Chloride Channel (Clc)-5 Is Necessary for Exocytic Trafficking of Na+/H+ Exchanger 3 (NHE3)
2011; Elsevier BV; Volume: 286; Issue: 26 Linguagem: Inglês
10.1074/jbc.m111.224998
ISSN1083-351X
AutoresZhihong Lin, Shi Jin, Xiaohong Duan, Tong Wang, Sabrina V. Martini, Phuson Hulamm, Boyoung Cha, Ann L. Hubbard, Mark Donowitz, Sandra E. Guggino,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knock-out mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca2+ (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO3−, measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3. ClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knock-out mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca2+ (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO3−, measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3. IntroductionSignificant information about the function of the intracellular voltage-dependent chloride/proton exchanger ClC-5 5The abbreviations used are: ClCchloride channelClC-5protein in human, mouse, and opossum kidney cell lineCLCN5human geneClcn5mouse geneOK cellsopossum kidney cellsNHE3Na+/H+ exchanger 3Nhe3mouse Na+/H+ exchange 3Npt2amouse sodium-phosphate cotransporter type IIaPTHparathyroid hormoneDEXdexamethasonescscrambled constructshshRNAKDknockdownBBbrush borderNHSN-hydroxysuccinimideTMAtetramethylammoniumROIregions of interestshRNAishRNA interference. has come from the characterization of patients with Dent disease and knock-out mouse models of Clcn5 that have the renal characteristics of this disease (1Pook M.A. Wrong O. Wooding C. Norden A.G. Feest T.G. Thakker R.V. Hum. Mol. Genet. 1993; 2: 2129-2134Crossref PubMed Scopus (88) Google Scholar, 2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar, 3Wang S.S. Devuyst O. Courtoy P.J. Wang X.T. Wang H. Wang Y. Thakker R.V. Guggino S. Guggino W.B. Hum. Mol. Genet. 2000; 9: 2937-2945Crossref PubMed Scopus (271) Google Scholar, 4Cebotaru V. Kaul S. Devuyst O. Cai H. Racusen L. Guggino W.B. Guggino S.E. Kidney Int. 2005; 68: 642-652Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 5Guggino S.E. Nat. Clin. Pract. Nephrol. 2007; 3: 449-455Crossref PubMed Scopus (24) Google Scholar). Dent disease is caused by mutations of CLCN5 (6Fisher S.E. Black G.C. Lloyd S.E. Hatchwell E. Wrong O. Thakker R.V. Craig I.W. Hum. Mol. Genet. 1994; 3: 2053-2059PubMed Google Scholar, 7Lloyd S.E. Pearce S.H. Günther W. Kawaguchi H. Igarashi T. Jentsch T.J. Thakker R.V. J. Clin. 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Rehfeldt A. Steinmeyer K. Ann. N.Y. Acad. Sci. 1993; 707: 285-293Crossref PubMed Scopus (16) Google Scholar, 13Thiemann A. Gründer S. Pusch M. Jentsch T.J. Nature. 1992; 356: 57-60Crossref PubMed Scopus (502) Google Scholar, 14Zdebik A.A. Zifarelli G. Bergsdorf E.Y. Soliani P. Scheel O. Jentsch T.J. Pusch M. J. Biol. Chem. 2008; 283: 4219-4227Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 15Accardi A. Walden M. Nguitragool W. Jayaram H. Williams C. Miller C. J. Gen. Physiol. 2005; 126: 563-570Crossref PubMed Scopus (169) Google Scholar, 16Scheel O. Zdebik A.A. Lourdel S. Jentsch T.J. Nature. 2005; 436: 424-427Crossref PubMed Scopus (403) Google Scholar). ClC-5 is expressed in the proximal tubule, the thick ascending limb, and the intercalated cells of the collecting duct in the human nephron (10Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S. Am. J. Physiol. Renal Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar, 17Devuyst O. Christie P.T. Courtoy P.J. Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (249) Google Scholar). In the mouse proximal tubule, immunofluorescence results indicate that ClC-5 is colocalized with the H+-ATPase, indicating an endosomal location (10Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S. Am. J. Physiol. Renal Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar, 18Günther W. Lüchow A. Cluzeaud F. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8075-8080Crossref PubMed Scopus (380) Google Scholar). The pH of transferrin-positive endosomes of cultured cells from Clcn5 knock-out proximal tubule cells is more alkaline than that observed in WT controls (19Hara-Chikuma M. Wang Y. Guggino S.E. Guggino W.B. Verkman A.S. Biochem. Biophys. Res. Commun. 2005; 329: 941-946Crossref PubMed Scopus (107) Google Scholar). The role of ClC-5 is thought to be moving chloride as a counterion to the proton that is pumped by the H+-ATPase (17Devuyst O. Christie P.T. Courtoy P.J. Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (249) Google Scholar, 20Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pflugers Arch. 1999; 437: 783-795Crossref PubMed Scopus (288) Google Scholar, 21Günther W. Piwon N. Jentsch T.J. Pflugers Arch. 2003; 445: 456-462Crossref PubMed Scopus (163) Google Scholar), although a role for Cl− beyond charge has been suggested recently (22Novarino G. Weinert S. Rickheit G. Jentsch T.J. Science. 2010; 328: 1398-1401Crossref PubMed Scopus (125) Google Scholar).Detailed studies of trafficking proteins in ClC-5-negative tissues have not been reported. Piwon et al. (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar) demonstrated that the surface expression of Nhe3 and Npt2a, which are transporters regulated by trafficking and normally reside in the renal proximal tubule brush border membrane, was significantly decreased in the Clcn5 KO mouse, and total Npt2a expression was decreased. Likewise, the proximal tubule scavenger receptor megalin was decreased in total amount and even further reduced at the apical surface (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar, 23Christensen E.I. Devuyst O. Dom G. Nielsen R. Van der Smissen P. Verroust P. Leruth M. Guggino W.B. Courtoy P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8472-8477Crossref PubMed Scopus (277) Google Scholar). These results suggested that apical domain trafficking was probably abnormal in the Clcn5 KO proximal tubule. However, no systematic evaluation of the role of ClC-5 in apical domain endocytosis/exocytosis has been reported. In mice fed with a phosphate-depleted diet, parathyroid hormone-induced NHE3 endocytosis was slowed in the Clcn5 KO proximal tubule, indicating that the endocytosis in intact mice is regulated in a ClC-5-dependent manner (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). However, slowed endocytosis would not explain the decreased steady-state surface distribution of Nhe3 in the Clcn5 KO proximal tubule. Consequently, Jentsch and co-workers (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar) hypothesized that in the Clcn5 KO mouse this was caused by a high concentration of tubular parathyroid hormone (PTH) caused by decreased reabsorption of PTH via megalin. Based on these observations, we believe that, due to the presence of both primary and secondary effects of ClC-5 in animal models, it may not be possible to fully dissect the direct role of ClC-5 in trafficking of apical membrane proteins by only using animal models. For example, in Clcn5 KO mice, changed luminal tubule levels of PTH might mask the direct function of ClC-5 on NHE3 surface expression. To further understand the effects of ClC-5 on NHE3 function and distribution, we characterized the ClC-5 dependence of basal and regulated Nhe3 activity in mouse proximal tubules and then studied basal and stimulated apical distribution and rates of endocytosis and exocytosis of NHE3 in the proximal tubule-like opossum kidney (OK) cell line using short hairpin RNA (shRNA) to silence ClC-5. We show evidence that ClC-5 is necessary for NHE3 exocytosis but not endocytosis and that decreased ClC-5 lowers the amount of NHE3 on the proximal tubule apical membrane in the absence of elevated luminal PTH.DISCUSSIONSeveral striking abnormalities have been documented in the proximal tubule of the Clcn5 KO mice. In the face of normal gross structure in the young mice studied (4Cebotaru V. Kaul S. Devuyst O. Cai H. Racusen L. Guggino W.B. Guggino S.E. Kidney Int. 2005; 68: 642-652Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), there are markedly reduced apical amounts of Nhe3, Npt2a, and megalin along with reduced total Npt2a and megalin (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). Although the increased urinary loss of sodium and phosphate is explained by the reduced apical expression of Nhe3 and Npt2a, the major proteins involved in the bulk of Na+ and phosphate absorption from the kidney, the mechanisms underlying the abnormal apical distribution and consequently the reduced function of the transporters have not been determined.We used two epithelial models to determine the consequences in renal proximal tubule of either absent or greatly reduced expression of ClC-5 on the activity of a transport protein, NHE3, which cycles between the endosomal system and the brush border under basal conditions and is acutely regulated primarily by changes in the rates of its endocytosis and/or exocytosis (35Donowitz M. Li X. Physiol. Rev. 2007; 87: 825-872Crossref PubMed Scopus (153) Google Scholar). Both in native murine proximal tubule and in a non-malignant proximal tubule cell line (OK cells), our results showed that ClC-5 was necessary to maintain basal NHE3 activity as well as for stimulation of NHE3 by short time exposure to DEX, whereas ClC-5 was not necessary for acute cAMP, elevated Ca2+, and PTH inhibition of NHE3. These changes in transport were all due to abnormal trafficking of NHE3 when examined in polarized OK cells with reduced NHE3 surface expression under basal conditions due to reduced exocytosis and a similarly reduced DEX stimulation due to failure of DEX to stimulate exocytosis of NHE3 in the absence of ClC-5. In contrast to a role for ClC-5 in basal and stimulated exocytosis, both cAMP and elevated Ca2+, both of which inhibit NHE3 at least in part by stimulating its endocytosis, caused normal percent inhibition of NHE3 in ClC-5-null mouse proximal tubule. Also PTH, which affects NHE3 by elevating both Ca2+ and cAMP in proximal tubule, caused a similar rate of endocytosis of NHE3 in WT and OK cells in which ClC-5 was knocked down. Moreover, given the prolonged half-life of NHE3 of ∼14 h (36Cavet M.E. Akhter S. Murtazina R. Sanchez de Medina F. Tse C.M. Donowitz M. Am. J. Physiol. Cell Physiol. 2001; 281: C2039-C2048Crossref PubMed Google Scholar), the changes described here, which occurred over much shorter times, indicate that changes in brush border (BB) NHE3 were not related to changes in plasma membrane delivery of newly synthesized NHE3. We conclude that ClC-5 is necessary for basal and DEX-stimulated exocytosis but not for endocytosis of NHE3 in these epithelial cells.This is the first detailed examination in renal proximal tubule of the dependence on ClC-5 of trafficking of Nhe3 and examination of effects on exocytosis, although the initial study of the renal phenotype of Clcn5 KO mice identified a role for ClC-5 in proximal tubule trafficking (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). There were multiple reasons for attention to be paid first to potential effects of ClC-5 on endocytosis. Clcn5 KO mouse renal cortex exhibited a reduced BB amount of the receptor protein megalin along with reduced total expression of megalin; megalin is the major renal proximal tubule BB receptor for endocytosis (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar, 23Christensen E.I. Devuyst O. Dom G. Nielsen R. Van der Smissen P. Verroust P. Leruth M. Guggino W.B. Courtoy P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8472-8477Crossref PubMed Scopus (277) Google Scholar). Also, the clinical manifestations of Dent disease are consistent with reduced proximal tubule endocytosis; there is reduced renal absorption of glucose, amino acids, and low molecular weight proteins, accounting for the glucosuria, aminoaciduria, and low molecular weight proteinuria that are part of this syndrome (5Guggino S.E. Nat. Clin. Pract. Nephrol. 2007; 3: 449-455Crossref PubMed Scopus (24) Google Scholar). Moreover, knocking out ClC-5 reduced proximal tubule receptor-mediated endocytosis (β2-microglobulin and albumin) and fluid phase endocytosis (dextran and horseradish peroxidase) (37Wang Y. Cai H. Cebotaru L. Hryciw D.H. Weinman E.J. Donowitz M. Guggino S.E. Guggino W.B. Am. J. Physiol. Renal Physiol. 2005; 289: F850-F862Crossref PubMed Scopus (55) Google Scholar). Lastly, because NaPi2a is regulated only by endocytosis and does not undergo exocytosis, it was logical that emphasis would be on the role of ClC-5 in its endocytosis (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar).However, it was also recognized that reduced endocytosis would increase, not decrease, BB NHE3 and NaPi2a; this is the opposite of what was observed (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). Thus, it was suggested that this aspect of the pathophysiology of the lack of ClC-5 in KO mice kidney is caused by reduced PTH endocytosis/increased luminal PTH because this process is dependent on surface megalin. Thus, reduced surface NHE3 and NaPi2a were considered secondary to reduced PTH endocytosis (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). However, there were inconsistencies in the data supporting this hypothesis. For instance, other animal models in which megalin (38Bachmann S. Schlichting U. Geist B. Mutig K. Petsch T. Bacic D. Wagner C.A. Kaissling B. Biber J. Murer H. Willnow T.E. J. Am. Soc. Nephrol. 2004; 15: 892-900Crossref PubMed Scopus (83) Google Scholar) and its chaperone receptor-associated protein (39Bacic D. Capuano P. Gisler S.M. Pribanic S. Christensen E.I. Biber J. Loffing J. Kaissling B. Wagner C.A. Murer H. Pflugers Arch. 2003; 446: 475-484Crossref PubMed Scopus (38) Google Scholar) were knocked out had unaltered surface expression of NHE3. These contradictory results led us to speculate that, besides the low surface expression of megalin and consequently high concentration of tubular PTH, additional mechanism(s) might contribute to the reduced NHE3 surface expression observed in the animal model lacking ClC-5.Recent studies focusing on molecular mechanisms of ClC-5 actions indicated that ClC-5 might have a direct role in regulation of NHE3 trafficking. Drugs that abrogate vacuolar acidification (as occurs with ClC-5 KO) did not affect the rate of endocytic uptake but inhibited recycling or transfer to lysosomes (40Devuyst O. Jouret F. Auzanneau C. Courtoy P.J. Nephron Physiol. 2005; 99: p69-p73Crossref PubMed Scopus (39) Google Scholar). In addition, ClC-5 directly interacts with NHERF2, a PDZ domain-containing protein that is essential for both endocytosis and exocytosis of NHE3 (32Hryciw D.H. Ekberg J. Ferguson C. Lee A. Wang D. Parton R.G. Pollock C.A. Yun C.C. Poronnik P. J. Biol. Chem. 2006; 281: 16068-16077Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 35Donowitz M. Li X. Physiol. Rev. 2007; 87: 825-872Crossref PubMed Scopus (153) Google Scholar, 41Lee-Kwon W. Kawano K. Choi J.W. Kim J.H. Donowitz M. J. Biol. Chem. 2003; 278: 16494-16501Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Moreover, NHE3 has a function similar to that of ClC-5 in respect to acidification of some endosomes (37Wang Y. Cai H. Cebotaru L. Hryciw D.H. Weinman E.J. Donowitz M. Guggino S.E. Guggino W.B. Am. J. Physiol. Renal Physiol. 2005; 289: F850-F862Crossref PubMed Scopus (55) Google Scholar). Finally, in proximal tubules and intercalated ducts in several patients with Dent disease, the H+-ATPase appeared on the basolateral membrane rather than in the normal BB location (42Moulin P. Igarashi T. Van der Smissen P. Cosyns J.P. Verroust P. Thakker R.V. Scheinman S.J. Courtoy P.J. Devuyst O. Kidney Int. 2003; 63: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). This appears to be an example of altered polarity of a specific protein, whereas the polar distribution of multiple other proteins was normal (42Moulin P. Igarashi T. Van der Smissen P. Cosyns J.P. Verroust P. Thakker R.V. Scheinman S.J. Courtoy P.J. Devuyst O. Kidney Int. 2003; 63: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). These observations suggested that ClC-5, NHE3, and H+-ATPase might localize (at least partially) in a common compartment, and ClC-5 might regulate NHE3 trafficking. This evidence led us to examine in detail the role of ClC-5 in the trafficking of NHE3. Our data showed that under basal conditions (which is in the absence of high luminal PTH) surface expression of NHE3 is decreased in OK cells with reduced expression of ClC-5, which is similar to what is seen with megalin, suggesting that it is likely that ClC-5 plays a similar role in the regulation of trafficking of NHE3 and megalin. Furthermore, our direct measurements of NHE3 exocytosis and endocytosis demonstrate that, at least in OK cells, it was exocytosis and not endocytosis of NHE3 that depended on ClC-5.The similarity of the ClC-5 dependence of NHE3 trafficking in intact mouse proximal tubules and OK cells is surprising because it has been suggested that in proximal tubules NHE3 inhibition by trafficking from the microvilli is primarily to a domain just superior to the intermicrovillar clefts and does not cycle to the clathrin-coated pit area and endosomes as occurs in other epithelia, including Caco-2 cells and OK cells (43McDonough A.A. Biemesderfer D. Curr. Opin. Nephrol. Hypertens. 2003; 12: 533-541Crossref PubMed Scopus (41) Google Scholar). Nonetheless, we suggest that ClC-5 is serving the same function in exocytosis in both proximal tubule and OK cells, although the nature of that comportment has not been identified in either.Concerning the mechanism by which reduced or absent ClC-5 reduces trafficking, it has been assumed that ClC-5, acting as a Cl−/H+ antiporter, neutralizes the electropositivity generated by the H+-ATPase pumping H+ ions into intracellular vesicles, thus allowing further acidification of the intracellular organelle in which both occur. Importantly, that this was the only role for ClC-5 was recently challenged by the demonstration of a role for Cl− separate from charge neutralization as well as a role for intraorganellar cations in establishing intracellular organelle acid pH (22Novarino G. Weinert S. Rickheit G. Jentsch T.J. Science. 2010; 328: 1398-1401Crossref PubMed Scopus (125) Google Scholar, 44Steinberg B.E. Huynh K.K. Brodovitch A. Jabs S. Stauber T. Jentsch T.J. Grinstein S. J. Cell Biol. 2010; 189: 1171-1186Crossref PubMed Scopus (198) Google Scholar). Of note, the identity of the intracellular organelle involved in NHE3 exocytosis has not been determined. Our results suggest that an NHE3-containing recycling endosome is likely to be affected by ClC-5 reduction.As an alternative explanation to direct effects of ClC-5 on intracellular organelle function, because the NHERF multi-PDZ domain scaffolding proteins are known to be involved in inhibition of NHE3 by cAMP and Ca2+ and in dexamethasone stimulation (31Bobulescu I.A. Dwarakanath V. Zou L. Zhang J. Baum M. Moe O.W. Am. J. Physiol. Renal Physiol. 2005; 289: F685-F691Crossref PubMed Scopus (76) Google Scholar, 41Lee-Kwon W. Kawano K. Choi J.W. Kim J.H. Donowitz M. J. Biol. Chem. 2003; 278: 16494-16501Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 45Cinar A. Chen M. Riederer B. Bachmann O. Wiemann M. Manns M. Kocher O. Seidler U. J. Physiol. 2007; 581: 1235-1246Crossref PubMed Scopus (55) Google Scholar, 46Sarker R. Valkhoff V.E. Zachos N.C. Lin R. Cha B. Chen T.E. Guggino S. Zizak M. de Jonge H. Hogema B. Donowitz M. Am. J. Physiol. Cell Physiol. 2011; 300: C771-C782Crossref PubMed Scopus (41) Google Scholar), it was determined whether there were any changes in the level of these proteins in the Clcn5 KO. By immunoblots, there was no change in the amounts of NHERF1, -2, and -3 in Clcn5 KO lysates of renal cortex (data not shown), indicating that changes in their levels did not account for abnormal Nhe3 activity in the Clcn5 KO.A surprising result of these studies was the apparently normal rate of both basal and stimulated NHE3 endocytosis in proximal tubule of both Clcn5 KO and OK cells with ClC-5 expression knocked down. As reviewed above, there is strong evidence that multiple examples of endocytosis are ClC-5-dependent in renal proximal tubule, although many may be due to the reduced apical megalin and/or to the consequently elevated luminal PTH (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). However, there has recently been recognition that there are many forms of apical endocytosis in addition to receptor-mediated/clathrin-dependent and lipid raft-dependent forms. The mechanisms by which NHE3 is endocytosed have been shown to include clathrin-dependent and clathrin-independent forms, including via lipid rafts (47Li X. Galli T. Leu S. Wade J.B. Weinman E.J. Leung G. Cheong A. Louvard D. Donowitz M. J. Physiol. 2001; 537: 537-552Crossref PubMed Scopus (118) Google Scholar, 48Murtazina R. Kovbasnjuk O. Donowitz M. Li X. J. Biol. Chem. 2006; 281: 17845-17855Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 49Chow C.W. Khurana S. Woodside M. Grinstein S. Orlowski J. J. Biol. Chem. 1999; 274: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Thus, it is possible that NHE3 endocytosis is not affected by reduced ClC-5, even though ClC-5 affects multiple forms of endocytosis, because NHE3 is taken up by some ClC-5-independent endocytic pathway(s).In summary, these studies have established a role for ClC-5 in exocytosis of at least one renal proximal tubule apical membrane transporter (NHE3) as well as showing that although ClC-5 is necessary for multiple example of endocytosis it is not necessary for endocytosis of all proximal tubule proteins that are regulated partially by stimulated endocytosis. Demonstration of the importance of ClC-5 in the regulation of NHE3 trafficking should allow determination of the specificity of the dependence on ClC-5 in regulated exocytosis and endocytosis of additional apical proteins as well as providing a probe to isolate and identify the intracellular compartment in which ClC-5 is involved in NHE3 exocytosis. IntroductionSignificant information about the function of the intracellular voltage-dependent chloride/proton exchanger ClC-5 5The abbreviations used are: ClCchloride channelClC-5protein in human, mouse, and opossum kidney cell lineCLCN5human geneClcn5mouse geneOK cellsopossum kidney cellsNHE3Na+/H+ exchanger 3Nhe3mouse Na+/H+ exchange 3Npt2amouse sodium-phosphate cotransporter type IIaPTHparathyroid hormoneDEXdexamethasonescscrambled constructshshRNAKDknockdownBBbrush borderNHSN-hydroxysuccinimideTMAtetramethylammoniumROIregions of interestshRNAishRNA interference. has come from the characterization of patients with Dent disease and knock-out mouse models of Clcn5 that have the renal characteristics of this disease (1Pook M.A. Wrong O. Wooding C. Norden A.G. Feest T.G. Thakker R.V. Hum. Mol. Genet. 1993; 2: 2129-2134Crossref PubMed Scopus (88) Google Scholar, 2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. 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Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (249) Google Scholar). In the mouse proximal tubule, immunofluorescence results indicate that ClC-5 is colocalized with the H+-ATPase, indicating an endosomal location (10Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S. Am. J. Physiol. Renal Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar, 18Günther W. Lüchow A. Cluzeaud F. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8075-8080Crossref PubMed Scopus (380) Google Scholar). The pH of transferrin-positive endosomes of cultured cells from Clcn5 knock-out proximal tubule cells is more alkaline than that observed in WT controls (19Hara-Chikuma M. Wang Y. Guggino S.E. Guggino W.B. Verkman A.S. Biochem. Biophys. Res. Commun. 2005; 329: 941-946Crossref PubMed Scopus (107) Google Scholar). The role of ClC-5 is thought to be moving chloride as a counterion to the proton that is pumped by the H+-ATPase (17Devuyst O. Christie P.T. Courtoy P.J. Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (249) Google Scholar, 20Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pflugers Arch. 1999; 437: 783-795Crossref PubMed Scopus (288) Google Scholar, 21Günther W. Piwon N. Jentsch T.J. Pflugers Arch. 2003; 445: 456-462Crossref PubMed Scopus (163) Google Scholar), although a role for Cl− beyond charge has been suggested recently (22Novarino G. Weinert S. Rickheit G. Jentsch T.J. Science. 2010; 328: 1398-1401Crossref PubMed Scopus (125) Google Scholar).Detailed studies of trafficking proteins in ClC-5-negative tissues have not been reported. Piwon et al. (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar) demonstrated that the surface expression of Nhe3 and Npt2a, which are transporters regulated by trafficking and normally reside in the renal proximal tubule brush border membrane, was significantly decreased in the Clcn5 KO mouse, and total Npt2a expression was decreased. Likewise, the proximal tubule scavenger receptor megalin was decreased in total amount and even further reduced at the apical surface (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar, 23Christensen E.I. Devuyst O. Dom G. Nielsen R. Van der Smissen P. Verroust P. Leruth M. Guggino W.B. Courtoy P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8472-8477Crossref PubMed Scopus (277) Google Scholar). These results suggested that apical domain trafficking was probably abnormal in the Clcn5 KO proximal tubule. However, no systematic evaluation of the role of ClC-5 in apical domain endocytosis/exocytosis has been reported. In mice fed with a phosphate-depleted diet, parathyroid hormone-induced NHE3 endocytosis was slowed in the Clcn5 KO proximal tubule, indicating that the endocytosis in intact mice is regulated in a ClC-5-dependent manner (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar). However, slowed endocytosis would not explain the decreased steady-state surface distribution of Nhe3 in the Clcn5 KO proximal tubule. Consequently, Jentsch and co-workers (2Piwon N. Günther W. Schwake M. Bösl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (480) Google Scholar) hypothesized that in the Clcn5 KO mouse this was caused by a high concentration of tubular parathyroid hormone (PTH) caused by decreased reabsorption of PTH via megalin. Based on these observations, we believe that, due to the presence of both primary and secondary effects of ClC-5 in animal models, it may not be possible to fully dissect the direct role of ClC-5 in trafficking of apical membrane proteins by only using animal models. For example, in Clcn5 KO mice, changed luminal tubule levels of PTH might mask the direct function of ClC-5 on NHE3 surface expression. To further understand the effects of ClC-5 on NHE3 function and distribution, we characterized the ClC-5 dependence of basal and regulated Nhe3 activity in mouse proximal tubules and then studied basal and stimulated apical distribution and rates of endocytosis and exocytosis of NHE3 in the proximal tubule-like opossum kidney (OK) cell line using short hairpin RNA (shRNA) to silence ClC-5. We show evidence that ClC-5 is necessary for NHE3 exocytosis but not endocytosis and that decreased ClC-5 lowers the amount of NHE3 on the proximal tubule apical membrane in the absence of elevated luminal PTH.
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