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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.


Mutations of C229 and/or H231 suppress the Cd2+ effect in shifting the Po-V curve of I225W/V490W and WT CLC-0. (A–C) Steady-state Po-V curves of I225W/V490W with the C229S single mutation (A), the H231Y single mutation (B), or the C229S/H231Y double mutations (C) in the presence of various [Cd2+]. Notice that the shift of the Po-V curve by Cd2+ is abolished, whereas the inhibition of the maximal Po at high concentrations of Cd2+ is still present. (D) Steady-state Po-V curves of the C229S mutant on the WT background in different [Cd2+].
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fig7: Mutations of C229 and/or H231 suppress the Cd2+ effect in shifting the Po-V curve of I225W/V490W and WT CLC-0. (A–C) Steady-state Po-V curves of I225W/V490W with the C229S single mutation (A), the H231Y single mutation (B), or the C229S/H231Y double mutations (C) in the presence of various [Cd2+]. Notice that the shift of the Po-V curve by Cd2+ is abolished, whereas the inhibition of the maximal Po at high concentrations of Cd2+ is still present. (D) Steady-state Po-V curves of the C229S mutant on the WT background in different [Cd2+].

Mentions: Among the 12 endogenous cysteines in CLC-0, we have shown previously that C229, which is located at the dimer interface near the intracellular end of the transmembrane domain, can be modified by intracellular MTS reagents (Zhang et al., 2010). Interestingly, a histidine residue, H231, is located nearby, raising the possibility that it can act with C229 to coordinate Cd2+ as a bidentate ligand (Puljung and Zagotta, 2011). We thus tested whether C229 and H231 could both be involved in the formation of the Cd2+-binding site. Mutating C229 (C229S), H231 (H231Y), or both (C229S/H231Y) in the background of I225W/V490W indeed suppressed the ability of Cd2+ to shift the steady-state activation curve (Fig. 7, A–C, respectively). When the C229S mutation was made in the WT CLC-0, the Cd2+ inhibition effect was also significantly reduced (Fig. 7 D). Likewise, the mutation H231Y also reduced the sensitivity of Cd2+ inhibition on WT CLC-0 (not depicted). However, the reduction of maximal channel activity by high [Cd2+] still existed in these mutants (Fig. 7, A–D), suggesting that it is mediated by a different mechanism. Our results thus demonstrate that C229 and H231 form the high affinity Cd2+-binding site, where binding of Cd2+ shifts the steady-state activation curve of I225W/V490W. It is likely that C229 and H231 from both CLC-0 subunits together coordinate Cd2+ binding, as these residues are located near the dimer interface and therefore are close to each other (see Fig. 1). The conclusion that C229 and H231 form the Cd2+-binding site is further supported by experiments from CLC-1. These two residues are not conserved in CLC-1, which is not sensitive to intracellular Cd2+ modulation (Figs. 8 A and 9 A). When cysteine and histidine were introduced to the corresponding positions in CLC-1 (S294C/Y296H), a Cd2+-binding site was created, and the activation of CLC-1 could be modulated by intracellular Cd2+ (Figs. 8 B and 9, A and B). Like the MTS modification of the endogenous cysteine (C229) in CLC-0, modification of the introduced cysteine (S294C) abolished Cd2+ inhibition on CLC-1 containing the engineered Cd2+-binding site (Fig. 9 C).


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

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

Mutations of C229 and/or H231 suppress the Cd2+ effect in shifting the Po-V curve of I225W/V490W and WT CLC-0. (A–C) Steady-state Po-V curves of I225W/V490W with the C229S single mutation (A), the H231Y single mutation (B), or the C229S/H231Y double mutations (C) in the presence of various [Cd2+]. Notice that the shift of the Po-V curve by Cd2+ is abolished, whereas the inhibition of the maximal Po at high concentrations of Cd2+ is still present. (D) Steady-state Po-V curves of the C229S mutant on the WT background in different [Cd2+].
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fig7: Mutations of C229 and/or H231 suppress the Cd2+ effect in shifting the Po-V curve of I225W/V490W and WT CLC-0. (A–C) Steady-state Po-V curves of I225W/V490W with the C229S single mutation (A), the H231Y single mutation (B), or the C229S/H231Y double mutations (C) in the presence of various [Cd2+]. Notice that the shift of the Po-V curve by Cd2+ is abolished, whereas the inhibition of the maximal Po at high concentrations of Cd2+ is still present. (D) Steady-state Po-V curves of the C229S mutant on the WT background in different [Cd2+].
Mentions: Among the 12 endogenous cysteines in CLC-0, we have shown previously that C229, which is located at the dimer interface near the intracellular end of the transmembrane domain, can be modified by intracellular MTS reagents (Zhang et al., 2010). Interestingly, a histidine residue, H231, is located nearby, raising the possibility that it can act with C229 to coordinate Cd2+ as a bidentate ligand (Puljung and Zagotta, 2011). We thus tested whether C229 and H231 could both be involved in the formation of the Cd2+-binding site. Mutating C229 (C229S), H231 (H231Y), or both (C229S/H231Y) in the background of I225W/V490W indeed suppressed the ability of Cd2+ to shift the steady-state activation curve (Fig. 7, A–C, respectively). When the C229S mutation was made in the WT CLC-0, the Cd2+ inhibition effect was also significantly reduced (Fig. 7 D). Likewise, the mutation H231Y also reduced the sensitivity of Cd2+ inhibition on WT CLC-0 (not depicted). However, the reduction of maximal channel activity by high [Cd2+] still existed in these mutants (Fig. 7, A–D), suggesting that it is mediated by a different mechanism. Our results thus demonstrate that C229 and H231 form the high affinity Cd2+-binding site, where binding of Cd2+ shifts the steady-state activation curve of I225W/V490W. It is likely that C229 and H231 from both CLC-0 subunits together coordinate Cd2+ binding, as these residues are located near the dimer interface and therefore are close to each other (see Fig. 1). The conclusion that C229 and H231 form the Cd2+-binding site is further supported by experiments from CLC-1. These two residues are not conserved in CLC-1, which is not sensitive to intracellular Cd2+ modulation (Figs. 8 A and 9 A). When cysteine and histidine were introduced to the corresponding positions in CLC-1 (S294C/Y296H), a Cd2+-binding site was created, and the activation of CLC-1 could be modulated by intracellular Cd2+ (Figs. 8 B and 9, A and B). Like the MTS modification of the endogenous cysteine (C229) in CLC-0, modification of the introduced cysteine (S294C) abolished Cd2+ inhibition on CLC-1 containing the engineered Cd2+-binding site (Fig. 9 C).

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.