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pH‐Dependent Interactions of Cd2+ and a Carboxylate Blocker with the Rat ClC‐1 Chloride Channel and Its R304E Mutant in the Sf‐9 Insect Cell Line

1 Gating of the skeletal muscle chloride channel (ClC‐1) is sensitive to extracellular pH. In this study, whole‐cell recording of currents from wild‐type (WT) ClC‐1 and a mutant, R304E, expressed in the Sf‐9 insect cell line was used to investigate further the nature of the pH‐sensitive residues. 2 Extracellular Cd2+ produced a concentration‐dependent block of WT ClC‐1 with an IC50 of 1.0 ± 0.1 mm and a Hill coefficient of 2.0 ± 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 ± 0.1 and a Hill coefficient for proton binding of 3.0 ± 0.3. Anthracene‐9‐carboxylate (A‐9‐C) block of WT ClC‐1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 ± 0.1 and a Hill coefficient for proton binding of 1.0 ± 0.2. 3 Compared with WT ClC‐1, R304E had a lower affinity for Cd2+ (IC50, 3.0 ± 0.3 mm) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 ± 0.3. In contrast, the A‐9‐C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC‐1. 4 ClC‐1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A‐9‐C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A‐9‐C binding site to affect its characteristics, but it does alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5 The block of ClC‐1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A‐9‐C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC‐1.