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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 gate open probabilities. (A) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a 10-ms repolarization to −140 mV. The initial part of the 200-ms pulse to −140 mV is not shown. Dashed line represents zero current. (B) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a short 200-μs pulse to +200 mV, and then by a 10-ms repolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Dashed line represents zero current. (C) Open probabilities for the fast (▴) and slow (○) gating processes. Continuous lines are the fits of the open probabilities with . The obtained values are: P0f = 0.16, V1/2f = −77 mV; and zf = 1.03, P0s = 0.65, V1/2s = −51 mV, and zs = 0.81.
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Figure 6: Separation of fast and slow gate open probabilities. (A) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a 10-ms repolarization to −140 mV. The initial part of the 200-ms pulse to −140 mV is not shown. Dashed line represents zero current. (B) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a short 200-μs pulse to +200 mV, and then by a 10-ms repolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Dashed line represents zero current. (C) Open probabilities for the fast (▴) and slow (○) gating processes. Continuous lines are the fits of the open probabilities with . The obtained values are: P0f = 0.16, V1/2f = −77 mV; and zf = 1.03, P0s = 0.65, V1/2s = −51 mV, and zs = 0.81.

Mentions: We used the pulse protocols described in materials and methods to separate the voltage dependence of the fast and slow gates. Fig. 6 A shows typical tail currents recorded when, after 200-ms pulses to various Vp, the voltage is immediately stepped to −140 mV. In Fig. 6 B are shown the currents recorded from the same patch when the voltage is stepped to +200 mV for 200 μs to fully activate the fast gate before the −140-mV repolarization. With these protocols, we are able to separate the steady state open probabilities of the fast and slow gating processes at Vp (see materials and methods). We fitted the open probabilities obtained in this way (Fig. 6 C) to a Boltzmann function with an offset ().


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 gate open probabilities. (A) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a 10-ms repolarization to −140 mV. The initial part of the 200-ms pulse to −140 mV is not shown. Dashed line represents zero current. (B) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a short 200-μs pulse to +200 mV, and then by a 10-ms repolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Dashed line represents zero current. (C) Open probabilities for the fast (▴) and slow (○) gating processes. Continuous lines are the fits of the open probabilities with . The obtained values are: P0f = 0.16, V1/2f = −77 mV; and zf = 1.03, P0s = 0.65, V1/2s = −51 mV, and zs = 0.81.
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Figure 6: Separation of fast and slow gate open probabilities. (A) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a 10-ms repolarization to −140 mV. The initial part of the 200-ms pulse to −140 mV is not shown. Dashed line represents zero current. (B) Currents recorded when 200-ms pulses of increasing voltages from −140 to +100 mV are followed by a short 200-μs pulse to +200 mV, and then by a 10-ms repolarization to −140 mV. The initial part of the 120-ms pulse to −140 mV is not shown. Dashed line represents zero current. (C) Open probabilities for the fast (▴) and slow (○) gating processes. Continuous lines are the fits of the open probabilities with . The obtained values are: P0f = 0.16, V1/2f = −77 mV; and zf = 1.03, P0s = 0.65, V1/2s = −51 mV, and zs = 0.81.
Mentions: We used the pulse protocols described in materials and methods to separate the voltage dependence of the fast and slow gates. Fig. 6 A shows typical tail currents recorded when, after 200-ms pulses to various Vp, the voltage is immediately stepped to −140 mV. In Fig. 6 B are shown the currents recorded from the same patch when the voltage is stepped to +200 mV for 200 μs to fully activate the fast gate before the −140-mV repolarization. With these protocols, we are able to separate the steady state open probabilities of the fast and slow gating processes at Vp (see materials and methods). We fitted the open probabilities obtained in this way (Fig. 6 C) to a Boltzmann function with an offset ().

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