Activation of swelling-activated chloride current by tumor necrosis factor-alpha requires ClC-3-dependent endosomal reactive oxygen production.

Journal Article

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 (ICl(swell)), but overexpression of ClC-3 can activate currents that resemble ICl(swell). Because H(2)O(2) activates ICl(swell) directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates ICl(swell). 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-alpha (TNF-alpha) (10 ng/ml) activated outwardly rectifying Cl(-) currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 microm) and by hypertonicity (340 mosm) identified them as ICl(swell). ICl(swell) was also activated by H(2)O(2) (500 microm), and the effect of TNF-alpha was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced ICl(swell) in ClC-3 null cells in the absence of swelling or TNF-alpha, and this effect was also blocked by catalase. ICl(swell) 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-alpha-mediated activation of ICl(swell). Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H(2)O(2)-dependent activation of ICl(swell), but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl(swell) by TNF-alpha requires ClC-3-dependent endosomal H(2)O(2) production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl(swell).

Full Text

Duke Authors

Cited Authors

  • Matsuda, JJ; Filali, MS; Moreland, JG; Miller, FJ; Lamb, FS

Published Date

  • July 23, 2010

Published In

Volume / Issue

  • 285 / 30

Start / End Page

  • 22864 - 22873

PubMed ID

  • 20479003

Pubmed Central ID

  • 20479003

Electronic International Standard Serial Number (EISSN)

  • 1083-351X

Digital Object Identifier (DOI)

  • 10.1074/jbc.M109.099838


  • eng

Conference Location

  • United States