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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.

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.


Suppressing the Cd2+ effect in the S294C/Y296H mutant of CLC-1 by intracellular MTS reagents. (A) Micromolar concentrations of intracellular Cd2+ have little effect on the WT CLC-1. The current was monitored continuously by a pulse of 60 mV followed by a voltage step at −100 mV. Intracellular Cd2+ was applied (downward arrows) and washed out (upward arrows) as indicated. Inset shown on the right panel is a comparison of two original recording traces taken before (black) and after (red) the application of 100 µM Cd2+. (B) Intracellular Cd2+ potentiates the current of the S294C/Y296H double mutant of CLC-1. Inset on the right is a comparison of two original recording traces taken before (black) and after (red) 100 µM [Cd2+]. Notice that the current deactivation kinetics at the hyperpolarization voltage caused by the closure of the fast gate is not altered by Cd2+, indicating that the effect is not on the fast gating of the channel. (C) Potentiation of the S294C/Y296H mutant by intracellular Cd2+ is abolished after modifying the channel with intracellular MTSET, indicating that the introduced cysteine and histidine at the positions of S294 and Y296 form a Cd2+-binding site in CLC-1 as well.
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fig9: Suppressing the Cd2+ effect in the S294C/Y296H mutant of CLC-1 by intracellular MTS reagents. (A) Micromolar concentrations of intracellular Cd2+ have little effect on the WT CLC-1. The current was monitored continuously by a pulse of 60 mV followed by a voltage step at −100 mV. Intracellular Cd2+ was applied (downward arrows) and washed out (upward arrows) as indicated. Inset shown on the right panel is a comparison of two original recording traces taken before (black) and after (red) the application of 100 µM Cd2+. (B) Intracellular Cd2+ potentiates the current of the S294C/Y296H double mutant of CLC-1. Inset on the right is a comparison of two original recording traces taken before (black) and after (red) 100 µM [Cd2+]. Notice that the current deactivation kinetics at the hyperpolarization voltage caused by the closure of the fast gate is not altered by Cd2+, indicating that the effect is not on the fast gating of the channel. (C) Potentiation of the S294C/Y296H mutant by intracellular Cd2+ is abolished after modifying the channel with intracellular MTSET, indicating that the introduced cysteine and histidine at the positions of S294 and Y296 form a Cd2+-binding site in CLC-1 as well.

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)

Suppressing the Cd2+ effect in the S294C/Y296H mutant of CLC-1 by intracellular MTS reagents. (A) Micromolar concentrations of intracellular Cd2+ have little effect on the WT CLC-1. The current was monitored continuously by a pulse of 60 mV followed by a voltage step at −100 mV. Intracellular Cd2+ was applied (downward arrows) and washed out (upward arrows) as indicated. Inset shown on the right panel is a comparison of two original recording traces taken before (black) and after (red) the application of 100 µM Cd2+. (B) Intracellular Cd2+ potentiates the current of the S294C/Y296H double mutant of CLC-1. Inset on the right is a comparison of two original recording traces taken before (black) and after (red) 100 µM [Cd2+]. Notice that the current deactivation kinetics at the hyperpolarization voltage caused by the closure of the fast gate is not altered by Cd2+, indicating that the effect is not on the fast gating of the channel. (C) Potentiation of the S294C/Y296H mutant by intracellular Cd2+ is abolished after modifying the channel with intracellular MTSET, indicating that the introduced cysteine and histidine at the positions of S294 and Y296 form a Cd2+-binding site in CLC-1 as well.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: Suppressing the Cd2+ effect in the S294C/Y296H mutant of CLC-1 by intracellular MTS reagents. (A) Micromolar concentrations of intracellular Cd2+ have little effect on the WT CLC-1. The current was monitored continuously by a pulse of 60 mV followed by a voltage step at −100 mV. Intracellular Cd2+ was applied (downward arrows) and washed out (upward arrows) as indicated. Inset shown on the right panel is a comparison of two original recording traces taken before (black) and after (red) the application of 100 µM Cd2+. (B) Intracellular Cd2+ potentiates the current of the S294C/Y296H double mutant of CLC-1. Inset on the right is a comparison of two original recording traces taken before (black) and after (red) 100 µM [Cd2+]. Notice that the current deactivation kinetics at the hyperpolarization voltage caused by the closure of the fast gate is not altered by Cd2+, indicating that the effect is not on the fast gating of the channel. (C) Potentiation of the S294C/Y296H mutant by intracellular Cd2+ is abolished after modifying the channel with intracellular MTSET, indicating that the introduced cysteine and histidine at the positions of S294 and Y296 form a Cd2+-binding site in CLC-1 as well.
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.