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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.

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Introducing a cysteine and a histidine residue to positions in CLC-1 that correspond to C229 and H231 of CLC-0 enables a modulation of CLC-1 by micromolar [Cd2+]. (A) The overall channel open probability (Po; top) and the common-gate open probability (Poc; bottom) of WT CLC-1 as a function of voltage in the absence (control, black) and presence of 100 µM Cd2+ (blue). (B) The Po–V and Poc–V curves of the S294C/Y296H mutant of CLC-1 in various intracellular [Cd2+]. C229 and H231 of CLC-0 are not conserved in CLC-1, and as expected, 100 µM Cd2+ has nearly no effect on WT CLC-1 (Fahlke et al., 1998). However, 10 µM Cd2+ significantly increases the open probability of the common gate (Poc) of the CLC-1 mutant S294C/Y296H. Notice the opposite effect of Cd2+ on the open probability of CLC-1 (potentiation) compared with that of CLC-0 (inhibition).
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fig8: Introducing a cysteine and a histidine residue to positions in CLC-1 that correspond to C229 and H231 of CLC-0 enables a modulation of CLC-1 by micromolar [Cd2+]. (A) The overall channel open probability (Po; top) and the common-gate open probability (Poc; bottom) of WT CLC-1 as a function of voltage in the absence (control, black) and presence of 100 µM Cd2+ (blue). (B) The Po–V and Poc–V curves of the S294C/Y296H mutant of CLC-1 in various intracellular [Cd2+]. C229 and H231 of CLC-0 are not conserved in CLC-1, and as expected, 100 µM Cd2+ has nearly no effect on WT CLC-1 (Fahlke et al., 1998). However, 10 µM Cd2+ significantly increases the open probability of the common gate (Poc) of the CLC-1 mutant S294C/Y296H. Notice the opposite effect of Cd2+ on the open probability of CLC-1 (potentiation) compared with that of CLC-0 (inhibition).

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)

Introducing a cysteine and a histidine residue to positions in CLC-1 that correspond to C229 and H231 of CLC-0 enables a modulation of CLC-1 by micromolar [Cd2+]. (A) The overall channel open probability (Po; top) and the common-gate open probability (Poc; bottom) of WT CLC-1 as a function of voltage in the absence (control, black) and presence of 100 µM Cd2+ (blue). (B) The Po–V and Poc–V curves of the S294C/Y296H mutant of CLC-1 in various intracellular [Cd2+]. C229 and H231 of CLC-0 are not conserved in CLC-1, and as expected, 100 µM Cd2+ has nearly no effect on WT CLC-1 (Fahlke et al., 1998). However, 10 µM Cd2+ significantly increases the open probability of the common gate (Poc) of the CLC-1 mutant S294C/Y296H. Notice the opposite effect of Cd2+ on the open probability of CLC-1 (potentiation) compared with that of CLC-0 (inhibition).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: Introducing a cysteine and a histidine residue to positions in CLC-1 that correspond to C229 and H231 of CLC-0 enables a modulation of CLC-1 by micromolar [Cd2+]. (A) The overall channel open probability (Po; top) and the common-gate open probability (Poc; bottom) of WT CLC-1 as a function of voltage in the absence (control, black) and presence of 100 µM Cd2+ (blue). (B) The Po–V and Poc–V curves of the S294C/Y296H mutant of CLC-1 in various intracellular [Cd2+]. C229 and H231 of CLC-0 are not conserved in CLC-1, and as expected, 100 µM Cd2+ has nearly no effect on WT CLC-1 (Fahlke et al., 1998). However, 10 µM Cd2+ significantly increases the open probability of the common gate (Poc) of the CLC-1 mutant S294C/Y296H. Notice the opposite effect of Cd2+ on the open probability of CLC-1 (potentiation) compared with that of CLC-0 (inhibition).
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.


Related in: MedlinePlus