Oxidant-Resistant LRRC8A/C Anion Channels Support Superoxide Production by Nox1

Tumor necrosis factor- (TNF) activates NADPH Oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2*-) required for subsequent signaling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNF. LRRC8 channel currents are modulated by oxidants, suggesting that oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNF-induced extracellular and endosomal O2*- production, receptor endocytosis, NF-{kappa}B activation, and proliferation. In contrast, siLRRC8D potentiated NF-{kappa}B activation. Nox1 co-immunoprecipitated with 8C and 8D, and co-localized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric 8C and 8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited LRRC8C, but potently inhibited 8D currents. ChlorT exposure also greatly reduced subsequent current block by DCPIB, implicating external sites of oxidation. Substitution of the extracellular loop domains (EL1, EL2) of 8D onto 8C conferred significantly stronger ChlorT-dependent inhibition. 8A/C channel activity is thus more effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNF. LRRC8A/C channel downregulation represents a novel strategy to reduce TNF-induced inflammation. Key PointsO_LILRRC8A-containing anion channels associate with Nox1 and regulate superoxide production and TNF signaling. Here we show that .LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells. C_LIO_LILRRC8C knockdown inhibited TNF-induced O2*- production, receptor endocytosis, NF-{kappa}B activation, and proliferation while LRRC8D knockdown enhanced NF-{kappa}B activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation. C_LIO_LIThe oxidant chloramine-T (ChlorT, 1 mM) weakly ([~]25%) inhibited 8C currents but potently ([~]80%) inhibited 8D currents. Substitution of the two extracellular loop (EL) domains of 8D onto 8C conferred significantly stronger ChlorT-dependent inhibition. C_LIO_LIChlorT also impaired current block by DCPIB, which occurs through interaction with EL1, further implicating external sites of oxidation. C_LIO_LI8A/C channels most effectively maintain activity in an oxidized microenvironment, as is expected to result from Nox1 activity at the plasma membrane. C_LI