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 language | English (US) |
|---|---|
| Pages (from-to) | 43-58 |
| Number of pages | 16 |
| Journal | Journal of General Physiology |
| Volume | 133 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 2009 |
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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 journal › Article
}
TY - JOUR
T1 - Blocking pore-open mutants of CLC-0 by amphiphilic blockers
AU - Zhang, Xiao Dong
AU - Tseng, Pang Yen
AU - Yu, Wei Ping
AU - Chen, Tsung Yu
PY - 2009/1
Y1 - 2009/1
N2 - 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.
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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UR - http://www.scopus.com/inward/citedby.url?scp=60549111078&partnerID=8YFLogxK
U2 - 10.1085/jgp.200810004
DO - 10.1085/jgp.200810004
M3 - Article
C2 - 19088381
AN - SCOPUS:60549111078
VL - 133
SP - 43
EP - 58
JO - Journal of General Physiology
JF - Journal of General Physiology
SN - 0022-1295
IS - 1
ER -