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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.

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Study of the voltage dependence of CPB-inhibition. Two different pulse protocols are used to study the voltage dependence of on (A) and off (B) CPB inhibition. Recordings obtained from the same patch in 5 mM CPB with protocol A or with protocol B are shown in C and D, respectively. For clarity, only a few current traces are shown at the test voltages indicated. (C) After full removal by the prepulse to +60 mV, the on kinetics and steady-state level of CPB inhibition are measured for various steps to more negative voltages, Vp; the current relaxations at Vp are fitted by double exponential functions (smooth lines) with a fast time constant, τf, very close to that of normal deactivation, and a much larger time constant, τs, reflecting CPB-binding kinetics. At the onset of the following tail pulse to +60 mV, all the noninhibited channels open within ∼1 ms (Accardi and Pusch 2000), yielding an “instantaneous” current proportional to the unbound probability, pU(Vp). (D) After strong inhibition by a prepulse to −140 mV, the conductance increase for various steps to more positive voltages is fitted by a double exponential function for Vp < −60 mV and by a single exponential function for larger Vp's (smooth lines). (E) Shown the pU = punbound values of the same patch shown in C for the single experiment using the pulse data shown in C. pU was calculated as described in the text. The solid lines are fits of , and are shown only for clarity.
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Figure 8: Study of the voltage dependence of CPB-inhibition. Two different pulse protocols are used to study the voltage dependence of on (A) and off (B) CPB inhibition. Recordings obtained from the same patch in 5 mM CPB with protocol A or with protocol B are shown in C and D, respectively. For clarity, only a few current traces are shown at the test voltages indicated. (C) After full removal by the prepulse to +60 mV, the on kinetics and steady-state level of CPB inhibition are measured for various steps to more negative voltages, Vp; the current relaxations at Vp are fitted by double exponential functions (smooth lines) with a fast time constant, τf, very close to that of normal deactivation, and a much larger time constant, τs, reflecting CPB-binding kinetics. At the onset of the following tail pulse to +60 mV, all the noninhibited channels open within ∼1 ms (Accardi and Pusch 2000), yielding an “instantaneous” current proportional to the unbound probability, pU(Vp). (D) After strong inhibition by a prepulse to −140 mV, the conductance increase for various steps to more positive voltages is fitted by a double exponential function for Vp < −60 mV and by a single exponential function for larger Vp's (smooth lines). (E) Shown the pU = punbound values of the same patch shown in C for the single experiment using the pulse data shown in C. pU was calculated as described in the text. The solid lines are fits of , and are shown only for clarity.

Mentions: The experiments discussed above show that both at large negative and at large positive voltages the interaction of the C212S channels with CPB is characterized by relatively slow relaxations of the macroscopic currents that are clearly distinct from the normal gating of drug-free channels. A quantitative dissection of the two processes is in fact possible at all voltages, and was performed using the two types of tail protocols illustrated in Fig. 8. In the first protocol (Fig. 8 A), the channels are first almost completely freed from inhibition and fully activated by a prepulse to +60 mV; they are then allowed to relax for 2 s toward their steady-state condition at a variable test potential, Vp. Finally, the voltage is stepped back to +60 mV. This protocol allows measurements of the voltage dependencies of CPB “on-binding” relaxations (during the conditioning to Vp) and of the steady-state CPB-binding probabilities (from the early currents after the last step to +60 mV; see below). Sample recordings obtained with this protocol from a patch exposed to 5 mM CPB are shown in Fig. 8 C. The second protocol (Fig. 8 B and sample recordings from the same patch are shown in Fig. 8 D) was designed to characterize “off-binding” relaxations. In this case, the channels were first almost maximally inhibited by a pulse to −140 mV, and then allowed to recover from inhibition at various more positive “tail” potentials.


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)

Study of the voltage dependence of CPB-inhibition. Two different pulse protocols are used to study the voltage dependence of on (A) and off (B) CPB inhibition. Recordings obtained from the same patch in 5 mM CPB with protocol A or with protocol B are shown in C and D, respectively. For clarity, only a few current traces are shown at the test voltages indicated. (C) After full removal by the prepulse to +60 mV, the on kinetics and steady-state level of CPB inhibition are measured for various steps to more negative voltages, Vp; the current relaxations at Vp are fitted by double exponential functions (smooth lines) with a fast time constant, τf, very close to that of normal deactivation, and a much larger time constant, τs, reflecting CPB-binding kinetics. At the onset of the following tail pulse to +60 mV, all the noninhibited channels open within ∼1 ms (Accardi and Pusch 2000), yielding an “instantaneous” current proportional to the unbound probability, pU(Vp). (D) After strong inhibition by a prepulse to −140 mV, the conductance increase for various steps to more positive voltages is fitted by a double exponential function for Vp < −60 mV and by a single exponential function for larger Vp's (smooth lines). (E) Shown the pU = punbound values of the same patch shown in C for the single experiment using the pulse data shown in C. pU was calculated as described in the text. The solid lines are fits of , and are shown only for clarity.
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Related In: Results  -  Collection

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

Figure 8: Study of the voltage dependence of CPB-inhibition. Two different pulse protocols are used to study the voltage dependence of on (A) and off (B) CPB inhibition. Recordings obtained from the same patch in 5 mM CPB with protocol A or with protocol B are shown in C and D, respectively. For clarity, only a few current traces are shown at the test voltages indicated. (C) After full removal by the prepulse to +60 mV, the on kinetics and steady-state level of CPB inhibition are measured for various steps to more negative voltages, Vp; the current relaxations at Vp are fitted by double exponential functions (smooth lines) with a fast time constant, τf, very close to that of normal deactivation, and a much larger time constant, τs, reflecting CPB-binding kinetics. At the onset of the following tail pulse to +60 mV, all the noninhibited channels open within ∼1 ms (Accardi and Pusch 2000), yielding an “instantaneous” current proportional to the unbound probability, pU(Vp). (D) After strong inhibition by a prepulse to −140 mV, the conductance increase for various steps to more positive voltages is fitted by a double exponential function for Vp < −60 mV and by a single exponential function for larger Vp's (smooth lines). (E) Shown the pU = punbound values of the same patch shown in C for the single experiment using the pulse data shown in C. pU was calculated as described in the text. The solid lines are fits of , and are shown only for clarity.
Mentions: The experiments discussed above show that both at large negative and at large positive voltages the interaction of the C212S channels with CPB is characterized by relatively slow relaxations of the macroscopic currents that are clearly distinct from the normal gating of drug-free channels. A quantitative dissection of the two processes is in fact possible at all voltages, and was performed using the two types of tail protocols illustrated in Fig. 8. In the first protocol (Fig. 8 A), the channels are first almost completely freed from inhibition and fully activated by a prepulse to +60 mV; they are then allowed to relax for 2 s toward their steady-state condition at a variable test potential, Vp. Finally, the voltage is stepped back to +60 mV. This protocol allows measurements of the voltage dependencies of CPB “on-binding” relaxations (during the conditioning to Vp) and of the steady-state CPB-binding probabilities (from the early currents after the last step to +60 mV; see below). Sample recordings obtained with this protocol from a patch exposed to 5 mM CPB are shown in Fig. 8 C. The second protocol (Fig. 8 B and sample recordings from the same patch are shown in Fig. 8 D) was designed to characterize “off-binding” relaxations. In this case, the channels were first almost maximally inhibited by a pulse to −140 mV, and then allowed to recover from inhibition at various more positive “tail” potentials.

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