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Fast and slow gating relaxations in the muscle chloride channel CLC-1.

Accardi A, Pusch M - J. Gen. Physiol. (2000)

Bottom Line: Biophys.J. 71:695-706).Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, Via de Marini 6, I-16149 Genova, Italy.

ABSTRACT
Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from -160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to -140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, tau(f), was steeper than that of the slow one, tau(s) (apparent activation valences were z(f) approximately -0. 79 and z(s) approximately -0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (tau(f) approximately 16 micros, tau(s) approximately 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero "offsets" at negative voltages, indicating that the two "gates" never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pH(int), and [Cl](ext) dependence, and the dominant myotonia inducing mutation, I290M. Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.

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Separation of fast and slow gating process of CLC-1. (A) A 120-ms pulse to −140 mV is followed by a short pulse of varying duration to +100 mV, increasing its duration in 10-μs steps. The patch is then hyperpolarized to −140 mV for 15 ms. The thick line is a double-exponential fit of the initial currents recorded upon hyperpolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Only every second trace is shown on the graph. Dashed line represents zero current. (B) Instantaneous currents recorded upon repolarization at −140 mV plotted as a function of the prepulse duration. The continuous line is a two-exponential fit with time constants of 80 μs and 3.4 ms.
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Figure 2: Separation of fast and slow gating process of CLC-1. (A) A 120-ms pulse to −140 mV is followed by a short pulse of varying duration to +100 mV, increasing its duration in 10-μs steps. The patch is then hyperpolarized to −140 mV for 15 ms. The thick line is a double-exponential fit of the initial currents recorded upon hyperpolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Only every second trace is shown on the graph. Dashed line represents zero current. (B) Instantaneous currents recorded upon repolarization at −140 mV plotted as a function of the prepulse duration. The continuous line is a two-exponential fit with time constants of 80 μs and 3.4 ms.

Mentions: Fig. 2 A shows an example of typical currents recorded with an envelope protocol with Vp = 100 mV (see material and methods). The flat trace corresponds to the case where tp = 0 ms. As can be seen, even a brief, 20 μs step to 100 mV is sufficient to induce a relatively large change in the initial current. Increasing the duration of the depolarizing pulse increases the initial current recorded at −140 mV. In Fig. 2 B are shown the initial current values as a function of tp together with a double exponential fit (a single exponential fit is not adequate). The fast time constant in this case (100 mV) was 80 μs. To correctly evaluate the slower time constant, we used protocols similar to those described in Fig. 2, increasing prepulse duration in 100-μs steps instead of 10 μs. An example for Vp = 200 mV is shown in Fig. 3. The first trace in Fig. 3 A corresponds to tp = 40 μs, a pulse duration that is sufficient to saturate the fast gating process at this voltage. In Fig. 3 B, the initial values of the currents recorded upon repolarization (•) are shown as a function of tp. They can be well fitted with a single exponential (continuous line) with a time constant, τs, of 1.1 ms.


Fast and slow gating relaxations in the muscle chloride channel CLC-1.

Accardi A, Pusch M - J. Gen. Physiol. (2000)

Separation of fast and slow gating process of CLC-1. (A) A 120-ms pulse to −140 mV is followed by a short pulse of varying duration to +100 mV, increasing its duration in 10-μs steps. The patch is then hyperpolarized to −140 mV for 15 ms. The thick line is a double-exponential fit of the initial currents recorded upon hyperpolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Only every second trace is shown on the graph. Dashed line represents zero current. (B) Instantaneous currents recorded upon repolarization at −140 mV plotted as a function of the prepulse duration. The continuous line is a two-exponential fit with time constants of 80 μs and 3.4 ms.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233683&req=5

Figure 2: Separation of fast and slow gating process of CLC-1. (A) A 120-ms pulse to −140 mV is followed by a short pulse of varying duration to +100 mV, increasing its duration in 10-μs steps. The patch is then hyperpolarized to −140 mV for 15 ms. The thick line is a double-exponential fit of the initial currents recorded upon hyperpolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Only every second trace is shown on the graph. Dashed line represents zero current. (B) Instantaneous currents recorded upon repolarization at −140 mV plotted as a function of the prepulse duration. The continuous line is a two-exponential fit with time constants of 80 μs and 3.4 ms.
Mentions: Fig. 2 A shows an example of typical currents recorded with an envelope protocol with Vp = 100 mV (see material and methods). The flat trace corresponds to the case where tp = 0 ms. As can be seen, even a brief, 20 μs step to 100 mV is sufficient to induce a relatively large change in the initial current. Increasing the duration of the depolarizing pulse increases the initial current recorded at −140 mV. In Fig. 2 B are shown the initial current values as a function of tp together with a double exponential fit (a single exponential fit is not adequate). The fast time constant in this case (100 mV) was 80 μs. To correctly evaluate the slower time constant, we used protocols similar to those described in Fig. 2, increasing prepulse duration in 100-μs steps instead of 10 μs. An example for Vp = 200 mV is shown in Fig. 3. The first trace in Fig. 3 A corresponds to tp = 40 μs, a pulse duration that is sufficient to saturate the fast gating process at this voltage. In Fig. 3 B, the initial values of the currents recorded upon repolarization (•) are shown as a function of tp. They can be well fitted with a single exponential (continuous line) with a time constant, τs, of 1.1 ms.

Bottom Line: Biophys.J. 71:695-706).Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, Via de Marini 6, I-16149 Genova, Italy.

ABSTRACT
Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from -160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to -140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, tau(f), was steeper than that of the slow one, tau(s) (apparent activation valences were z(f) approximately -0. 79 and z(s) approximately -0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (tau(f) approximately 16 micros, tau(s) approximately 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero "offsets" at negative voltages, indicating that the two "gates" never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pH(int), and [Cl](ext) dependence, and the dominant myotonia inducing mutation, I290M. Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.

Show MeSH
Related in: MedlinePlus