Oxidant-mediated activation and inhibition of LRRC8C and LRRC8D channel currents require N-terminal and Transmembrane 1 pore domains

Leucine Rich Repeat Containing 8C (LRRC8C) anion channels modulate NADPH oxidase 1 activity and allow extracellular superoxide influx promoting inflammatory signaling. Here we studied chimeric 8C/8D channels and identified oxidant-dependent current modulation within the N-terminus (NT) and first transmembrane domain (TM1). Chloramine-T (ChlT) elicited inhibitory and activating current responses, whereas other redox agents had comparatively little impact. ChlT moderately inhibited wild-type (WT) 8C current and abrogated block by DCPIB. Substitution of the 8D NT (8D1-22) conferred ChlT-dependent current activation, as did 8D2-4, 8D5-11, I2F, and I2Y substitution. M48T (distal TM1) substitution enhanced WT 8C current inhibition and impaired activation in NT mutants. An M48D mutation diminished 8C current block by DCPIB by [~]50%. WT 8D currents were potently inhibited by ChlT. Substitution of the 8C first extracellular loop (EL1) weakened inhibition, while 8C EL1 + TM145-49 substitution produced ChlT-mediated current activation. 8C45-49 or T48M substitutions in 8D resulted in rapid disruption and loss of initial current inhibition, and a progressive increase of non-rectifying current. These results provide evidence that NT2-4, particularly I2/F2, in combination with M48 are primary determinants of activating vs. inhibitory current modulation by ChlT. M48 oxidation limits 8C inhibition and is required for activating responses, while T48 and 8D EL1 promote 8D signature current inhibition. ChlT exposure disrupts subsequent or preexisting channel block by DCPIB, consistent with a common site of interaction. Thus, factors that alter NT pore stability and mobility may regulate inhibition vs. activation of LRRC8C by redox stress.