title: pH-dependent interactions of Cd2+ and a carboxylate blocker with the rat C1C-1 chloride channel and its R304E mutant in the Sf-9 insect cell line.
abstract: 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.
authors.affiliation: Department of Physiology, University of Adelaide, Australia.
authors.forename: G Y
authors.initials: GY
authors.lastname: Rychkov
authors.forename: D S
authors.initials: DS
authors.lastname: Astill
authors.forename: B
authors.initials: B
authors.lastname: Bennetts
authors.forename: B P
authors.initials: BP
authors.lastname: Hughes
authors.forename: A H
authors.initials: AH
authors.lastname: Bretag
authors.forename: M L
authors.initials: ML
authors.lastname: Roberts
mesh_terms: D000818:Animals; D002104:Cadmium / Q000494:pharmacology*; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000502:physiology; D008670:Metals / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D018408:Patch-Clamp Techniques; D017354:Point Mutation / Q000502:physiology; D051381:Rats
chemical_list: D018118:Chloride Channels; D008670:Metals; D002104:Cadmium