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

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 H₂O₂ 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-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 ICl_swell. ICl_swell was also activated by H₂O₂ (500 μm), and the effect of TNF-α 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-α, 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-α-mediated activation of ICl_swell. Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H₂O₂-dependent activation of ICl_swell, but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl_swell by TNF-α requires ClC-3-dependent endosomal H₂O₂ production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl_swell.