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

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.

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.

Figure 1

Limitations of the envelope protocols at high voltages. (A) Capacity artifact recorded when stepping from a holding potential of −140 to +200 mV for 1 ms due to a patch of a noninjected oocyte, the patch pipette and the holder (solid line), or from the pipette holder only (dashed line). (B) Typical current recorded with an envelope protocol to +200 mV, with t _p = 180 μs. Cursor positions used for the fitting are indicated by arrows: A = 0 (beginning of the repolarization pulse), B = 225 μs, C = 470 μs, D = 610 μs, and E = 4,775 μs. Three single-exponential fits were performed (that were superimposable). In the first (continuous line), the trace was fitted between B and E and extrapolated to A (set 1); in the second (thick dashed line, indistinguishable from the thick continuous line), the fit was done between D and E and extrapolated to C (set 2); in the third (not shown), the trace was fitted between D and E and extrapolated to A (set 3). (C) Initial currents recorded upon repolarization for a whole envelope protocol using set 1 of the cursor positions (○), set 2 (□) or set 3 (▵). The continuous lines are double-exponential fits of the initial currents recorded upon repolarization in all three cases. Only two curves are clearly distinguishable since the traces corresponding to sets 1 and 3 are almost superimposable (and make up the thicker line), while the thinner visible curve corresponds to set 2. The time constants found are: τ_f(set 1) = 16 μs, τ_s(set 1) = 467 μs; τ_f(set 2) = 17 μs, τ_s(set 2) = 456 μs; τ_f(set 3) = 17 μs, τ_s(set 3) = 462 μs. (D) Double (continuous line) and the single (dashed line) exponential fit of the initial currents evaluated with set 1 of cursor positions but without the first three points (those with t _p = 10, 20, and 30 μs) to avoid contamination from phenomena of partial charging of the membrane. Clearly, a single-exponential function (dashed line) is inadequate to fit the data. The time constants obtained are τ_f = 19 μs and τ_s = 590 μs.

Figure 3

Determination of the slow time constant of CLC-1. (A) A 120-ms pulse to −140 mV is followed by a pulse of varying duration to +200 mV, increasing its duration in 100-μs steps starting from a minimum of 40 μs. The patch is then repolarized to −140 mV for 10 ms. 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 fast gate is maximally activated by the prepulse, thus the recorded currents reflect only the slow gate activation. A single exponential fit gives a time constant of 1.1 ms.

Figure 4

Fast and slow time constants as evaluated with envelope protocols are plotted as a function of voltage for different experimental conditions. Circles, standard conditions; squares, low external chloride; triangles, low internal pH (see materials and methods); filled symbols, fast gate time constants; open symbols, slow gate time constants. Error bars indicate SEM. Continuous lines are exponential fits () of the fast and slow gate time constants in standard conditions yielding activation gating charges z ^f _standard = −0.79 and z ^s _standard = −0.42. Similar analysis was performed for low chloride and low pH also, but the fits are not shown. The dashed horizontal line indicates the limit of 30 μs. Data points below this value were not included in the fitting procedures because of possible limitations caused by a limited voltage clamp rise time (see materials and methods for details).

Figure 5

Evaluation of the fast gate time constant of CLC-0 with envelope protocols. (A) A 120-ms pulse to −120 mV followed by a pulse of varying duration to +80 mV is applied to the patch, increasing its duration in 80-μs steps. The patch is then hyperpolarized to −120 mV for 18 ms. The thick line is the single exponential fit of the initial values recorded upon repolarization. Its intercept with the current-trace reflects the best estimate of the initial current recorded upon repolarization. The downward “peak” of the individual traces is caused by the capacitive transient and thus does not faithfully reflect the ionic current flowing through the open channels (see also Fig. 1 B). The initial part of the 120-ms pulse to −120 mV is not shown. The dashed line represents zero current. (B) Time constants of the fast gate of CLC-0 measured both with envelope protocols (□) and by direct fitting of the current during the activation pulse (▵). The continuous line is a single exponential fit () of the fast gate time constants that yields z ^f _CLC-0 = −0.55.

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: P ₀ ^f = 0.16, V_1/2 ^f = −77 mV; and z ^f = 1.03, P ₀ ^s = 0.65, V_1/2 ^s = −51 mV, and z ^s = 0.81.

Figure 8

Currents evoked with protocols similar to those described in Fig. 6A and Fig. B, in different experimental conditions. The stimulation protocol in the left column is a 200-ms pulse at voltages varying from −140 to +100 mV in 20-mV steps, followed by a short activating pulse at +200 mV whose duration is sufficient to fully activate the fast gate in the different conditions, and then followed by a 10-ms repolarization to −140 mV. The stimulation protocol in the right column is the same, except that there is no activating pulse at +200 mV. The initial part of the 200-ms pulse to the varying potential is not shown. Dashed line represents zero current. (A) Standard conditions. (B) Low external chloride solution, 20 mM. (C) Low internal pH 6.5. (D) Dominant myotonia inducing mutation, I290M.

Figure 7

Double-barreled model for CLC-1. Channel opening is governed by two types of gates, one common pore gate (slow gate) and two protopore gates (fast gates). States S₀–S₂ are nonconducting, because the slow gate is closed. State S₃ is nonconducting because both fast gates are closed. The conductance of state S₅ is twice that of S₄. The fast gates are independent of each other with the slow gate open, whereas all other transition rates depend on the channel state.

Figure 9

Fast and slow gate open probabilities in different experimental conditions. (A) Boltzmann fits (continuous lines) of the mean values of the fast (filled symbols) and (empty symbols) slow gating process open probabilities in different conditions. Circles, standard solution; squares, low extracellular chloride; triangles, low intracellular pH; inverted triangles, I290M mutation. (B) Half activation potentials for the fast and slow gate in the different experimental conditions described above. (C) Residual open probability at most negative voltages for the fast and slow gate in the different experimental conditions described above. Shaded histograms relative to fast gate parameters and empty histograms relative to slow gate parameters.