Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of mutations at the intracellular pore entrance on the kon and koff of the CPA block. All mutants were with the E166C background mutation. (A) Recording traces of the E166C (black trace) and the E166C/K519E (red trace) mutants. On the left are the original current traces of these two mutants obtained by stepping the membrane voltage from +80 to −160 mV. On the right, the initial current amplitude (after the capacitance spike) of the E166C/K519E mutant at −160 mV is scaled to the same initial value as that of the E166C mutant. The inverse of the time constant of the current inhibition course is plotted against [CPA] in the bottom panel to evaluate kon and koff according to Eq. 3. (B) Analysis of the apparent kon and koff of the CPA block at −160 mV among three channels in which K, M, and E are at position 519 of the E166C mutant, respectively. The fitted kon and koff values were: E166C/K519: 6.3 × 105 M−1s−1 and 17.2 s−1; E166C/K519M: 1.7 × 105 M−1s−1 and 10.5 s−1; and E166C/K519E: 1.1 × 105 M−1s−1 and 9.5 s−1. Notice that both kon and koff are reduced. Thus, the effects of these mutations on the apparent blocker affinity (shown in Fig. 6) are small. (C) Comparison of the apparent kon and koff of the CPA block at −160 mV among four E127Q/K519M combinatorial mutations in the E166C background. The kon and koff values were: E166C/E127Q: 7.6 × 105 M−1s−1 and 17.9 s−1; and E166C/E127Q/K519M: 4.6 × 105 M−1s−1 and 11.4 s−1.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Effects of mutations at the intracellular pore entrance on the kon and koff of the CPA block. All mutants were with the E166C background mutation. (A) Recording traces of the E166C (black trace) and the E166C/K519E (red trace) mutants. On the left are the original current traces of these two mutants obtained by stepping the membrane voltage from +80 to −160 mV. On the right, the initial current amplitude (after the capacitance spike) of the E166C/K519E mutant at −160 mV is scaled to the same initial value as that of the E166C mutant. The inverse of the time constant of the current inhibition course is plotted against [CPA] in the bottom panel to evaluate kon and koff according to Eq. 3. (B) Analysis of the apparent kon and koff of the CPA block at −160 mV among three channels in which K, M, and E are at position 519 of the E166C mutant, respectively. The fitted kon and koff values were: E166C/K519: 6.3 × 105 M−1s−1 and 17.2 s−1; E166C/K519M: 1.7 × 105 M−1s−1 and 10.5 s−1; and E166C/K519E: 1.1 × 105 M−1s−1 and 9.5 s−1. Notice that both kon and koff are reduced. Thus, the effects of these mutations on the apparent blocker affinity (shown in Fig. 6) are small. (C) Comparison of the apparent kon and koff of the CPA block at −160 mV among four E127Q/K519M combinatorial mutations in the E166C background. The kon and koff values were: E166C/E127Q: 7.6 × 105 M−1s−1 and 17.9 s−1; and E166C/E127Q/K519M: 4.6 × 105 M−1s−1 and 11.4 s−1.

Mentions: Previous experiments showed that the K519E mutation in the background of E166A mutant renders the inhibition process slower (Traverso et al., 2003). Therefore, we also examined the effects of the K519/E127 combinatorial mutations on the CPA inhibition kinetics. Consistent with the previous report, the K519E mutation indeed slows down the CPA inhibition kinetics (Fig. 7 A, top). Analysis of the CPA inhibition rate (1/τ) between E166C and E166C/K519E mutants (Fig. 7 A, bottom) shows that the K519E mutation reduces the apparent kon and koff by approximately six- and twofold, respectively, at −160 mV. Fig. 7 (B and C) displays similar comparisons among various combinatorial mutations of K519 and E127, all in the background of the E166C mutation. It can be seen that the major effect caused by the mutations of E127 or K519 is a change of kon. These mutations also change the koff of CPA block, although such a change is not easily discerned at the very negative voltage of −160 mV (Fig. 7, B and C).


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

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

Effects of mutations at the intracellular pore entrance on the kon and koff of the CPA block. All mutants were with the E166C background mutation. (A) Recording traces of the E166C (black trace) and the E166C/K519E (red trace) mutants. On the left are the original current traces of these two mutants obtained by stepping the membrane voltage from +80 to −160 mV. On the right, the initial current amplitude (after the capacitance spike) of the E166C/K519E mutant at −160 mV is scaled to the same initial value as that of the E166C mutant. The inverse of the time constant of the current inhibition course is plotted against [CPA] in the bottom panel to evaluate kon and koff according to Eq. 3. (B) Analysis of the apparent kon and koff of the CPA block at −160 mV among three channels in which K, M, and E are at position 519 of the E166C mutant, respectively. The fitted kon and koff values were: E166C/K519: 6.3 × 105 M−1s−1 and 17.2 s−1; E166C/K519M: 1.7 × 105 M−1s−1 and 10.5 s−1; and E166C/K519E: 1.1 × 105 M−1s−1 and 9.5 s−1. Notice that both kon and koff are reduced. Thus, the effects of these mutations on the apparent blocker affinity (shown in Fig. 6) are small. (C) Comparison of the apparent kon and koff of the CPA block at −160 mV among four E127Q/K519M combinatorial mutations in the E166C background. The kon and koff values were: E166C/E127Q: 7.6 × 105 M−1s−1 and 17.9 s−1; and E166C/E127Q/K519M: 4.6 × 105 M−1s−1 and 11.4 s−1.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Effects of mutations at the intracellular pore entrance on the kon and koff of the CPA block. All mutants were with the E166C background mutation. (A) Recording traces of the E166C (black trace) and the E166C/K519E (red trace) mutants. On the left are the original current traces of these two mutants obtained by stepping the membrane voltage from +80 to −160 mV. On the right, the initial current amplitude (after the capacitance spike) of the E166C/K519E mutant at −160 mV is scaled to the same initial value as that of the E166C mutant. The inverse of the time constant of the current inhibition course is plotted against [CPA] in the bottom panel to evaluate kon and koff according to Eq. 3. (B) Analysis of the apparent kon and koff of the CPA block at −160 mV among three channels in which K, M, and E are at position 519 of the E166C mutant, respectively. The fitted kon and koff values were: E166C/K519: 6.3 × 105 M−1s−1 and 17.2 s−1; E166C/K519M: 1.7 × 105 M−1s−1 and 10.5 s−1; and E166C/K519E: 1.1 × 105 M−1s−1 and 9.5 s−1. Notice that both kon and koff are reduced. Thus, the effects of these mutations on the apparent blocker affinity (shown in Fig. 6) are small. (C) Comparison of the apparent kon and koff of the CPA block at −160 mV among four E127Q/K519M combinatorial mutations in the E166C background. The kon and koff values were: E166C/E127Q: 7.6 × 105 M−1s−1 and 17.9 s−1; and E166C/E127Q/K519M: 4.6 × 105 M−1s−1 and 11.4 s−1.
Mentions: Previous experiments showed that the K519E mutation in the background of E166A mutant renders the inhibition process slower (Traverso et al., 2003). Therefore, we also examined the effects of the K519/E127 combinatorial mutations on the CPA inhibition kinetics. Consistent with the previous report, the K519E mutation indeed slows down the CPA inhibition kinetics (Fig. 7 A, top). Analysis of the CPA inhibition rate (1/τ) between E166C and E166C/K519E mutants (Fig. 7 A, bottom) shows that the K519E mutation reduces the apparent kon and koff by approximately six- and twofold, respectively, at −160 mV. Fig. 7 (B and C) displays similar comparisons among various combinatorial mutations of K519 and E127, all in the background of the E166C mutation. It can be seen that the major effect caused by the mutations of E127 or K519 is a change of kon. These mutations also change the koff of CPA block, although such a change is not easily discerned at the very negative voltage of −160 mV (Fig. 7, B and C).

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