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

Xiao Dong Zhang, Pang Yen Tseng, Wei Ping Yu, Tsung Yu Chen

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 affi nity. 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 fi nd that the CPA-blocking affi nities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affi nity 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 affi nity. 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 affi nity 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 fi rst 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 fi t between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.

Original languageEnglish (US)
Pages (from-to)43-58
Number of pages16
JournalJournal of General Physiology
Volume133
Issue number1
DOIs
StatePublished - Jan 2009

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Acetates
Alkanesulfonates
Fatty Acids
Voltage-Gated Potassium Channels
Chloride Channels
Hydrophobic and Hydrophilic Interactions
Ammonium Compounds
Anions
Cations
Ions
Amino Acids
Peptides
Mutation

ASJC Scopus subject areas

  • Physiology
  • Medicine(all)

Cite this

Blocking pore-open mutants of CLC-0 by amphiphilic blockers. / Zhang, Xiao Dong; Tseng, Pang Yen; Yu, Wei Ping; Chen, Tsung Yu.

In: Journal of General Physiology, Vol. 133, No. 1, 01.2009, p. 43-58.

Research output: Contribution to journalArticle

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title = "Blocking pore-open mutants of CLC-0 by amphiphilic blockers",
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 affi nity. 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 fi nd that the CPA-blocking affi nities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affi nity 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 affi nity. 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 affi nity 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 fi rst 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 fi t 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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AB - 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 affi nity. 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 fi nd that the CPA-blocking affi nities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affi nity 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 affi nity. 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 affi nity 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 fi rst 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 fi t 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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