1. Grating 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 pK(a) 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 pK(a) 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 pK(a), of this block, together with the apparent pK(a), for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.
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