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Activation of Swelling-activated Chloride Current by Tumor Necrosis Factor-α Requires ClC-3-dependent Endosomal Reactive Oxygen Production

2010; Elsevier BV; Volume: 285; Issue: 30 Linguagem: Inglês

10.1074/jbc.m109.099838

ISSN

1083-351X

Autores

James J. Matsuda, Mohammed Filali, Jessica G. Moreland, Francis J. Miller, Fred S. Lamb,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

ClC-3 is a Cl−/H+ antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (IClswell), but overexpression of ClC-3 can activate currents that resemble IClswell. Because H2O2 activates IClswell directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates IClswell. Whole-cell perforated patch clamp methods were used to record Cl− currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-α (TNF-α) (10 ng/ml) activated outwardly rectifying Cl− currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 μm) and by hypertonicity (340 mosm) identified them as IClswell. IClswell was also activated by H2O2 (500 μm), and the effect of TNF-α was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced IClswell in ClC-3 null cells in the absence of swelling or TNF-α, and this effect was also blocked by catalase. IClswell activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-α-mediated activation of IClswell. Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H2O2-dependent activation of IClswell, but this effect was not ClC-3- or Rab5-dependent. Thus, activation of IClswell by TNF-α requires ClC-3-dependent endosomal H2O2 production. This demonstrates a functional link between two distinct anion currents, ClC-3 and IClswell. ClC-3 is a Cl−/H+ antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (IClswell), but overexpression of ClC-3 can activate currents that resemble IClswell. Because H2O2 activates IClswell directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates IClswell. Whole-cell perforated patch clamp methods were used to record Cl− currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-α (TNF-α) (10 ng/ml) activated outwardly rectifying Cl− currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 μm) and by hypertonicity (340 mosm) identified them as IClswell. IClswell was also activated by H2O2 (500 μm), and the effect of TNF-α was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced IClswell in ClC-3 null cells in the absence of swelling or TNF-α, and this effect was also blocked by catalase. IClswell activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-α-mediated activation of IClswell. Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H2O2-dependent activation of IClswell, but this effect was not ClC-3- or Rab5-dependent. Thus, activation of IClswell by TNF-α requires ClC-3-dependent endosomal H2O2 production. This demonstrates a functional link between two distinct anion currents, ClC-3 and IClswell. IntroductionClC-3 is a member of the CLC family of Cl− channels and Cl−/H+ antiporters. When expressed in HEK293 cells, it behaves as an antiporter at neutral pH (1.Matsuda J.J. Filali M.S. Volk K.A. Collins M.M. Moreland J.G. Lamb F.S. Am. J. Physiol. Cell Physiol. 2008; 294: C251-C262Crossref PubMed Scopus (73) Google Scholar). However, at high extracellular proton concentrations, transport becomes uncoupled, and ClC-3 behaves as an anion-selective pore (2.Matsuda J.J. Filali M.S. Collins M.M. Volk K.A. Lamb F.S. J. Biol. Chem. 2010; 285: 2569-2579Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This mode of ClC-3 activity may account for the native acid-activated current in HEK293 cells (3.Lambert S. Oberwinkler J. J. Physiol. 2005; 567: 191-213Crossref PubMed Scopus (62) Google Scholar). The basic biophysical properties of ClC-3 currents are similar under both coupled and uncoupled conditions. They display very sharp outward rectification and time-dependent activation. Mutation of the extracellular fast gate (E224A) alters both of these characteristics, and inhibition of wild type ClC-3 current by alkanethiolation with methanethiosulfonate can be prevented by deletion of cysteine residues in the first extracellular loop of the protein. These properties clearly associate the currents induced by ClC-3 expression with ClC-3 protein (1.Matsuda J.J. Filali M.S. Volk K.A. Collins M.M. Moreland J.G. Lamb F.S. Am. J. Physiol. Cell Physiol. 2008; 294: C251-C262Crossref PubMed Scopus (73) Google Scholar, 2.Matsuda J.J. Filali M.S. Collins M.M. Volk K.A. Lamb F.S. J. Biol. Chem. 2010; 285: 2569-2579Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).However, heterologous expression of ClC-3 has also been repeatedly associated with a less sharply outwardly rectifying anion current that displays time-dependent inactivation instead of activation. Currents induced in NIH3T3 cells by plasmid-mediated ClC-3 overexpression appeared indistinguishable from the swelling-activated anion current (IClswell), 2The abbreviations used are: IClswellswelling-induced anion currentCamKIIcalcium-calmodulin kinase IIGFPgreen florescent proteinH2O2hydrogen peroxideROSreactive oxygen speciesTNF-αtumor necrosis factor-αVSMvascular smooth muscleWTwild typeILinterleukinEGFepidermal growth factorpFpicofarad. and it was suggested that ClC-3 was an IClswell (4.Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). This idea was supported by a demonstration that anti-ClC-3 antibodies placed within the patch pipette block IClswell activation under hypotonic conditions (5.Duan D. Zhong J. Hermoso M. Satterwhite C.M. Rossow C.F. Hatton W.J. Yamboliev I. Horowitz B. Hume J.R. J. Physiol. 2001; 531: 437-444Crossref PubMed Scopus (82) Google Scholar, 6.Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W.J. Duan D. Horowitz T.L. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (82) Google Scholar) and by the fact that N-terminal deletion mutants of ClC-3 yielded constitutively active, IClswell-like currents (7.Rossow C.F. Duan D. Hatton W.J. Britton F. Hume J.R. Horowitz B. Acta. Physiol. 2006; 187: 5-19Crossref PubMed Scopus (13) Google Scholar). Unfortunately, other investigators have either been unable to express ClC-3 currents (8.Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 9.Jentsch T.J. Günther W. Pusch M. Schwappach B. J. Physiol. 1995; 482: 19S-25SCrossref PubMed Scopus (201) Google Scholar, 10.Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) or have observed currents that were insensitive to changes in cell volume (10.Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 11.Huang P. Liu J. Di A. Robinson N.C. Musch M.W. Kaezel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12.Ogura T. Furukawa T. Toyozaki T. Yamada K. Zheng Y.J. Katayama Y. Nakaya H. Inagaki N. FASEB J. 2002; 16: 863-865Crossref PubMed Scopus (73) Google Scholar, 13.Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar, 14.Li X. Shimada K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; 275: 35994-35998Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Importantly, ClC-3 null cells have consistently exhibited normal appearing whole-cell (15.Gong W. Xu H. Shimizu T. Morishima S. Tanabe S. Tachibe T. Uchida S. Sasaki S. Okada Y. Cell Physiol. Biochem. 2004; 14: 213-224Crossref PubMed Scopus (65) Google Scholar, 16.Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bösl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 17.Arreola J. Begenisich T. Nehrke K. Nguyen H.V. Park K. Richardson L. Yang B. Schutte B.C. Lamb F.S. Melvin J.E. J. Physiol. 2002; 545: 207-216Crossref PubMed Scopus (87) Google Scholar) and single channel (18.Wang J. Xu H. Morishima S. Tanabe S. Jishage K. Uchida S. Sasaki S. Okada Y. Shimizu T. Jpn. J. Physiol. 2005; 55: 379-383Crossref PubMed Scopus (25) Google Scholar) IClswell currents. The regulation of IClswell was, however, clearly altered in ClC-3 null cells (6.Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W.J. Duan D. Horowitz T.L. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (82) Google Scholar). Other investigators have identified a calcium-calmodulin kinase II (CamKII)-dependent Cl− current in cells expressing ClC-3 (11.Huang P. Liu J. Di A. Robinson N.C. Musch M.W. Kaezel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These currents, which look like IClswell, were not responsive to changes in cell volume and were absent in smooth muscle cells (19.Robinson N.C. Huang P. Kaetzel M.A. Lamb F.S. Nelson D.J. J. Physiol. 2004; 556: 353-368Crossref PubMed Scopus (68) Google Scholar) and hippocampal neurons from ClC-3 null mice (20.Wang X.Q. Deriy L.V. Foss S. Huang P. Lamb F.S. Kaetzel M.A. Bindokas V. Marks J.D. Nelson D.J. Neuron. 2006; 52: 321-333Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We hypothesized that these apparently disparate data might be reconciled if ClC-3 is required for regulation of IClswell.Multiple lines of evidence have established a relationship between ClC-3, NADPH oxidase activity, and signaling via reactive oxygen species (ROS). Nox1 and ClC-3 co-localize to early endosomes in vascular smooth muscle (VSM) cells, and both are required for tumor necrosis factor (TNF-α) and interleukin (IL-1β)-dependent ROS generation and for the subsequent activation of the transcription factor nuclear factor κB (NF-κB) (21.Miller Jr., F.J. Filali M. Huss G.J. Stanic B. Chamseddine A. Barna T.J. Lamb F.S. Circ. Res. 2007; 101: 663-671Crossref PubMed Scopus (172) Google Scholar). Intracellular but not extracellular Nox2 activity in neutrophils also requires ClC-3. The intracellular oxidative burst (22.Moreland J.G. Davis A.P. Bailey G. Nauseef W.M. Lamb F.S. J. Biol. Chem. 2006; 281: 12277-12288Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), ROS-dependent endotoxin signaling (23.Moreland J.G. Davis A.P. Matsuda J.J. Hook J.S. Bailey G. Nauseef W.M. Lamb F.S. J. Biol. Chem. 2007; 282: 33958-33967Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and shape change during chemotaxis (24.Volk A.P. Heise C.K. Hougen J.L. Artman C.M. Volk K.A. Wessels D. Soll D.R. Nauseef W.M. Lamb F.S. Moreland J.G. J. Biol. Chem. 2008; 283: 34315-34326Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) are all impaired in ClC-3-deficient neutrophils. We have proposed that ClC-3 provides charge neutralization of electron flow through NADPH oxidase, into intracellular vesicles (1.Matsuda J.J. Filali M.S. Volk K.A. Collins M.M. Moreland J.G. Lamb F.S. Am. J. Physiol. Cell Physiol. 2008; 294: C251-C262Crossref PubMed Scopus (73) Google Scholar, 25.Lamb F.S. Moreland J.G. Miller Jr., F.J. Antioxid. Redox Signal. 2009; 11: 1335-1347Crossref PubMed Scopus (36) Google Scholar), but this relationship remains to be directly demonstrated experimentally.H2O2 activates IClswell under isotonic conditions and provides the signaling mechanism by which epidermal growth factor (EGF) stimulates IClswell in HeLa cells (26.Varela D. Simon F. Riveros A. Jørgensen F. Stutzin A. J. Biol. Chem. 2004; 279: 13301-13304Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 27.Ren Z. Raucci Jr., F.J. Browe D.M. Baumgarten C.M. Cardiovasc. Res. 2008; 77: 73-80Crossref PubMed Scopus (55) Google Scholar, 28.Browe D.M. Baumgarten C.M. J. Gen. Physiol. 2006; 127: 237-251Crossref PubMed Scopus (72) Google Scholar). Activation of IClswell in ventricular myocytes exposed to hypotonicity (27.Ren Z. Raucci Jr., F.J. Browe D.M. Baumgarten C.M. Cardiovasc. Res. 2008; 77: 73-80Crossref PubMed Scopus (55) Google Scholar) or stretch (29.Browe D.M. Baumgarten C.M. J. Gen. Physiol. 2004; 124: 273-287Crossref PubMed Scopus (90) Google Scholar) also requires H2O2 production that is triggered via a sequential pathway that involves angiotensin II and EGF receptor activation (28.Browe D.M. Baumgarten C.M. J. Gen. Physiol. 2006; 127: 237-251Crossref PubMed Scopus (72) Google Scholar, 29.Browe D.M. Baumgarten C.M. J. Gen. Physiol. 2004; 124: 273-287Crossref PubMed Scopus (90) Google Scholar, 30.Ren Z. Baumgarten C.M. Am. J. Physiol. Heart Circ. Physiol. 2005; 288: H2628-H2636Crossref PubMed Scopus (28) Google Scholar). Hypoosmotic stress has also been linked to Nox activation and subsequent ROS production in cultured brain astrocytes (31.Reinehr R. Görg B. Becker S. Qvartskhava N. Bidmon H.J. Selbach O. Haas H.L. Schliess F. Häussinger D. Glia. 2007; 55: 758-771Crossref PubMed Scopus (159) Google Scholar). Finally, the protein kinase inhibitor staurosporine has been shown to activate IClswell via Nox activation in HeLa cells (32.Shimizu T. Numata T. Okada Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6770-6773Crossref PubMed Scopus (205) Google Scholar).We tested the hypothesis that TNF-α-dependent generation of H2O2 activates IClswell in VSM cells. Because TNF-α-induced H2O2 production is ClC-3-dependent, the Cl−/H+ antiporter would be expected to be required for the process. The results of these studies provide evidence that endosomal ClC-3-dependent ROS production is required for cytokine-mediated activation of IClswell.DISCUSSIONIn VSM cells, TNF-α activates Cl− currents that are indistinguishable from those elicited by hypotonic conditions. The response to TNF-α is absent in ClC-3 null cells and is blocked in WT cells by scavenging of H2O2 or impairment of either early endosome processing or endosome recycling to the plasma membrane. Thrombin and cell swelling also cause H2O2-dependent IClswell activation but via pathways that are ClC-3-independent.The fact that TNF-α activates IClswell is novel but perhaps not so surprising. Cytokines are well known to cause ROS generation (21.Miller Jr., F.J. Filali M. Huss G.J. Stanic B. Chamseddine A. Barna T.J. Lamb F.S. Circ. Res. 2007; 101: 663-671Crossref PubMed Scopus (172) Google Scholar, 39.Galkina E. Ley K. Annu. Rev. Immunol. 2009; 27: 165-197Crossref PubMed Scopus (1108) Google Scholar), and activation of IClswell by hypotonic conditions is H2O2-dependent (26.Varela D. Simon F. Riveros A. Jørgensen F. Stutzin A. J. Biol. Chem. 2004; 279: 13301-13304Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 27.Ren Z. Raucci Jr., F.J. Browe D.M. Baumgarten C.M. Cardiovasc. Res. 2008; 77: 73-80Crossref PubMed Scopus (55) Google Scholar, 28.Browe D.M. Baumgarten C.M. J. Gen. Physiol. 2006; 127: 237-251Crossref PubMed Scopus (72) Google Scholar). The most significant observation in this study is that TNF-α ROS signaling requires ClC-3, whereas responses to hypotonicity and thrombin do not. This suggests that VSM cells have at least two ROS-generating mechanisms that operate in parallel with respect to activation of IClswell. It is therefore perhaps not surprising that the roles of ClC-3 and IClswell have been difficult to discriminate. ClC-3 independence of the response to hypotonicity is in accordance with the work of multiple other investigators who have also seen no reduction in the magnitude of IClswell in various ClC-3 null cells (6.Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W.J. Duan D. Horowitz T.L. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (82) Google Scholar, 16.Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bösl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 17.Arreola J. Begenisich T. Nehrke K. Nguyen H.V. Park K. Richardson L. Yang B. Schutte B.C. Lamb F.S. Melvin J.E. J. Physiol. 2002; 545: 207-216Crossref PubMed Scopus (87) Google Scholar). Thus, in VSM cells, swelling-induced ROS production most likely occurs somewhere other than in early endosomes, where Nox1 activity is ClC-3-dependent (21.Miller Jr., F.J. Filali M. Huss G.J. Stanic B. Chamseddine A. Barna T.J. Lamb F.S. Circ. Res. 2007; 101: 663-671Crossref PubMed Scopus (172) Google Scholar, 22.Moreland J.G. Davis A.P. Bailey G. Nauseef W.M. Lamb F.S. J. Biol. Chem. 2006; 281: 12277-12288Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Like TNF-α, thrombin also activates Nox1 (34.Miller Jr., F.J. Chu X. Stanic B. Tian X. Sharma R.V. Davisson R.L. Lamb F.S. Antioxid. Redox. Signal. 2010; 12: 583-593Crossref PubMed Scopus (67) Google Scholar) and causes proinflammatory responses that are very similar to those linked to TNF-α (40.Martorell L. Martínez-González J. Rodríguez C. Gentile M. Calvayrac O. Badimon L. Thromb. Haemost. 2008; 99: 305-315Crossref PubMed Scopus (164) Google Scholar). However, the effects of thrombin are endocytosis-independent (34.Miller Jr., F.J. Chu X. Stanic B. Tian X. Sharma R.V. Davisson R.L. Lamb F.S. Antioxid. Redox. Signal. 2010; 12: 583-593Crossref PubMed Scopus (67) Google Scholar), and accordingly, IClswell activation by thrombin is not impaired in ClC-3 null cells (see Fig. 9 for a schematic overview). Like thrombin, hypotonicity-induced ROS production may utilize Nox1 in the plasma membrane. Alternatively Nox4 has been shown to be activated by hypotonic stress, and the ROS produced in turn can activate IClswell in NIH3T3 cells (41.Friis M.B. Vorum K.G. Lambert I.H. Am. J. Physiol. Cell Physiol. 2008; 294: C1552-C1565Crossref PubMed Scopus (26) Google Scholar).Links between ClC-3 Expression and IClswellClC-3 overexpression activates anion currents with biophysical properties very similar to those of IClswell. Observations linking ClC-3 to IClswell include 1) enhanced current in ClC-3-expressing cells (4.Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar, 11.Huang P. Liu J. Di A. Robinson N.C. Musch M.W. Kaezel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 37.Zhou J.G. Ren J.L. Qiu Q.Y. He H. Guan Y.Y. J. Biol. Chem. 2005; 280: 7301-7308Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 42.Yin Z. Tong Y. Zhu H. Watsky M.A. Am. J. Physiol. Cell Physiol. 2008; 294: C535-C542Crossref PubMed Scopus (46) Google Scholar), 2) altered current regulation in ClC-3 null cells (6.Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W.J. Duan D. Horowitz T.L. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (82) Google Scholar), 3) inhibition of current by anti-ClC-3 antibodies (37.Zhou J.G. Ren J.L. Qiu Q.Y. He H. Guan Y.Y. J. Biol. Chem. 2005; 280: 7301-7308Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 38.Wang G.X. Hatton W.J. Wang G.L. Zhong J. Yamboliev I. Duan D. Hume J.R. Am. J. Physiol. Heart Circ. Physiol. 2003; 285: H1453-H1463Crossref PubMed Scopus (73) Google Scholar), and 4) increased magnitude of IClswell induced by a constitutively active mutant of ClC-3 (7.Rossow C.F. Duan D. Hatton W.J. Britton F. Hume J.R. Horowitz B. Acta. Physiol. 2006; 187: 5-19Crossref PubMed Scopus (13) Google Scholar). However, as shown in Fig. 4, IClswell has repeatedly been found to be unaltered in cells lacking ClC-3 (6.Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W.J. Duan D. Horowitz T.L. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (82) Google Scholar, 16.Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bösl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 17.Arreola J. Begenisich T. Nehrke K. Nguyen H.V. Park K. Richardson L. Yang B. Schutte B.C. Lamb F.S. Melvin J.E. J. Physiol. 2002; 545: 207-216Crossref PubMed Scopus (87) Google Scholar). In previous work, we have characterized ClC-3 currents that are very different from IClswell. They have time-dependent activation rather than inactivation and are much more sharply outwardly rectifying. ClC-3 currents are similar to those produced by ClC-4 and ClC-5 except that the time constants of activation are slower (1.Matsuda J.J. Filali M.S. Volk K.A. Collins M.M. Moreland J.G. Lamb F.S. Am. J. Physiol. Cell Physiol. 2008; 294: C251-C262Crossref PubMed Scopus (73) Google Scholar, 2.Matsuda J.J. Filali M.S. Collins M.M. Volk K.A. Lamb F.S. J. Biol. Chem. 2010; 285: 2569-2579Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). These currents are inhibited by the same anti-ClC-3 antibody that was previously shown to inhibit IClswell (supplemental Fig. 2). Reconciliation of the existence of unique ClC-3 currents with the many observations linking ClC-3 to IClswell is obviously challenging. We believe that much of the association between ClC-3 expression and IClswell can be attributed to effects of altered ClC-3 abundance or function on endosomal ROS signaling, which in turn controls the activity of a distinct anion conductance, IClswell.TNF-α and IL-1β both failed to activate IClswell in ClC-3 null VSM cells (Fig. 1), yet H2O2 (Fig. 2), swelling (Fig. 4), and thrombin (Fig. 7) all elicited normal responses. Overexpression of ClC-3, in the absence of any other stimulus, activated IClswell in ClC-3 null VSM cells (Fig. 3). Both the current induced by cytokines and by ClC-3 overexpression were blocked by scavenging of H2O2 with catalase. Furthermore, as demonstrated by the relatively small increments in current caused by TNF-α in ClC-3-overexpressing cells and by hypotonic conditions in TNF-α stimulated cells, these three currents were clearly not additive. Taken together, our findings suggest that all three stimuli increase ROS production and thereby activate the independent anion conductance that produces IClswell.In addition to TNF-α, IL-1β, and thrombin, multiple other receptor-coupled stimuli, including angiotensin II, growth factors (28.Browe D.M. Baumgarten C.M. J. Gen. Physiol. 2006; 127: 237-251Crossref PubMed Scopus (72) Google Scholar, 43.Clempus R.E. Griendling K.K. Cardiovasc. Res. 2006; 71: 216-225Crossref PubMed Scopus (284) Google Scholar), and lysophosphatidic acid (41.Friis M.B. Vorum K.G. Lambert I.H. Am. J. Physiol. Cell Physiol. 2008; 294: C1552-C1565Crossref PubMed Scopus (26) Google Scholar, 42.Yin Z. Tong Y. Zhu H. Watsky M.A. Am. J. Physiol. Cell Physiol. 2008; 294: C535-C542Crossref PubMed Scopus (46) Google Scholar), have been shown to cause ROS production and also activate IClswell. Although not all have been tested, the current activated by lysophosphatidic acid has been shown to be ClC-3 expression-dependent (42.Yin Z. Tong Y. Zhu H. Watsky M.A. Am. J. Physiol. Cell Physiol. 2008; 294: C535-C542Crossref PubMed Scopus (46) Google Scholar). Many of these agonists mobilize intracellular calcium, which can activate CamKII. This enzyme has been linked to regulation of a chloride conductance that shares many basic properties (rectification, time-dependent inactivation, ion selectivity) with IClswell. Activation of this current is dependent upon ClC-3 expression (11.Huang P. Liu J. Di A. Robinson N.C. Musch M.W. Kaezel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and is absent in ClC-3 null cells (19.Robinson N.C. Huang P. Kaetzel M.A. Lamb F.S. Nelson D.J. J. Physiol. 2004; 556: 353-368Crossref PubMed Scopus (68) Google Scholar). Furthermore, a specific serine on the N terminus of ClC-3 has been identified as the site of CamKII regulation (19.Robinson N.C. Huang P. Kaetzel M.A. Lamb F.S. Nelson D.J. J. Physiol. 2004; 556: 353-368Crossref PubMed Scopus (68) Google Scholar). It will be important to determine if CamKII phosphorylation of ClC-3 is required for endosomal H2O2 production and thereby IClswell activation. CamKII activation itself is regulated by ROS in a complex manner (44.Trebak M. Ginnan R. Singer H.A. Jourd'heuil D. Antioxid. Redox Signal. 2010; 12: 657-674Crossref PubMed Scopus (103) Google Scholar), and both ROS-dependent NF-κB stimulation and CamKII activation are required for induction of vascular cell adhesion molecule expression by IL-1β (45.Luo S.F. Chang C.C. Lee I.T. Lee C.W. Lin W.N. Lin C.C. Yang C.M. Toxicol. Appl. Pharmacol. 2009; 237: 8-21Crossref PubMed Scopus (38) Google Scholar). Unfortunately, the molecular mechanism by which H2O2 actually activates IClswell has not received much attention. In rat hepatoma cells, the mechanism has been proposed to be indirect and involve phospholipase Cγ1 phosphorylation and calcium mobilization (46.Varela D. Simon F. Olivero P. Armisén R. Leiva-Salcedo E. Jørgensen F. Sala F. Stutzin A. Cell Physiol. Biochem. 2007; 20: 773-780Crossref PubMed Scopus (31) Google Scholar).It is important to note that we see a very different response to ClC-3 overexpression in HEK293 cells, where adenoviral expression of ClC-3 results in enough ClC-3 being present in the plasma membrane to allow us to measure ClC-3 current at pH 7.35 (1.Matsuda J.J. Filali M.S. Volk K.A. Collins M.M. Moreland J.G. Lamb F.S. Am. J. Physiol. Cell Physiol. 2008; 294: C251-C262Crossref PubMed Scopus (73) Google Scholar, 2.Matsuda J.J. Filali M.S. Collins M.M. Volk K.A. Lamb F.S. J. Biol. Chem. 2010; 285: 2569-2579Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) but does not activate IClswell. We can only speculate on the reason for this important difference, but it may be related to the relative amount of ClC-3 and/or IClswell that cycles thought the plasma membrane. This is not to say that there is no ClC-3 in the plasma membrane of VSM cells. The ability of low pH to activate endogenous ClC-3 current (Fig. 8 and Ref. 2.Matsuda J.J. Filali M.S. Collins M.M. Volk K.A. Lamb F.S. J. Biol. Chem. 2010; 285: 2569-2579Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) demonstrates that ClC-3 is present but is not detectable at physiologic pH and is clearly distinct from IClswell. The fact that ClC-3 expression activates IClswell in VSM cells, but not in HEK293 cells, highlights what may be very significant cell type-dependent variability in the interdependence of ClC-3 and IClswell. This may complicate the reconciliation of all data related to ClC-3 expression and IClswell.Endosome Dependence of IClswell ActivationRab guanosine triphosphatases control budding and fission of vesicles and localize to specific intracellular compartments. Rab5 regulates the biogenesis and trafficking of early endosomes (35.Dinneen J.L. Ceresa B.P. Exp. Cell Res. 2004; 294: 509-522Crossref PubMed Scopus (50) Google Scholar). Dominant negative S34N Rab5 inhibits early endosomal fusion (47.Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (767) Google Scholar) and impairs proper processing of both transferrin and epidermal growth factor receptors (35.Dinneen J.L. Ceresa B.P. Exp. Cell Res. 2004; 294: 509-522Crossref PubMed Scopus (50) Google Scholar). Rab11 controls movement of proteins through a recycling compartment that forms via processing of early endosomes and mediates movement of endocytosed proteins back to the plasma membrane (48.Seachrist J.L. Ferguson S.S. Life Sci. 2003; 74: 225-235Crossref PubMed Scopus (174) Google Scholar, 49.Maxfield F.R. McGraw T.E. Nat. Rev. Mol. Cell Biol. 2004; 5: 121-132Crossref PubMed Scopus (1461) Google Scholar). S25N Rab11 disrupts this process and impairs both processing of and signaling via G-protein-coupled receptors, including the lysophosphatidic acid receptor (48.Seachrist J.L. Ferguson S.S. Life Sci. 2003; 74: 225-235Crossref PubMed Scopus (174) Google Scholar, 50.García-Regalado A. Guzmán-Hernández M.L. Ramírez-Rangel I. Robles-Molina E. Balla T. Vázquez-Prado J. Reyes-Cruz G. Mol. Biol. Cell. 2008; 19: 4188-4200Crossref PubMed Scopus (57) Google Scholar). Both S34N Rab5 and S25N Rab11 profoundly impaired the ability of TNF-α (Fig. 6), but not thrombin (Fig. 7) or hypotonic buffer (Fig. 6A), to activate IClswell. There are two potential interpretations of these data. Either proper processing or positioning of H2O2-producing endosomes is required for IClswell activation, or IClswell is activated within endosomes and recycled back to the plasma membrane in an active state. ROS-producing endosomes are Rab5-positive (21.Miller Jr., F.J. Filali M. Huss G.J. Stanic B. Chamseddine A. Barna T.J. Lamb F.S. Circ. Res. 2007; 101: 663-671Crossref PubMed Scopus (172) Google Scholar), and the Rab5 compartment is "upstream" of Rab11. Therefore, the ability of the Rab11 mutant, which does not impede function until after early endosome f

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