Limits...
Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(p-chlorophenoxy)butyric acid (CPB).

Pusch M, Accardi A, Liantonio A, Ferrera L, De Luca A, Camerino DC, Conti F - J. Gen. Physiol. (2001)

Bottom Line: CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state.Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV.As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, I-6149 Genova, Italy. pusch@barolo.icb.ge.cnr.it

ABSTRACT
We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl(-)channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate "locked open" (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V = 60 mV) with an IC50 of approximately 30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with Cl(-) for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.

Show MeSH

Related in: MedlinePlus

Binomial analysis of the single pore block. The occupancy of each of the three conductance levels (0, 1 “pore” open, 2 “pores” open) obtained from the Gaussian fits of Fig. 2 are plotted as bars for each of the four conditions of Fig. 2 ([A] V =−80mV, no CPB; [B] V =−80 mV, 1 mM S(−)-CPB; [C] −100 mV, no CPB; [D] −100 mV, 1 mM S(−)-CPB). The squares in each graph indicate the best fit assuming an independent gating of two identical protopores according to  ([A] p = 0.37; [B] p = 0.15; [C] p = 0.21; [D] p = 0.06). Mean values (n ≥ 3, ±SD) were as follows: for −80 mV, 0 CPB, p = 0.41 ± 0.03; for −80 mV, 1 mM CPB, p = 0.19 ± 0.03; for −100 mV, 0 CPB: p = 0.25 ± 0.03; for −100 mV, 1 mM CPB, p = 0.09 ± 0.02; for −120 mV, 0 CPB, p = 0.18 ± 0.01; and for −120 mV, 1 mM CPB, p = 0.05 ± 0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233749&req=5

Figure 3: Binomial analysis of the single pore block. The occupancy of each of the three conductance levels (0, 1 “pore” open, 2 “pores” open) obtained from the Gaussian fits of Fig. 2 are plotted as bars for each of the four conditions of Fig. 2 ([A] V =−80mV, no CPB; [B] V =−80 mV, 1 mM S(−)-CPB; [C] −100 mV, no CPB; [D] −100 mV, 1 mM S(−)-CPB). The squares in each graph indicate the best fit assuming an independent gating of two identical protopores according to ([A] p = 0.37; [B] p = 0.15; [C] p = 0.21; [D] p = 0.06). Mean values (n ≥ 3, ±SD) were as follows: for −80 mV, 0 CPB, p = 0.41 ± 0.03; for −80 mV, 1 mM CPB, p = 0.19 ± 0.03; for −100 mV, 0 CPB: p = 0.25 ± 0.03; for −100 mV, 1 mM CPB, p = 0.09 ± 0.02; for −120 mV, 0 CPB, p = 0.18 ± 0.01; and for −120 mV, 1 mM CPB, p = 0.05 ± 0.01.

Mentions: CPB could inhibit the permeation of the single protopores of the double-barreled channel, or it could somehow shut down the conduction of both protopores simultaneously; e.g., by blocking a common pathway or by overcoming and reversing the effect of the C212S mutation and keeping the slow gate of ClC-0 closed. This question can be answered by single-channel recordings such as those illustrated in Fig. 2. The figure shows short stretches of recordings of a single C212S channel at −80 mV (top traces) and −100 mV (bottom traces) in the absence of CPB (left traces) and with 1 mM CPB present (right traces). The double-barreled appearance of the channel is evident by the presence of two equally spaced nonzero current levels that are never observed singly (see also Lin et al. 1999). CPB clearly decreases the mean time spent by the channel in any of the two conductive states, but it has no significant effect on their conductance (Fig. 2, dashed lines). From the latter observation, we can conclude that on-off kinetics does not cause flickering on the time scale of channel openings, i.e., the drug is not a fast open-pore blocker. Both in the absence and in the presence of CPB, the amplitude histograms of the recordings (shown next to the traces) could be well fitted with the sum of three Gaussian distributions from which the occupation probabilities of the three conductance levels, p0, p1, and p2, were obtained (Fig. 3, bars). In the absence of CPB, these could be very well approximated by a binomial distribution of the form: 6\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\begin{matrix}{\mathrm{p}}_{0}= \left \left(1-{\mathrm{p}}\right) \right ^{2}\\ {\mathrm{p}}_{1}=2{\mathrm{p}} \left \left(1-{\mathrm{p}}\right) \right \\ {\mathrm{p}}_{2}={\mathrm{p}}^{2}{\mathrm{,}}\end{matrix}\end{equation*}\end{document} as expected if the measured current arises from the random openings of two independent pores with open probability p = 0.37 at −80 mV and p = 0.21 at −100 mV (Fig. 3A and Fig. C, squares). Also in the presence of CPB, the occupation probabilities could be very well described by a binomial distribution (Fig. 3B and Fig. D, squares, also see legend), but using reduced single-pore open probabilities, p′= 0.15 at −80 mV and p′= 0.06 at −100 mV. This result strongly supports the idea that CPB acts independently on single protopores, because if CPB prevented the opening or the permeation of both pores simultaneously, the probability distribution would change quite differently. In that case, the current would be zero while the channel binds CPB, whereas the relative probability of the two current levels while the channel is drug-free would remain the same and so would the observed ratio, p2/p1. The experimental data are in strong contrast with this prediction: in the experiment of Fig. 2, in the absence of CPB, the estimated values of p2/p1 were 0.27 at −80 mV and 0.11 at −100 mV, whereas in the presence of 1 mM CPB, the corresponding estimates were 0.10 and 0.02. A more direct qualitative demonstration that CPB acts on single protopores is the observation under drug application of numerous relatively long epochs containing only openings to the low conductance level (Fig. 4 B). We interpret these epochs as periods during which only one of the two protopores is inhibited by CPB. The comparison with recordings from the same channel before CPB addition (Fig. 4 A) shows that their spontaneous occurrence in the unaffected channel has a vanishingly small probability, and a simultaneous effect of CPB on both protopores cannot explain their appearance.


Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(p-chlorophenoxy)butyric acid (CPB).

Pusch M, Accardi A, Liantonio A, Ferrera L, De Luca A, Camerino DC, Conti F - J. Gen. Physiol. (2001)

Binomial analysis of the single pore block. The occupancy of each of the three conductance levels (0, 1 “pore” open, 2 “pores” open) obtained from the Gaussian fits of Fig. 2 are plotted as bars for each of the four conditions of Fig. 2 ([A] V =−80mV, no CPB; [B] V =−80 mV, 1 mM S(−)-CPB; [C] −100 mV, no CPB; [D] −100 mV, 1 mM S(−)-CPB). The squares in each graph indicate the best fit assuming an independent gating of two identical protopores according to  ([A] p = 0.37; [B] p = 0.15; [C] p = 0.21; [D] p = 0.06). Mean values (n ≥ 3, ±SD) were as follows: for −80 mV, 0 CPB, p = 0.41 ± 0.03; for −80 mV, 1 mM CPB, p = 0.19 ± 0.03; for −100 mV, 0 CPB: p = 0.25 ± 0.03; for −100 mV, 1 mM CPB, p = 0.09 ± 0.02; for −120 mV, 0 CPB, p = 0.18 ± 0.01; and for −120 mV, 1 mM CPB, p = 0.05 ± 0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233749&req=5

Figure 3: Binomial analysis of the single pore block. The occupancy of each of the three conductance levels (0, 1 “pore” open, 2 “pores” open) obtained from the Gaussian fits of Fig. 2 are plotted as bars for each of the four conditions of Fig. 2 ([A] V =−80mV, no CPB; [B] V =−80 mV, 1 mM S(−)-CPB; [C] −100 mV, no CPB; [D] −100 mV, 1 mM S(−)-CPB). The squares in each graph indicate the best fit assuming an independent gating of two identical protopores according to ([A] p = 0.37; [B] p = 0.15; [C] p = 0.21; [D] p = 0.06). Mean values (n ≥ 3, ±SD) were as follows: for −80 mV, 0 CPB, p = 0.41 ± 0.03; for −80 mV, 1 mM CPB, p = 0.19 ± 0.03; for −100 mV, 0 CPB: p = 0.25 ± 0.03; for −100 mV, 1 mM CPB, p = 0.09 ± 0.02; for −120 mV, 0 CPB, p = 0.18 ± 0.01; and for −120 mV, 1 mM CPB, p = 0.05 ± 0.01.
Mentions: CPB could inhibit the permeation of the single protopores of the double-barreled channel, or it could somehow shut down the conduction of both protopores simultaneously; e.g., by blocking a common pathway or by overcoming and reversing the effect of the C212S mutation and keeping the slow gate of ClC-0 closed. This question can be answered by single-channel recordings such as those illustrated in Fig. 2. The figure shows short stretches of recordings of a single C212S channel at −80 mV (top traces) and −100 mV (bottom traces) in the absence of CPB (left traces) and with 1 mM CPB present (right traces). The double-barreled appearance of the channel is evident by the presence of two equally spaced nonzero current levels that are never observed singly (see also Lin et al. 1999). CPB clearly decreases the mean time spent by the channel in any of the two conductive states, but it has no significant effect on their conductance (Fig. 2, dashed lines). From the latter observation, we can conclude that on-off kinetics does not cause flickering on the time scale of channel openings, i.e., the drug is not a fast open-pore blocker. Both in the absence and in the presence of CPB, the amplitude histograms of the recordings (shown next to the traces) could be well fitted with the sum of three Gaussian distributions from which the occupation probabilities of the three conductance levels, p0, p1, and p2, were obtained (Fig. 3, bars). In the absence of CPB, these could be very well approximated by a binomial distribution of the form: 6\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\begin{matrix}{\mathrm{p}}_{0}= \left \left(1-{\mathrm{p}}\right) \right ^{2}\\ {\mathrm{p}}_{1}=2{\mathrm{p}} \left \left(1-{\mathrm{p}}\right) \right \\ {\mathrm{p}}_{2}={\mathrm{p}}^{2}{\mathrm{,}}\end{matrix}\end{equation*}\end{document} as expected if the measured current arises from the random openings of two independent pores with open probability p = 0.37 at −80 mV and p = 0.21 at −100 mV (Fig. 3A and Fig. C, squares). Also in the presence of CPB, the occupation probabilities could be very well described by a binomial distribution (Fig. 3B and Fig. D, squares, also see legend), but using reduced single-pore open probabilities, p′= 0.15 at −80 mV and p′= 0.06 at −100 mV. This result strongly supports the idea that CPB acts independently on single protopores, because if CPB prevented the opening or the permeation of both pores simultaneously, the probability distribution would change quite differently. In that case, the current would be zero while the channel binds CPB, whereas the relative probability of the two current levels while the channel is drug-free would remain the same and so would the observed ratio, p2/p1. The experimental data are in strong contrast with this prediction: in the experiment of Fig. 2, in the absence of CPB, the estimated values of p2/p1 were 0.27 at −80 mV and 0.11 at −100 mV, whereas in the presence of 1 mM CPB, the corresponding estimates were 0.10 and 0.02. A more direct qualitative demonstration that CPB acts on single protopores is the observation under drug application of numerous relatively long epochs containing only openings to the low conductance level (Fig. 4 B). We interpret these epochs as periods during which only one of the two protopores is inhibited by CPB. The comparison with recordings from the same channel before CPB addition (Fig. 4 A) shows that their spontaneous occurrence in the unaffected channel has a vanishingly small probability, and a simultaneous effect of CPB on both protopores cannot explain their appearance.

Bottom Line: CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state.Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV.As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, I-6149 Genova, Italy. pusch@barolo.icb.ge.cnr.it

ABSTRACT
We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl(-)channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate "locked open" (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V = 60 mV) with an IC50 of approximately 30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with Cl(-) for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.

Show MeSH
Related in: MedlinePlus