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

ABSTRACT: 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).

SUBMITTER: Matsuda JJ

PROVIDER: S-EPMC2906278 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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

Matsuda James J JJ Filali Mohammed S MS Moreland Jessica G JG Miller Francis J FJ Lamb Fred S FS

The Journal of biological chemistry 20100517 30

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 i ...[more]

PMID: 20479003

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Project description:The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

Dataset Information

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

Publications

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

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