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Blocking pore-open mutants of CLC-0 by amphiphilic blockers.

Zhang XD, Tseng PY, Yu WP, Chen TY - J. Gen. Physiol. (2008)

Bottom Line: We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants.In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism.These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore.

View Article: PubMed Central - PubMed

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

ABSTRACT
The blockade of CLC-0 chloride channels by p-chlorophenoxy acetate (CPA) has been thought to be state dependent; the conformational change of the channel pore during the "fast gating" alters the CPA binding affinity. Here, we examine the mechanism of CPA blocking in pore-open mutants of CLC-0 in which the residue E166 was replaced by various amino acids. We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants. On the other hand, mutations at the intracellular pore entrance, although affecting the association and dissociation rates of the CPA block, generate only a modest effect on the steady-state blocking affinity. In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism. The blocking affinity of fatty acids and alkyl sulfonates increases with the length of these amphiphilic blockers, a phenomenon similar to the block of the Shaker K(+) channel by long-chain quaternary ammonium (QA) ions. These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore. This blocking mechanism appears to be very general because the block does not require a precise structural fit between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.

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The effect of octanoate in shifting the fast gate Po-V curve of the wild-type CLC-0 channel.
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fig13: The effect of octanoate in shifting the fast gate Po-V curve of the wild-type CLC-0 channel.

Mentions: Because the octanoate block of pore-open mutants of CLC-0 appears to be similar to the CPA block, we investigated if octanoate, like CPA, can shift the fast gate Po-V curve of wild-type CLC-0. The fast gate Po-V curves of wild-type CLC-0 in the absence and presence of various concentrations of octanoate were constructed from measuring the initial tail current following various tested voltages as described in previous studies (Chen and Chen, 2001; Zhang et al., 2006). Fig. 13 shows that octanoate indeed shifts the fast gate Po-V curve toward more depolarized voltages, and the degree of shift is roughly similar to that obtained by using the same concentration of CPA. These results suggest that the effect of shifting the fast gate Po-V curve of the wild-type CLC-0 is likely related to the pore-blocking mechanism.


Blocking pore-open mutants of CLC-0 by amphiphilic blockers.

Zhang XD, Tseng PY, Yu WP, Chen TY - J. Gen. Physiol. (2008)

The effect of octanoate in shifting the fast gate Po-V curve of the wild-type CLC-0 channel.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2606940&req=5

fig13: The effect of octanoate in shifting the fast gate Po-V curve of the wild-type CLC-0 channel.
Mentions: Because the octanoate block of pore-open mutants of CLC-0 appears to be similar to the CPA block, we investigated if octanoate, like CPA, can shift the fast gate Po-V curve of wild-type CLC-0. The fast gate Po-V curves of wild-type CLC-0 in the absence and presence of various concentrations of octanoate were constructed from measuring the initial tail current following various tested voltages as described in previous studies (Chen and Chen, 2001; Zhang et al., 2006). Fig. 13 shows that octanoate indeed shifts the fast gate Po-V curve toward more depolarized voltages, and the degree of shift is roughly similar to that obtained by using the same concentration of CPA. These results suggest that the effect of shifting the fast gate Po-V curve of the wild-type CLC-0 is likely related to the pore-blocking mechanism.

Bottom Line: We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants.In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism.These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore.

View Article: PubMed Central - PubMed

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

ABSTRACT
The blockade of CLC-0 chloride channels by p-chlorophenoxy acetate (CPA) has been thought to be state dependent; the conformational change of the channel pore during the "fast gating" alters the CPA binding affinity. Here, we examine the mechanism of CPA blocking in pore-open mutants of CLC-0 in which the residue E166 was replaced by various amino acids. We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants. On the other hand, mutations at the intracellular pore entrance, although affecting the association and dissociation rates of the CPA block, generate only a modest effect on the steady-state blocking affinity. In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism. The blocking affinity of fatty acids and alkyl sulfonates increases with the length of these amphiphilic blockers, a phenomenon similar to the block of the Shaker K(+) channel by long-chain quaternary ammonium (QA) ions. These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore. This blocking mechanism appears to be very general because the block does not require a precise structural fit between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.

Show MeSH
Related in: MedlinePlus