CHGB conducts F− and Cl− better than other anions. CHGB conducts F− and Cl− better than other anions. (A). Light scattering–signals measured from vesicles with (red and blue for pH 7.4 and 5.5, respectively) and without (black) CHGB. A 30-s pause for adding DMSO or valinomycin was not included. (B) The CHGB proteoliposomes were treated with different concentrations of the DIDS for 5 min at RT before experiment. DIDS inhibits CHGB-mediated Cl− flux. Red arrows indicate the addition of DMSO and valinomycin. Inset: change in the scattering was plotted against the DIDS concentration. Fitting with a Hill equation yielded a kD = 0.9 μM; n = 1.0. (C) Flux assay from vesicles with and without CHGB-MIF under different conditions in comparison with CHGB vesicles (black). (D) Flux assays of liposomes with and without CHGBΔMIF at two different pH's. Wild-type control in red. (E) Flux assays of liposomes containing CHGA, CHGB, and CHGB mutant proteins. Residues E545, E552, or E558 in the loop (L1) between helix 2 and helix 3 were mutated to alanine. (F) Relative anion selectivity of CHGB channel based on the flux assay. Error bars represent SD from three different sets of experiments. (G). Instead of 300 mM K-isethionate, 300 mM KF, KBr or KI was introduced to the extravesicular side against 300 mM KCl–loaded vesicles. Br− or I− supported significant flux signal. But, 300 mM F− abolished the flux. Error bars: SD, n = 3. (H). Calculated correction factor for valinomycin-mediated K+ transport because of steady state Nernst potential for 300 mM KCl–loaded CHGB vesicles with 0–5 mM KCl outside. (I) Flux assay from 300 mM KCl–loaded CHGB vesicles in the presence of 0–10 mM KCl and 300 mM K-isethionate in the extravesicular side. Vesicles had 1:10,000 PLR of CHGB: egg PC. Three independent sets of vesicles prepared from separate cell cultures (n = 3) were used to ensure high statistical power. Inset: change in the scattering was plotted against the KCl concentration and fitting with a Hill equation yielded a kD = 0.49 mM and n = 1.0. Gaya P Yadav et al. LSA 2018;1:e201800139 © 2018 Yadav et al.