Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S.
Journal - The Journal of physiology (England )
1. Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. 2. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(-) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. 5. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel.
|ISSN : ||0022-3751|
|Mesh Heading : ||Animals Chloride Channels Chlorides Clofibric Acid Humans Hydrogen-Ion Concentration Muscle, Skeletal Mutation Oocytes Osmolar Concentration Time Factors Xenopus laevis pharmacology analogs & derivatives pharmacology|
|Mesh Heading Relevant : ||Ion Channel Gating genetics metabolism metabolism physiology|
Fast and slow gating relaxations in the muscle chloride channel CLC-1.
Journal - The Journal of general physiology (UNITED STATES )
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.
|ISSN : ||0022-1295|
|Mesh Heading : ||Animals Chloride Channels Chlorides Female Humans Hydrogen-Ion Concentration Ion Channel Gating Kinetics Membrane Potentials Muscle, Skeletal Oocytes Point Mutation Recombinant Proteins Xenopus genetics metabolism metabolism metabolism genetics metabolism|
|Mesh Heading Relevant : ||metabolism|