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ClC‐3 anion current is required for superoxide production in early endosomes and subsequent NF‐ κ B activation by TNF α
Author(s) -
Filali Mohammed S,
Matsuda James J,
Gross Jordan R,
Storm Sarah K,
Moreland Jessica G,
Miller Francis J,
Lamb Fred S
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.937.17
Subject(s) - endosome , reactive oxygen species , chemistry , microbiology and biotechnology , nox1 , nadph oxidase , superoxide , hek 293 cells , nf κb , biochemistry , signal transduction , biology , intracellular , receptor , enzyme
ClC‐3 is an early endosomal Cl − ‐H + antiporter required for endosomal reactive oxygen species (ROS) generation in response to cytokines in vascular smooth muscle cells. To further define the role of ClC‐3 we quantified NADPH‐induced ROS production by affinity purified (Nox1‐immunoprecipitated) early endosomes from HEK293T cells using lucigenin‐enhanced chemiluminescence. We also assayed NF‐κB activity using an Ad‐NF‐κB luciferase reporter. Pretreatment of cells with TNFα enhanced ROS production by purified endosomes 2.7 ± 0.6 fold (n = 13). TNFα also caused a 9.5 ± 2.2 fold increase in NF‐κB activity (n = 18) which was Nox1 and ROS‐dependent (inhibited by Ad‐Nox1anti‐sense, Ad‐N17Rac1, and anti‐oxidants). Cl − channel inhibitors (DIDS and niflumic acid) blocked both ROS production and NF‐κB activation. Mutations of two different glutamate residues required for H + permeation of ClC‐3 (E224A or E281Q) did not impair NF‐κB activation. Surprisingly, the double mutant (E224A, E281Q) disrupted NF‐κB activation, but only when the long N‐terminal isoform of ClC‐3 was used. Expression of this clone also impaired ROS production by endosomes. Patch clamp recording revealed that the E224A, E281Q double mutant lacks both H + and Cl − current and acts as a dominant negative protein to disrupt endogenous ClC‐3 current. We conclude that ClC‐3 anion current is required for ROS production by Nox1 in early endosomes. Supported by HL06248.