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. 2018 Jun 20;293(35):13440–13451. doi: 10.1074/jbc.RA118.002853

Figure 2.

Figure 2.

Cysteine-scanning mutagenesis of LRRC8A/C N termini. WT and mutated LRRC8A and -C were coexpressed in LRRC8−/− HCT116 cells, and ICl,vol were elicited by hypotonicity as in Fig. 1B. A, mean maximum ICl,vol current densities (current normalized to respective cell capacitance) of LRRC8A/C channels carrying cysteine substitutions in both LRRC8A and -C. Mutations are indicated on the vertical axis with native residues in LRRC8A and -C separated by commas. The dashed line indicates mean ICl,vol density from WT/WT LRRC8A/C channels (WT measurements identical to Fig. 1F). B and C, typical effects of MTSEA on ICl,vol mediated by WT/WT (B) or Y9C/Q9C (C) LRRC8A/C channels. Left, representative time course of currents at −80 mV taken from voltage ramps applied every 15 s. Each circle represents an individual measurement. Red and blue bars indicate application of 25% hypotonic solution and hypotonic solution containing 200 μm MTSEA, respectively. Right, typical current traces elicited by these ramps (shown below the upper panel) in isotonic solution (Iso; a), full activation by hypotonic solution (Hypo; b), and at the end of the ∼ 4-min perfusion of MTSEA (c). Capacitance of the cells in B and C: 18.2 and 18.8 picofarads (pF), respectively. D, mean effect of MTSEA on maximal ICl,vol currents of LRRC8A/C heteromers carrying cysteine mutations in both LRRC8A and -C. Currents are normalized to those before MTSEA application. The dashed line represents mean, normalized current density of MTSEA-exposed WT/WT channels. E, effect of 50 μm intracellular Cd2+ (applied in pipette solution) on ICl,vol density from WT and mutant LRRC8A/C heteromers. F, effect of 50 μm intracellular Cd2+ (applied in pipette solution) on ICl,vol density from WT and mutant LRRC8A/C heteromers (the values for WT and Y9C/Q9C are the same as in E) and for LRRC8AC139S/CC141S with and without Y9C/Q9C mutation. Error bars, S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus WT (in A and D, Kruskal–Wallis test, Dunn's post hoc test; in E and D, Mann–Whitney test, false-discovery rate controlled by Benjamini–Hochberg procedure).