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Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Yu Y, Tsai MF, Yu WP, Chen TY - J. Gen. Physiol. (2015)

Bottom Line: Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating.Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0.The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

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Affiliation: Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618 Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618.

No MeSH data available.


Disulfide bond linking two CLC subunits. (A) Inhibition of CLC-0 by Cd2+ binding to the binding site containing four cysteines in the WT CLC-0 background (top) and the dimer interface mutant background (DM; bottom). Excised inside-out patch recordings. The voltage pulse (60 mV followed by −100 mV) for monitoring the current was given every 2 s, and the current at the end of the −100-mV voltage was monitored. Dotted lines, zero current level. Horizontal bars indicate the application of 10 µM Cd2+. (B) I229W/V490W dimer interface mutations (DM) facilitate disulfide bond formation in copper phenanthroline (CuP) between the two C231 residues. “4C” indicates that all four positions at positions 229 and 231 from two subunits were cysteine, whereas “2C” contained cysteine only at position 231 but not at position 229 (C229S/H231C). Arrows indicate the monomer of CLC-0, which disappeared likely because of a disulfide bond formation between the two C231 residues.
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fig10: Disulfide bond linking two CLC subunits. (A) Inhibition of CLC-0 by Cd2+ binding to the binding site containing four cysteines in the WT CLC-0 background (top) and the dimer interface mutant background (DM; bottom). Excised inside-out patch recordings. The voltage pulse (60 mV followed by −100 mV) for monitoring the current was given every 2 s, and the current at the end of the −100-mV voltage was monitored. Dotted lines, zero current level. Horizontal bars indicate the application of 10 µM Cd2+. (B) I229W/V490W dimer interface mutations (DM) facilitate disulfide bond formation in copper phenanthroline (CuP) between the two C231 residues. “4C” indicates that all four positions at positions 229 and 231 from two subunits were cysteine, whereas “2C” contained cysteine only at position 231 but not at position 229 (C229S/H231C). Arrows indicate the monomer of CLC-0, which disappeared likely because of a disulfide bond formation between the two C231 residues.

Mentions: Because binding sites for transition metal cations can be formed by four cysteine residues (Yellen et al., 1994), we tested the effect of Cd2+ on CLC-0 with the H231C mutation. Such a CLC-0 construct contains cysteine at all four positions of 229 and 231 from two subunits (and thus will be called “4C” mutant). In the WT background, the 4C mutant becomes very sensitive to Cd2+, and the Cd2+ inhibition is nearly irreversible (Fig. 10 A, top). Cd2+ inhibition of the 4C mutant with I225W/V490W background mutations, however, is very different; the inhibition can be quickly washed out, but with the time of patch excision the channel is less and less sensitive to Cd2+ (Fig. 10 A, bottom). We suspected that this time-dependent reduction of Cd2+ sensitivity might result from the formation of a disulfide bond among these four cysteine residues after patch excision, which could change the oxidation/reduction condition of the channel (Zhang et al., 2008). Indeed, Western blots shown in Fig. 10 B demonstrate that the monomeric band of the 4C mutant in the I225W/V490W background disappeared after oxidation (Fig. 10 B, right). On the other hand, the majority of the 4C mutant in the WT CLC-0 background still remained as monomers in the SDS gel after copper phenanthroline treatment (Fig. 10 B, left). Similar results were observed in a “2C” mutant in which cysteine was present only at position 231 but not at position 229 (Fig. 10 B), suggesting that it is the two cysteines at position 231 that form a disulfide bond. Thus, through the differences in Cd2+ inhibition and disulfide bond formation, we demonstrate that like the WT Cd2+-binding site, the structure of the mutant Cd2+-binding site with four cysteines is changed by the I225W/V490W mutations.


Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Yu Y, Tsai MF, Yu WP, Chen TY - J. Gen. Physiol. (2015)

Disulfide bond linking two CLC subunits. (A) Inhibition of CLC-0 by Cd2+ binding to the binding site containing four cysteines in the WT CLC-0 background (top) and the dimer interface mutant background (DM; bottom). Excised inside-out patch recordings. The voltage pulse (60 mV followed by −100 mV) for monitoring the current was given every 2 s, and the current at the end of the −100-mV voltage was monitored. Dotted lines, zero current level. Horizontal bars indicate the application of 10 µM Cd2+. (B) I229W/V490W dimer interface mutations (DM) facilitate disulfide bond formation in copper phenanthroline (CuP) between the two C231 residues. “4C” indicates that all four positions at positions 229 and 231 from two subunits were cysteine, whereas “2C” contained cysteine only at position 231 but not at position 229 (C229S/H231C). Arrows indicate the monomer of CLC-0, which disappeared likely because of a disulfide bond formation between the two C231 residues.
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Related In: Results  -  Collection

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fig10: Disulfide bond linking two CLC subunits. (A) Inhibition of CLC-0 by Cd2+ binding to the binding site containing four cysteines in the WT CLC-0 background (top) and the dimer interface mutant background (DM; bottom). Excised inside-out patch recordings. The voltage pulse (60 mV followed by −100 mV) for monitoring the current was given every 2 s, and the current at the end of the −100-mV voltage was monitored. Dotted lines, zero current level. Horizontal bars indicate the application of 10 µM Cd2+. (B) I229W/V490W dimer interface mutations (DM) facilitate disulfide bond formation in copper phenanthroline (CuP) between the two C231 residues. “4C” indicates that all four positions at positions 229 and 231 from two subunits were cysteine, whereas “2C” contained cysteine only at position 231 but not at position 229 (C229S/H231C). Arrows indicate the monomer of CLC-0, which disappeared likely because of a disulfide bond formation between the two C231 residues.
Mentions: Because binding sites for transition metal cations can be formed by four cysteine residues (Yellen et al., 1994), we tested the effect of Cd2+ on CLC-0 with the H231C mutation. Such a CLC-0 construct contains cysteine at all four positions of 229 and 231 from two subunits (and thus will be called “4C” mutant). In the WT background, the 4C mutant becomes very sensitive to Cd2+, and the Cd2+ inhibition is nearly irreversible (Fig. 10 A, top). Cd2+ inhibition of the 4C mutant with I225W/V490W background mutations, however, is very different; the inhibition can be quickly washed out, but with the time of patch excision the channel is less and less sensitive to Cd2+ (Fig. 10 A, bottom). We suspected that this time-dependent reduction of Cd2+ sensitivity might result from the formation of a disulfide bond among these four cysteine residues after patch excision, which could change the oxidation/reduction condition of the channel (Zhang et al., 2008). Indeed, Western blots shown in Fig. 10 B demonstrate that the monomeric band of the 4C mutant in the I225W/V490W background disappeared after oxidation (Fig. 10 B, right). On the other hand, the majority of the 4C mutant in the WT CLC-0 background still remained as monomers in the SDS gel after copper phenanthroline treatment (Fig. 10 B, left). Similar results were observed in a “2C” mutant in which cysteine was present only at position 231 but not at position 229 (Fig. 10 B), suggesting that it is the two cysteines at position 231 that form a disulfide bond. Thus, through the differences in Cd2+ inhibition and disulfide bond formation, we demonstrate that like the WT Cd2+-binding site, the structure of the mutant Cd2+-binding site with four cysteines is changed by the I225W/V490W mutations.

Bottom Line: Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating.Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0.The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618 Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618.

No MeSH data available.