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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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Inhibition of the E166C, E166L, and E166K mutants by CPA. (A) Macroscopic current recordings of these mutants in the absence (top) and presence (bottom) of 0.3 mM CPA. The horizontal and vertical scale bars in each mutant are 50 ms and 1 nA, respectively. (B) Percentage of current inhibitions at different voltages in various concentrations of CPA. [CPA] was (in mM): E166C: 0.03, 0.1, 0.3, 1, and 3; E166L: 0.03, 0.1, 0.3, 1, and 3; and E166K: 0.3, 1, 3, 10, and 30.
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fig2: Inhibition of the E166C, E166L, and E166K mutants by CPA. (A) Macroscopic current recordings of these mutants in the absence (top) and presence (bottom) of 0.3 mM CPA. The horizontal and vertical scale bars in each mutant are 50 ms and 1 nA, respectively. (B) Percentage of current inhibitions at different voltages in various concentrations of CPA. [CPA] was (in mM): E166C: 0.03, 0.1, 0.3, 1, and 3; E166L: 0.03, 0.1, 0.3, 1, and 3; and E166K: 0.3, 1, 3, 10, and 30.

Mentions: The original recording trace and the percentage of CPA inhibition of the E166C, E166L, and E166K mutants are shown in Fig. 2 (A and B, respectively). Again, these mutants show different sensitivities to the CPA inhibition. At −160 mV, while 0.3 mM CPA blocks >90% of the E166C current, the same concentration of CPA blocks ∼60% of the E166L current and only ∼10% of the E166K current. To evaluate the apparent CPA-blocking affinity in various mutants of E166, we plotted the fraction of the unblocked current as a function of [CPA], and the half-blocking concentration (K1/2) was obtained by fitting the data points with a Langmüir function (Eq. 1). The K1/2's of the CPA block at various voltages in different mutants are summarized in Table I, and Fig. 3 A shows the dose-dependent inhibition curves of several mutants at −140 mV. It can be seen that all mutants show voltage-dependent block—the more hyperpolarized the membrane potential, the higher the CPA-blocking affinity (Table I). However, different mutants have very different sensitivities for the CPA block. Fig. 3 B shows a plot of the apparent CPA affinities as a function of the side chain volume of the residue placed at position 166. It appears that two factors determine the steady-state blocking affinity: the side chain volume and the hydrophobicity of the introduced residue. Roughly speaking, the larger the side chain volume is, the lower the blocking affinity is. Moreover, a hydrophobic residue at position 166 (such as in E166A, E166V, and E166I mutants) renders the channel sensitive to the CPA block. The numbers associated with the data points in Fig. 3 B are the hydrophobic index of the amino acid (Kyte and Doolittle, 1982)—the more positive the number, the higher the hydrophobicity of the amino acid. At −140 mV, the mutant with a hydrophobic side chain (positive hydrophobic index) shows a K1/2 of <200 μM. On the other hand, placing a hydrophilic residue with a bulky side chain (such as E166, E166K, and E166Q) renders the channel insensitive to the CPA block; the K1/2 is on the order of several mM for these three channels (Fig. 3 B). A hydrophilic residue with a small side chain (such as serine) can still make the mutant sensitive to the CPA block. Overall, these mutants can be roughly classified into two groups (encompassed by the magenta and cyan circles, respectively, in Fig. 3 B), except for the E166G mutant, for which the introduced glycine has only a hydrogen atom on the side chain. The block of E166G by CPA is unique and is described in this issue (see p. 59) by Zhang and Chen (2008). However, this classification of the CPA block for various mutants into two groups has exceptions. The amino acids histidine, tyrosine, and tryptophan have a negative hydrophobic index (hydrophilic side chains), yet the CPA affinities of E166H, E166Y, and E166W are relatively higher than the other mutants in the hydrophilic category. As will be shown later, the ring structure on the side chain of these three residues, and the other amino acid phenylalanine (shown by red solid squares in Fig. 3 B), may cause this anomaly. The observation that the blocking affinity depends upon the side chain of the residue suggests that perhaps the CPA molecule directly interacts with the introduced residue at position 166. That CPA interacts with the E166 side chain was also suggested in a previous mutation study (Estevez et al., 2003).


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

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

Inhibition of the E166C, E166L, and E166K mutants by CPA. (A) Macroscopic current recordings of these mutants in the absence (top) and presence (bottom) of 0.3 mM CPA. The horizontal and vertical scale bars in each mutant are 50 ms and 1 nA, respectively. (B) Percentage of current inhibitions at different voltages in various concentrations of CPA. [CPA] was (in mM): E166C: 0.03, 0.1, 0.3, 1, and 3; E166L: 0.03, 0.1, 0.3, 1, and 3; and E166K: 0.3, 1, 3, 10, and 30.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2606940&req=5

fig2: Inhibition of the E166C, E166L, and E166K mutants by CPA. (A) Macroscopic current recordings of these mutants in the absence (top) and presence (bottom) of 0.3 mM CPA. The horizontal and vertical scale bars in each mutant are 50 ms and 1 nA, respectively. (B) Percentage of current inhibitions at different voltages in various concentrations of CPA. [CPA] was (in mM): E166C: 0.03, 0.1, 0.3, 1, and 3; E166L: 0.03, 0.1, 0.3, 1, and 3; and E166K: 0.3, 1, 3, 10, and 30.
Mentions: The original recording trace and the percentage of CPA inhibition of the E166C, E166L, and E166K mutants are shown in Fig. 2 (A and B, respectively). Again, these mutants show different sensitivities to the CPA inhibition. At −160 mV, while 0.3 mM CPA blocks >90% of the E166C current, the same concentration of CPA blocks ∼60% of the E166L current and only ∼10% of the E166K current. To evaluate the apparent CPA-blocking affinity in various mutants of E166, we plotted the fraction of the unblocked current as a function of [CPA], and the half-blocking concentration (K1/2) was obtained by fitting the data points with a Langmüir function (Eq. 1). The K1/2's of the CPA block at various voltages in different mutants are summarized in Table I, and Fig. 3 A shows the dose-dependent inhibition curves of several mutants at −140 mV. It can be seen that all mutants show voltage-dependent block—the more hyperpolarized the membrane potential, the higher the CPA-blocking affinity (Table I). However, different mutants have very different sensitivities for the CPA block. Fig. 3 B shows a plot of the apparent CPA affinities as a function of the side chain volume of the residue placed at position 166. It appears that two factors determine the steady-state blocking affinity: the side chain volume and the hydrophobicity of the introduced residue. Roughly speaking, the larger the side chain volume is, the lower the blocking affinity is. Moreover, a hydrophobic residue at position 166 (such as in E166A, E166V, and E166I mutants) renders the channel sensitive to the CPA block. The numbers associated with the data points in Fig. 3 B are the hydrophobic index of the amino acid (Kyte and Doolittle, 1982)—the more positive the number, the higher the hydrophobicity of the amino acid. At −140 mV, the mutant with a hydrophobic side chain (positive hydrophobic index) shows a K1/2 of <200 μM. On the other hand, placing a hydrophilic residue with a bulky side chain (such as E166, E166K, and E166Q) renders the channel insensitive to the CPA block; the K1/2 is on the order of several mM for these three channels (Fig. 3 B). A hydrophilic residue with a small side chain (such as serine) can still make the mutant sensitive to the CPA block. Overall, these mutants can be roughly classified into two groups (encompassed by the magenta and cyan circles, respectively, in Fig. 3 B), except for the E166G mutant, for which the introduced glycine has only a hydrogen atom on the side chain. The block of E166G by CPA is unique and is described in this issue (see p. 59) by Zhang and Chen (2008). However, this classification of the CPA block for various mutants into two groups has exceptions. The amino acids histidine, tyrosine, and tryptophan have a negative hydrophobic index (hydrophilic side chains), yet the CPA affinities of E166H, E166Y, and E166W are relatively higher than the other mutants in the hydrophilic category. As will be shown later, the ring structure on the side chain of these three residues, and the other amino acid phenylalanine (shown by red solid squares in Fig. 3 B), may cause this anomaly. The observation that the blocking affinity depends upon the side chain of the residue suggests that perhaps the CPA molecule directly interacts with the introduced residue at position 166. That CPA interacts with the E166 side chain was also suggested in a previous mutation study (Estevez et al., 2003).

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