PH-DEPENDENT INTERACTIONS OF CD2-1 CHLORIDE CHANNEL AND ITS R304E MUTANT IN THE SF-9 INSECT-CELL LINE( AND A CARBOXYLATE BLOCKER WITH THE RAT CLC)

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 i nsect cell line was used to investigate further the nature of the pH-s ensitive residues. 2. Extracellular Cd2+ produced a concentration-depe ndent block of WT ClC-1 with an IC50 of 1.0 +/- 0.1 mM and a Hill coef ficient of 2.0 +/- 0.3. This block was sensitive to external pH, reduc ing at low pH, with an apparent pK(a) of 6.8 +/- 0.1 and a Hill coeffi cient 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, wit h an apparent pK(a) of 6.4 +/- 0.1 and a Hill coefficent for proton bi nding of 1.0 +/- 0.2. 3. Compared with WT ClC-1, R304E had a lower aff inity for Cd2+ (IC50, 3.0 +/- 0.3 mM) but it had a similar Hill coeffi cient for transition metal ion binding. The Hill coefficient for proto n binding to the Cd2+ binding site was reduced to 1.4 +/- 0.3. In cont rast, the A-9-C binding site in R304E showed the same pH sensitivity a nd 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 res idues which can be protonated. Binding of A-9-C is influenced by proto nation 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 carboxyl ates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pK(a) of this bloc, together with the app arent pK(a) for A-9-C block and gating are all compatible with the inv olvement of His residues in the pore and gate of ClC-1.