoenderop J.G. De Pont J.J. Bindels R.J. Willems P.H. Kidney Int. 1999; 55: 225-233Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17.de Groot T. Lee K. Langeslag M. Xi Q. Jalink K. Bindels R.J. Hoenderop J.G. J. Am. Soc. Nephrol. 2009; 20: 1693-1704Crossref PubMed Scopus (129) Google Scholar). Recently, we reported that TRPV5 undergoes constitutive caveolin-dependent endocytosis and that activation of PKC inhibits this process causing accumulation of the channel at the cell surface (18.Cha S.K. Wu T. Huang C.L. Am. J. Physiol. Renal Physiol. 2008; 294: F1212-F1221Crossref PubMed Scopus (90) Google Scholar). This inhibition of caveolar endocytosis of TRPV5 via PKC may be one of the mechanisms for acute regulation of the channel by PTH. In the present study, we report that WNK4 stimulates caveola-mediated endocytosis of TRPV5 and thereby increases the range of regulation of the channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca2+ reabsorption by PTH or tissue kallikrein in vivo.DISCUSSIONThe TRPV5 channel forms the luminal gate of transcellular Ca2+ reabsorption in the distal nephron of the kidney and is a target of regulation of renal Ca2+ transport by pH and hormones (13.Hoenderop J.G. Nilius B. Bindels R.J. Annu. Rev. Physiol. 2002; 64: 529-549Crossref PubMed Scopus (205) Google Scholar, 15.van Abel M. Hoenderop J.G. van der Kemp A.W. Friedlaender M.M. van Leeuwen J.P. Bindels R.J. Kidney Int. 2005; 68: 1708-1721Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The abundance of proteins at the cell surface is a balance between retrieval and insertion of the newly synthesized and recycled proteins. Recently, we reported that TRPV5 undergoes constitutive caveola-mediated endocytosis, and activation of PKC inhibits the endocytosis leading to accumulation of TRPV5 at the cell surface (18.Cha S.K. Wu T. Huang C.L. Am. J. Physiol. Renal Physiol. 2008; 294: F1212-F1221Crossref PubMed Scopus (90) Google Scholar). In the present study, we report that WNK4 kinase enhances the endocytosis of TRPV5. As would be expected, an increase in endocytosis decreases the plasma membrane abundance of TRPV5. The lower basal level of TRPV5 in the presence of WNK4 increases the range of stimulation of the channel by PKC via inhibition of endocytosis. Surprisingly, the rate of increase of TRPV5 stimulated by PKC is also enhanced by WNK4. Because an increase in endocytosis decreases the density of cargos at the cell surface, the number of channels internalized per unit time in the steady state should be the same irrespective of the rate of endocytosis. If so, one would expect that the rate of increase caused by decreasing endocytosis be the same with or without WNK4. One possible explanation for our finding may be related to the fact that there are three types of caveolae with different membrane dynamic activities (28.McMahon K.A. Zajicek H. Li W.P. Peyton M.J. Minna J.D. Hernandez V.J. Luby-Phelps K. Anderson R.G. EMBO J. 2009; 28: 1001-1015Crossref PubMed Scopus (158) Google Scholar). Type 1 caveolae are able to pinch off the plasma membrane forming endocytic vesicles and also travel to the deeper intracellular organelles, such as late endosomes. Type 2 caveolae are more superficially localized and involved in continuous rounds of fission and fusion without traveling deep below the cell surface. Type 3 is relatively uncharacterized. It is conceivable that WNK4 may alter the distribution of TRPV5 in caveolae and/or affect the dynamics of three caveolae differentially. Redistribution to a faster turnover caveolae compartment (such as type 2) may allow TRPV5 to reappear at the cell surface faster when endocytosis is inhibited.WNK4 kinase is abundantly expressed in the distal nephron including the distal convoluted tubule and the cortical connecting tubule, where transcellular Ca2+ reabsorption mediated by TRPV5 occurs (13.Hoenderop J.G. Nilius B. Bindels R.J. Annu. Rev. Physiol. 2002; 64: 529-549Crossref PubMed Scopus (205) Google Scholar). Tissue kallikrein is a serine protease produced in the cortical connecting tubule (29.Figueroa C.D. MacIver A.G. Mackenzie J.C. Bhoola K.D. Histochemistry. 1988; 89: 437-442Crossref PubMed Scopus (104) Google Scholar). Tissue kallikrein activates bradykinin-2 receptor either directly or indirectly by releasing kinin from kininogen (30.Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar, 31.Hecquet C. Tan F. Marcic B.M. Erdös E.G. Mol. Pharmacol. 2000; 58: 828-836Crossref PubMed Scopus (110) Google Scholar). A recent study reports that activation of PKC underlies the stimulation of TRPV5 and renal Ca2+ transport by tissue kallikrein (32.Gkika D. Topala C.N. Chang Q. Picard N. Thébault S. Houillier P. Hoenderop J.G. Bindels R.J. EMBO J. 2006; 25: 4707-4716Crossref PubMed Scopus (67) Google Scholar). Activation of PKC is also implicated as a molecular mechanism for stimulation of TRPV5 and renal Ca2+ transport by PTH (18.Cha S.K. Wu T. Huang C.L. Am. J. Physiol. Renal Physiol. 2008; 294: F1212-F1221Crossref PubMed Scopus (90) Google Scholar). Thus, our finding that WNK4 amplifies PKC stimulation of TRPV5 is likely important for physiological regulation of renal Ca2+ transport by tissue kallikrein and PTH. Further studies are required to determine how WNK4 enhances the rate of increase of TRPV5 by PKC and to quantify the importance of this effect in vivo.WNK kinases regulate many ion transport proteins by altering membrane trafficking. The mechanisms by which WNK kinases regulate these proteins are different. For example, WNK1 activates SGK1 to inhibit Nedd4-mediated internalization of the epithelial Na+ channel (33.Xu B.E. Stippec S. Chu P.Y. Lazrak A. Li X.J. Lee B.H. English J.M. Ortega B. Huang C.L. Cobb M.H. Proc. Natl. Acad. Sci. U. S.A. 2005; 102: 10315-10320Crossref PubMed Scopus (163) Google Scholar). WNK1 and -4 stimulate clathrin-mediated endocytosis of ROMK (19.He G. Wang H.R. Huang S.K. Huang C.L. J. Clin. Invest. 2007; 117: 1078-1087Crossref PubMed Scopus (128) Google Scholar, 20.Lazrak A. Liu Z. Huang C.L. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1615-1620Crossref PubMed Scopus (161) Google Scholar, 34.Kahle K.T. Wilson F.H. Leng Q. Lalioti M.D. O'Connell A.D. Dong K. Rapson A.K. MacGregor G.G. Giebisch G. Hebert S.C. Lifton R.P. Nat. Genet. 2003; 35: 372-376Crossref PubMed Scopus (340) Google Scholar, 35.Wade 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 (113) Google Scholar). WNK4 decreases cell-surface expression of the thiazide-sensitive NaCl cotransporter NCC by inhibiting forward trafficking (21.Subramanya A.R. Liu J. Ellison D.H. Wade J.B. Welling P.A. J. Biol. Chem. 2009; 284: 18471-18480Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). We now show that WNK4 stimulates caveola-mediated endocytosis of TRPV5. The manner by which WNK kinases regulate ion transporters via such diverse mechanisms remains elusive. We have shown previously that WNK4 interacts with SH3 domains of intersectin through a set of multiple proline-rich motifs residing between amino acids 545 and 558 (19.He G. Wang H.R. Huang S.K. Huang C.L. J. Clin. Invest. 2007; 117: 1078-1087Crossref PubMed Scopus (128) Google Scholar). Intersectin, in turn, recruits dynamin and other endocytic accessory proteins to participate in the formation of clathrin-coated vesicles and the separation of vesicles from the plasma membrane. Fission of caveolar vesicles also requires dynamin (36.Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3017) Google Scholar). The role of intersectin in the fission of caveolar vesicles and its importance in caveola-mediated endocytosis has been demonstrated (26.Predescu S.A. Predescu D.N. Timblin B.K. Stan R.V. Malik A.B. Mol. Biol. Cell. 2003; 14: 4997-5010Crossref PubMed Scopus (80) Google Scholar, 27.Klein I.K. Predescu D.N. Sharma T. Knezevic I. Malik A.B. Predescu S. J. Biol. Chem. 2009; 284: 25953-25961Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Our present study shows that intersectin is necessary for WNK4 stimulation of endocytosis of TRPV5 via caveolae. Moreover, the region of WNK4 involved in the endocytosis of TRPV5 is the same as that in the endocytosis of ROMK. Thus, WNK4 regulates both clathrin-mediated and caveola-mediated endocytosis by the same mechanism involving an interaction with intersectin. Our present results are in contrast to those by Jiang et al. (37.Jiang Y. Ferguson W.B. Peng J.B. Am. J. Physiol. Renal Physiol. 2007; 292: F545-F554Crossref PubMed Scopus (82) Google Scholar, 38.Jiang Y. Cong P. Williams S.R. Zhang W. Na T. Ma H.P. Peng J.B. Biochem. Biophys. Res. Commun. 2008; 375: 225-229Crossref PubMed Scopus (27) Google Scholar) reporting that WNK4 increases surface expression of TRPV5 and that it occurs by enhancing the secretory pathway. The precise reason(s) for these differences is unknown but may be related to differences in the experimental system employed (mammalian cells in our study versus Xenopus oocytes in their study (37.Jiang Y. Ferguson W.B. Peng J.B. Am. J. Physiol. Renal Physiol. 2007; 292: F545-F554Crossref PubMed Scopus (82) Google Scholar, 38.Jiang Y. Cong P. Williams S.R. Zhang W. Na T. Ma H.P. Peng J.B. Biochem. Biophys. Res. Commun. 2008; 375: 225-229Crossref PubMed Scopus (27) Google Scholar).Patients with PHA2 with WNK4 mutations develop hypercalciuria (5.Mayan H. Munter G. Shaharabany M. Mouallem M. Pauzner R. Holtzman E.J. Farfel Z. J. Clin. Endocrinol. Metab. 2004; 89: 4025-4030Crossref PubMed Scopus (85) Google Scholar). Studies using an expression system and in animals and humans, have provided compelling evidence that mutations of WNK4 that cause PHA2 increase the activity of NCC, causing Na+ retention and hypertension (39.Yang C.L. Angell J. Mitchell R. Ellison D.H. J. Clin. Invest. 2003; 111: 1039-1045Crossref PubMed Scopus (397) Google Scholar, 40.Wilson 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 (356) Google Scholar, 41.Lalioti M.D. Zhang J. Volkman H.M. Kahle K.T. Hoffmann K.E. Toka H.R. Nelson-Williams C. Ellison D.H. Flavell R. Booth C.J. Lu Y. Geller D.S. Lifton R.P. Nat. Genet. 2006; 38: 1124-1132Crossref PubMed Scopus (305) Google Scholar, 42.Yang S.S. Morimoto T. Rai T. Chiga M. Sohara E. Ohno M. Uchida K. Lin S.H. Moriguchi T. Shibuya H. Kondo Y. Sasaki S. Uchida S. Cell Metab. 2007; 5: 331-344Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Inhibition of NCC by thiazide diuretics or loss-of-function mutations of the NCC gene causes hypocalciuria (43.Ellison D.H. J. Am. Soc. Nephrol. 2003; 14: 538-540PubMed Google Scholar). Thus, hypercalciuria in patients with WNK4 mutations is probably, at least in part, due to the increase in the activity of NCC. In support of this idea, inhibition of NCC by thiazide diuretics ameliorates hypercalciuria in patients or animal models of PHA2 with WNK4 mutations (5.Mayan H. Munter G. Shaharabany M. Mouallem M. Pauzner R. Holtzman E.J. Farfel Z. J. Clin. Endocrinol. Metab. 2004; 89: 4025-4030Crossref PubMed Scopus (85) Google Scholar, 41.Lalioti M.D. Zhang J. Volkman H.M. Kahle K.T. Hoffmann K.E. Toka H.R. Nelson-Williams C. Ellison D.H. Flavell R. Booth C.J. Lu Y. Geller D.S. Lifton R.P. Nat. Genet. 2006; 38: 1124-1132Crossref PubMed Scopus (305) Google Scholar, 42.Yang S.S. Morimoto T. Rai T. Chiga M. Sohara E. Ohno M. Uchida K. Lin S.H. Moriguchi T. Shibuya H. Kondo Y. Sasaki S. Uchida S. Cell Metab. 2007; 5: 331-344Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, patents with PHA2 typically develop hypercalciuria before the onset of hypertension (5.Mayan H. Munter G. Shaharabany M. Mouallem M. Pauzner R. Holtzman E.J. Farfel Z. J. Clin. Endocrinol. Metab. 2004; 89: 4025-4030Crossref PubMed Scopus (85) Google Scholar). Moreover, transgenic mice overexpressing a bacterial artificial chromosome carrying the mouse NCC gene do not show hypercalciuria despite Na+ retention and hypertension, 3D. H. Ellison, personal communication. suggesting that other factors may also be involved. We and others (19.He G. Wang H.R. Huang S.K. Huang C.L. J. Clin. Invest. 2007; 117: 1078-1087Crossref PubMed Scopus (128) Google Scholar, 34.Kahle K.T. Wilson F.H. Leng Q. Lalioti M.D. O'Connell A.D. Dong K. Rapson A.K. MacGregor G.G. Giebisch G. Hebert S.C. Lifton R.P. Nat. Genet. 2003; 35: 372-376Crossref PubMed Scopus (340) Google Scholar) have shown that disease-causing mutant WNK4s exert a greater inhibition of ROMK than wild type WNK4, contributing to hyperkalemia in PHA2. This is due to an increased interaction of mutant WNK4's with intersectin (19.He G. Wang H.R. Huang S.K. Huang C.L. J. Clin. Invest. 2007; 117: 1078-1087Crossref PubMed Scopus (128) Google Scholar). Our present study shows that a disease mutant WNK4 exerts a greater inhibition of TRPV5 than wild type WNK4. These results raise the possibility that WNK4 regulation of TRPV5 may also be contributory to hypercalciuria in PHA2. Further studies of TRPV5 expression in PHA2 patients and/or animal models are required. IntroductionWNK (With-no-lysine (K)) kinases are large serine-threonine protein kinases characterized by an atypical placement of the catalytic lysine (1.Xu 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 (407) Google Scholar). Mammalian WNK kinases consist of four members, named WNK1–4 (1.Xu 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 (407) Google Scholar, 2.Veríssimo F. Jordan P. Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (222) Google Scholar, 3.Wilson 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 (1206) Google Scholar). Each WNK kinase is encoded by a separate gene. WNK1–4 are between 1,200- and 2,100-amino acids long. Each contains a ∼270-amino acid conserved kinase domain located near the amino terminus (1.Xu 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 (407) Google Scholar). In addition, they share an autoinhibitory domain, one to two coiled-coil domains, and multiple PXXP proline-rich motifs for potential protein-protein interaction (1.Xu 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 (407) Google Scholar, 2.Veríssimo F. Jordan P. Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (222) Google Scholar, 3.Wilson 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 (1206) Google Scholar). The remaining amino acid sequences of WNK1–4 are not conserved.Mutations in WNK1 and WNK4 cause the autosomal-dominant disease pseudohypoaldosteronism type 2 (PHA2) 2The abbreviations used are: PHA2pseudohypoaldosteronism type 2PKCprotein kinase CFLfull-lengthOAG1-oleoyl-acetyl-sn-glycerolPTHparathyroid hormoneGFPgreen fluorescent proteinNCCthiazide-sensitive NaCl co-transporterROMKrenal outer medullary K+ channelHEKhuman embryonic kidneyCCVclathrin-coated vesiclesiRNAsmall interference RNAPTH1Rtype 1 PTH receptorSH3Src homology-3WTwild type. (3.Wilson 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 (1206) Google Scholar). PHA2-causing mutations in WNK1 are large deletions within the first intron resulting in an increase in the abundance of transcript. Mutations in WNK4 are missense mutations in the coding sequence leading to altered protein function. PHA2 is characterized by hypertension and hyperkalemia (3.Wilson 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 (1206) Google Scholar, 4.Schambelan M. Sebastian A. Rector Jr., F.C. Kidney Int. 1981; 19: 716-727Abstract Full Text PDF PubMed Scopus (192) Google Scholar). Interestingly, patients with PHA2 caused by WNK4 mutations, but not patients with WNK1 mutations, also have abnormally high urinary calcium excretion (hypercalciuria) (5.Mayan H. Munter G. Shaharabany M. Mouallem M. Pauzner R. Holtzman E.J. Farfel Z. J. Clin. Endocrinol. Metab. 2004; 89: 4025-4030Crossref PubMed Scopus (85) Google Scholar, 6.Achard J.M. Warnock D.G. Disse-Nicodème S. Fiquet-Kempf B. Corvol P. Fournier A. Jeunemaitre X. Am. J. Med. 2003; 114: 495-498Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).WNK kinases are broadly distributed. WNK1 has multiple alternatively spliced isoforms, including an ubiquitous long isoform and a kidney-specific isoform lacking the kinase domain and predominantly expressed in distal tubules of the kidney (7.Delaloy 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 (138) Google Scholar, 8.O'Reilly M. Marshall E. Speirs H.J. Brown R.W. J. Am. Soc. Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (142) Google Scholar). WNK2 is predominantly expressed in heart, brain, and colon (7.Delaloy 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 (138) Google Scholar); WNK3 and WNK4 are mostly expressed in kidney, heart, and brain (7.Delaloy 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 (138) Google Scholar, 9.Holden S. Cox J. Raymond F.L. Gene. 2004; 335: 109-119Crossref PubMed Scopus (50) Google Scholar, 10.Kahle K.T. Gimenez I. Hassan H. Wilson F.H. Wong R.D. Forbush B. Aronson P.S. Lifton R.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2064-2069Crossref PubMed Scopus (149) Google Scholar). In addition, WNK4 is expressed in many other epithelial tissues (10.Kahle K.T. Gimenez I. Hassan H. Wilson F.H. Wong R.D. Forbush B. Aronson P.S. Lifton R.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2064-2069Crossref PubMed Scopus (149) Google Scholar). The distribution of WNK1 and WNK4 in the renal tubules, and that mutations in them cause hypertension and hyperkalemia, suggest that they play important roles in regulating renal ion transport. Indeed, WNK1 and WNK4 have been shown to regulate renal ion transport proteins, including paracellular tight junction proteins claudins; cation-chloride co-transporters NCC, KCl cotransporters, and Na+-K+-2Cl− cotransporters; epithelial Na+ channel; and renal K+ channel ROMK (11.Kahle K.T. Ring A.M. Lifton R.P. Annu. Rev. Physiol. 2008; 70: 329-355Crossref PubMed Scopus (195) Google Scholar, 12.Huang C.L. Yang S.S. Lin S.H. Curr. Opin. Nephrol. Hypertens. 2008; 17: 519-525Crossref PubMed Scopus (78) Google Scholar).TRPV5 (transient receptor potential vanilloid 5) channel is localized to the apical membrane of distal convoluted tubules and connecting tubules and functions as a gatekeeper for transcellular Ca2+ reabsorption in the kidney. Many hormones, including parathyroid hormone (PTH), regulate renal Ca2+ reabsorption at least partly via TRPV5 (13.Hoenderop J.G. Nilius B. Bindels R.J. Annu. Rev. Physiol. 2002; 64: 529-549Crossref PubMed Scopus (205) Google Scholar, 14.Lambers T.T. Bindels R.J. Hoenderop J.G. Kidney Int. 2006; 69: 650-654Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). One mechanism for PTH regulation of TRPV5 is by increasing the protein expression of TRPV5, providing a mechanism for long term regulation of renal Ca2+ reabsorption (15.van Abel M. Hoenderop J.G. van der Kemp A.W. Friedlaender M.M. van Leeuwen J.P. Bindels R.J. Kidney Int. 2005; 68: 1708-1721Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In addition, PTH can enhance renal Ca2+ reabsorption within 10–15 min of its administration. The mechanism of acute regulation of TRPV5 by PTH involves activation of protein kinase A and PKC (16.Hoenderop J.G. De Pont J.J. Bindels R.J. Willems P.H. Kidney Int. 1999; 55: 225-233Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17.de Groot T. Lee K. Langeslag M. Xi Q. Jalink K. Bindels R.J. Hoenderop J.G. J. Am. Soc. Nephrol. 2009; 20: 1693-1704Crossref PubMed Scopus (129) Google Scholar). Recently, we reported that TRPV5 undergoes constitutive caveolin-dependent endocytosis and that activation of PKC inhibits this process causing accumulation of the channel at the cell surface (18.Cha S.K. Wu T. Huang C.L. Am. J. Physiol. Renal Physiol. 2008; 294: F1212-F1221Crossref PubMed Scopus (90) Google Scholar). This inhibition of caveolar endocytosis of TRPV5 via PKC may be one of the mechanisms for acute regulation of the channel by PTH. In the present study, we report that WNK4 stimulates caveola-mediated endocytosis of TRPV5 and thereby increases the range of regulation of the channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca2+ reabsorption by PTH or tissue kallikrein in vivo.
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