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

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 pHsensitive residues. 2. Extracellular Cd¥ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1·0 ± 0·1 mÒ 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 Cd¥ (IC50, 3·0 ± 0·3 mÒ) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd¥ 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¥, 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¥ 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.

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle

“…First, these findings validate adequate space clamp uniformity even in naive fibers because ClC-1 channel gating was unaffected by loss of a significant fraction of cell capacitance originating from the T-tubule compartment. Second, previous studies demonstrated that ClC-1 gating is strongly influenced by chloride concentration (Rychkov et al, 1996(Rychkov et al, , 1997Accardi and Pusch, 2000;Chen and Chen, 2001), pH (Rychkov et al, 1996(Rychkov et al, , 1997Accardi and Pusch, 2000;Chen and Chen, 2001;Bennetts et al, 2007), nitric oxide (Lin et al, 2008), and with some controversy, the binding of adenosine derivatives (Bennetts et al, 2005Tseng et al, 2007;Zhang et al, 2008;Zifarelli and Pusch, 2008). Although we did not directly test if these factors modulate native ClC-1 channel activity in FDB fibers, our findings suggest that detubulation by formamide addition and withdrawal does not markedly perturb basal modulatory influences of these factors on ClC-1 gating.…”

“…(Fukuda and Kawa, 1977) at a concentration (250 µM) that had no effect on ClC-1 currents (Rychkov et al, 1997;unpublished data).…”

Identification of Three Cysteines as Targets for the Zn2+ Blockade of the Human Skeletal Muscle Chloride Channel

“…Studies on the pH dependence of ClC-1 current block by Cd 2ϩ , another group IIb cation with behavior similar to that of Zn 2ϩ , yielded a pK a value of 6.8, i.e. nearly identical to ours (23). The authors of that study suggested that histidine residues might be involved.…”

Relevance of the D13 Region to the Function of the Skeletal Muscle Chloride Channel, ClC-1

“…the R304E mutant (17). When we tested our present mutants, Cd 2ϩ was as effective on ⌬D13 as on the WT channel (IC 50 ϭ 1.1 Ϯ 0.1 mM (n ϭ 3) for ⌬D13 and 1.0 Ϯ 0.1 mM (n ϭ 3) for WT), whereas fs895X required higher concentrations of Cd 2ϩ to achieve the same level of block (IC 50 ϭ 2.7 Ϯ 0.2 mM, (n ϭ 5).…”

“…Data were collected, filtered at 3 kHz, and analyzed on an IBM-compatible PC using pCLAMP v6.0 software (Axon Instruments). Parameters such as time constants, apparent open probability, and IC 50 were determined as described previously (16,17). …”