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Elimination of the slow gating of ClC-0 chloride channel by a point mutation.

Lin YW, Lin CW, Chen TY - J. Gen. Physiol. (1999)

Bottom Line: With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition.At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced.These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.

ABSTRACT
The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn2+) from the extracellular side, leading to a Zn2+-induced current inhibition. To further explore the relation of Zn2+ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn2+ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn2+ inhibition. These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.

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(A) Positions of cysteine residues of ClC-0. The membrane topology of the top panel was drawn according to Schmidt-Rose and Jentsch 1997, whereas the bottom panel was drawn according to Fahlke et al. 1997. Placing the D8–D9 linker in intracellular side (bottom), however, violates the fact that there is a glycosylation site in this linker (Middleton et al. 1994). (B) Alignment of the amino acid sequences of D5 and D11 from several ClC channels. Stars on top of the sequences denote C212, C213 (in D5), and C480 (in D11) of ClC-0.
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Figure 1: (A) Positions of cysteine residues of ClC-0. The membrane topology of the top panel was drawn according to Schmidt-Rose and Jentsch 1997, whereas the bottom panel was drawn according to Fahlke et al. 1997. Placing the D8–D9 linker in intracellular side (bottom), however, violates the fact that there is a glycosylation site in this linker (Middleton et al. 1994). (B) Alignment of the amino acid sequences of D5 and D11 from several ClC channels. Stars on top of the sequences denote C212, C213 (in D5), and C480 (in D11) of ClC-0.

Mentions: We started with examining the expressions of various cysteine mutants as well as studying their slow-gating behaviors. Fig. 1 A shows the positions of the 12 cysteine residues of ClC-0 based on the putative membrane topology. Among these mutations, 9 of 12 cysteine mutants generated voltage-dependent chloride current similar to that of the wild-type channel. Fig. 2 A shows the quasi–steady state activation curve of the slow gate of wild-type ClC-0. Consistent with previous studies (Pusch et al. 1997; Chen 1998), the open probability of the slow gate is higher at more hyperpolarized membrane potentials. This voltage dependence of the slow gating is different in C212S, C213G, and C480S. These mutants reveal either no voltage dependence (C212S; Fig. 2 B), a reverse voltage dependence with lower open probability at negative potential (C213G; Fig. 2 C), or only slight voltage dependence (C480S; Fig. 2 D). As shown in Fig. 1 B, C212 and C213 of the Torpedo channel are also conserved in ClC-1 and ClC-2, but this conservation occurs only within this branch of the channel family. The channel in the closest branch, for example, ClC-Ka, already loses this conservation. C480 has less conservation, with a corresponding cysteine present only in ClC-1, but not ClC-2. The significance of the conservation of these cysteine residues remains to be determined.


Elimination of the slow gating of ClC-0 chloride channel by a point mutation.

Lin YW, Lin CW, Chen TY - J. Gen. Physiol. (1999)

(A) Positions of cysteine residues of ClC-0. The membrane topology of the top panel was drawn according to Schmidt-Rose and Jentsch 1997, whereas the bottom panel was drawn according to Fahlke et al. 1997. Placing the D8–D9 linker in intracellular side (bottom), however, violates the fact that there is a glycosylation site in this linker (Middleton et al. 1994). (B) Alignment of the amino acid sequences of D5 and D11 from several ClC channels. Stars on top of the sequences denote C212, C213 (in D5), and C480 (in D11) of ClC-0.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229640&req=5

Figure 1: (A) Positions of cysteine residues of ClC-0. The membrane topology of the top panel was drawn according to Schmidt-Rose and Jentsch 1997, whereas the bottom panel was drawn according to Fahlke et al. 1997. Placing the D8–D9 linker in intracellular side (bottom), however, violates the fact that there is a glycosylation site in this linker (Middleton et al. 1994). (B) Alignment of the amino acid sequences of D5 and D11 from several ClC channels. Stars on top of the sequences denote C212, C213 (in D5), and C480 (in D11) of ClC-0.
Mentions: We started with examining the expressions of various cysteine mutants as well as studying their slow-gating behaviors. Fig. 1 A shows the positions of the 12 cysteine residues of ClC-0 based on the putative membrane topology. Among these mutations, 9 of 12 cysteine mutants generated voltage-dependent chloride current similar to that of the wild-type channel. Fig. 2 A shows the quasi–steady state activation curve of the slow gate of wild-type ClC-0. Consistent with previous studies (Pusch et al. 1997; Chen 1998), the open probability of the slow gate is higher at more hyperpolarized membrane potentials. This voltage dependence of the slow gating is different in C212S, C213G, and C480S. These mutants reveal either no voltage dependence (C212S; Fig. 2 B), a reverse voltage dependence with lower open probability at negative potential (C213G; Fig. 2 C), or only slight voltage dependence (C480S; Fig. 2 D). As shown in Fig. 1 B, C212 and C213 of the Torpedo channel are also conserved in ClC-1 and ClC-2, but this conservation occurs only within this branch of the channel family. The channel in the closest branch, for example, ClC-Ka, already loses this conservation. C480 has less conservation, with a corresponding cysteine present only in ClC-1, but not ClC-2. The significance of the conservation of these cysteine residues remains to be determined.

Bottom Line: With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition.At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced.These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.

ABSTRACT
The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn2+) from the extracellular side, leading to a Zn2+-induced current inhibition. To further explore the relation of Zn2+ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn2+ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn2+ inhibition. These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.

Show MeSH
Related in: MedlinePlus