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pH‐Dependent Interactions of Cd 2+ and a Carboxylate Blocker with the Rat ClC‐1 Chloride Channel and Its R304E Mutant in the Sf‐9 Insect Cell Line
Author(s) -
Rychkov G. Y.,
Astill D.,
Bennetts B.,
Hughes B. P.,
Bretag A. H.,
Roberts M. L.
Publication year - 1997
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1997.355bn.x
Subject(s) - protonation , chemistry , carboxylate , binding site , stereochemistry , extracellular , biophysics , ion , biochemistry , organic chemistry , biology
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 Cd 2+ produced a concentration‐dependent block of WT ClC‐1 with an IC 50 of 1.0 ± 0.1 m m and a Hill coefficient of 2.0 ± 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent p K 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 p K 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 Cd 2+ (IC 50 , 3.0 ± 0.3 m m ) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd 2+ 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 Cd 2+ , 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 Cd 2+ 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 p K a of this block, together with the apparent p K 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.