Voltage-dependent activation of CLC-2 in the presence of intracellular, poorly permeant anions. (A–C and E and F) Whole-cell currents recorded from five cells dialyzed with solutions containing 140 mM Cl⁻ (A), 140 mM Glu (B), 140 mM methanesulfonate or 140 mM F⁻ (C), 140 mM acetate (E), and 140 mM sulfate (F). Cells were bathed in solution containing 140 mM Cl⁻ (pH_e = pH_i 7.3). B (inset) plots data obtained from the same cell dialyzed with internal solution containing 140 mM Cl⁻ and sequentially bathed in 140 mM Cl⁻ (reddish purple) and 140 mM Glu (black). I_Cl(t) was recorded between −200 and 40 mV in 20-mV increments, and I_tail(t) was recorded at 80 mV. (D) G_norm(V_m) curves obtained from cells dialyzed with solutions containing poorly permeant anions F⁻, Glu, gluconate (Gluc), and methanesulfonate (MeSO₃⁻). Continuous lines are fits to Boltzmann equation, yielding the following V_0.5 and z values: Cl⁻ (black squares), 79.9 ± 6.6 mV and −0.94 ± 0.01 (n = 28); F⁻ (vermilion triangles), −146.4 ± 3.3 mV and −0.96 ± 0.08 (n = 5); Glu (reddish purple triangles), −180.7 ± 2.3 mV and −1.32 ± 0.02 (n = 6); gluconate (sky blue circles), −189.6 ± 2.8 mV and −1.17 ± 0.05 (n = 8); methanesulfonate (blue triangles), −177.7 ± 3.6 mV and −1.16 ± 0.03 (n = 8). (Inset) Superposition of voltage–activation curves after subtracting corresponding V_0.5 values (listed above) under each ionic condition. Error bars represent mean ± SEM.