Figure 11.

The extracellular loop domains of LRRC8D confer enhanced inhibitory ChlorT response to LRRC8C. A, Currents evoked by 250 ms voltage ramps (−100 to +120 mV) in hypotonic saline, recorded from a cell expressing the LRR8C-8A IL protein substituted with both 8D extracellular loops (8C-8D EL1,2). Current is strongly inhibited by 1 mM ChlorT (green). DCPIB is ineffective in blocking residual current following ChlorT exposure (post-ox. DCPIB; orange). B, Summary of 8C-8D EL1,2 current inhibition by ChlorT and DCPIB. 1 mM ChlorT inhibits current by 69% (***P < 0.0001 vs hypotonic control current; paired ratio t-test, n = 7). Post-ox. DCPIB produces no further current block (P = 0.1431, n = 7). C, Comparison of current sensitivity to 1 mM ChlorT in LRRC8A^(−/−) mutant cells expressing various LRRC8A, 8C, and 8D chimeric constructs. Symbols represent individual recordings; values are normalized to current amplitude at +120 mV in control saline. LRRC8A (8A-8C EL1; n = 14) displays variable individual responses, but no overall oxidant sensitivity (P = 0.2837 vs. Control; same data as Fig. 8I). Homomeric LRRC8C-8A IL shows weak inhibition (Fig. 8G), while LRRC8D-8A IL shows strong inhibition (Fig. 8H, 1 mM ChlorT). Substitution of EL1 of LRRC8D onto 8C (8C-8D EL1) does not alter the response to ChlorT (P = 0.4308 vs 8C-8A IL; n = 4). Substitution of both EL1 and EL2 (8C-8D EL1,2) confers a strong inhibitory oxidant response (n = 7; as in B. ***P = 0.0006 vs. 8C-8A IL, **P = 0.0023 vs. 8D-8A IL; Mann-Whitney tests). Substitution of EL1 of LRRC8C onto 8D abolishes inhibition and results in mild augmentation of current amplitude (*P = 0.0109 vs. Control; n = 5).