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. 2001 Aug 1;534(Pt 3):745–752. doi: 10.1111/j.1469-7793.2001.00745.x

Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S

Alessio Accardi 1, Loretta Ferrera 1, Michael Pusch 1
PMCID: PMC2278749  PMID: 11483705

Abstract

  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.

The chloride channel from the electric organ of Torpedo, ClC-0, was the first protein of the ClC family to be cloned (Jentsch et al. 1990). Meanwhile many homologues have been identified in several species (Maduke et al. 2000). Although for many ClCs the physiological function is relatively unclear, it is known that ClC-1 (Steinmeyer et al. 1991) is responsible for the majority of the resting conductance of skeletal muscle and that impairing normal channel function can lead to dominant or recessive forms of myotonia congenita (Koch et al. 1992; Pusch et al. 1995; Wollnik et al. 1997).

Chloride channels are thought to be dimeric proteins with a double-barrelled structure: the channel comprises two independent permeation pathways, each delimited by a single subunit. Recent structural data from a prokaryotic channel (Mindell et al. 2001) strongly support the doublebarrelled structure of ClC-type channels. In ClC-0 two different gating mechanisms regulate the openings of the protopores. A slow common pore gate acts on both protopores simultaneously while two independent fast, single protopore gates regulate the openings of each permeation pathway (Miller, 1982; Middleton et al. 1996; Ludewig et al. 1996). For ClC-1 two different gating process could be easily identified at negative voltages (Rychkov et al. 1996) while for positive voltages envelope protocols were used to show that ClC-1 has two gating processes at all voltages (Accardi & Pusch, 2000). The two gating processes of ClC-1 have been tentatively identified with the fast and slow gate of ClC-0 (Saviane et al. 1999; Accardi & Pusch, 2000).

A single point mutation, C212S, has been shown to completely abolish the slow gating of ClC-0, apparently locking it in the open state at all investigated voltages while leaving the fast gate almost unaltered (Lin et al. 1999). The cysteine in position 212 of ClC-0 is conserved in ClC-1 (C277 in ClC-1). In this work we study the effect on ClC-1 of mutation C277S, and show that it drastically reduces the slow component of the macroscopic gating relaxations, increasing its minimal open probability while having only a minor effect on the fast component. This analogy of effect reinforces the molecular identification of the slow gating process of ClC-1 with the slow gate of ClC-0.

METHODS

Current recording

WT hClC-1 (Koch et al. 1992) and the mutant C277S were expressed in Xenopus oocytes and currents were measured at 18 °C 2-5 days after injection using the inside-out configuration of the patch clamp technique (Hamill et al. 1981) with an EPC-7 (List, Darmstadt, Germany) amplifier and the acquisition program Pulse (HEKA, Lambrecht/Pfalz, Germany).

Data analysis was performed using self-written software (Visual C++, Microsoft) and the SigmaPlot program (Jandel Scientific, Corte Madera).

The bath (internal) solution contained (mm): 100 N-methyl-d-glucamine (NMDG)-Cl, 2 MgCl2, 10 Hepes, 2 EGTA, pH 7.3. The standard extracellular (pipette) solution contained (mm): 100 NMDG-Cl, 5 MgCl2, 10 Hepes, pH 7.3. In the low external chloride solution 90 mm of NMDG-Cl was replaced with 90 mm of NMDG-glutamate. pH was adjusted with NMDG or HCl.

Molecular biology

Mutation C277S was introduced into ClC-1 by standard recombinant PCR mutagenesis. The final construct was verified by sequencing over the PCR generated fragment.

Envelope protocols

The use of envelope protocols to separate the fast and slow gating processes of ClC-1 was described previously (Accardi & Pusch, 2000). Briefly, to monitor the fast activation, patches were held at -140 mV for 200 ms to maximally deactivate the channels; the voltage was then stepped to different test potentials, Vp, for increasing amounts of time, tp, and finally repolarised to -140 mV. The dependence of the initial current recorded upon repolarising the patch to -140 mV, I0, on tp directly reflects the time course of activation at Vp. Due to technical limitations, time constants faster than 30 μs are not reliable and so were not used for quantitative fits (see Accardi & Pusch, 2000).

Deduction of fast and slow gating process open probability

Under the assumption that ClC-1 can conduct only if both gating processes are in the open state we have previously shown (Accardi & Pusch, 2000) that using a regular tail protocol (composed of a 200 ms pulse to a variable Vp followed by a repolarisation to -140 mV) and a tail protocol where a 200 μs pulse to +200 mV to fully activate the fast gating process, is applied prior to the -140 mV repolarisation it is possible to measure the open probability of the slow gating process and the conditional open probability of the fast process given that the slow gating process is open.

We fitted the open probabilities obtained in this way using a Boltzmann function with an offset:

graphic file with name tjp0534-0745-m1.jpg (1)

where P0 is the residual open probability at most negative voltages, V½ is the half-activation potential and z is the apparent gating charge.

Statistical treatment of data

All data are presented as means ± standard error of the mean (s.e.m.) and n indicates the number of experiments performed in each condition.

Statistical significance was evaluated by the standard Student's unpaired t test. The fast gate time constants and the values for the open probabilities of the two processes were taken from Accardi & Pusch (2000). We routinely performed control experiments with WT ClC-1, where we always found values in good agreement with the published data. However the number of experiments was not sufficient to guarantee a good statistical significance compared with the mutant. We therefore decided to use the previously published data to evaluate the effect of mutation C277S.

RESULTS

Mutating cysteine 212 to serine (C212S) locks the common pore gate of ClC-0 in the open state (Lin et al. 1999). To test the hypothesis that the slow gating relaxations seen in ClC-1 reflect the gating of the common pore gate of the presumably double-barrelled ClC-1, we mutated the cysteine in the equivalent position of ClC-1, C277, to a serine.

Qualitatively the currents induced by the mutant channel look similar to those of WT (Fig. 1B and C). They deactivate at negative voltages with kinetics in the ten millisecond range and display an instantaneous inward rectification. A more careful analysis, however, reveals a striking difference: at negative voltages the deactivation of the mutant can be well fitted with a single exponential function (Fig. 1E) while, as already known, gating of WT ClC-1 is best fitted with a double exponential function (Fig. 1D). To illustrate this difference we show the WT trace recorded at -120 mV from Fig. 1B in panel D on a double logarithmic scale and on a linear scale in the inset. A single (continuous line) and a double (dashed line) exponential fit were performed on the trace. A single exponential does not fit well the WT trace especially at the beginning and the end of the pulse where either the fast or the slow process is dominant. The time constants of the two processes identified in this manner are τf = 12.3 ms and τs = 85.8 ms and the ratio of the coefficients of the two exponentials is Cf/Cs = 3.2. On the other hand, the single and double exponential fits of the trace at -120 mV from Fig. 1C of the C277S mutant are almost superimposable (Fig. 1E). The time constant of the single exponential fit is 13.3 ms, close to the fast time constant for WT. The time constants obtained with a double exponential fit are τf = 11.8 ms and τs = 32.9 ms and the ratio of the coefficients is Cf/Cs = 8.7. Thus the slow gating process, if present, is greatly diminished in the C277S mutant while the fast gating process seems to be little affected.

Figure 1. Deactivation of mutant C277S fits a single exponential.

Figure 1

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A, I-V protocol. Following a 50 ms pulse to +60 mV, to fully activate the currents, the voltage was stepped to values increasing in 20 mV steps between -160 and +80 mV for 200 ms. Finally, the voltage was stepped to -140 mV for 20 ms to record the tail currents. B, WT ClC-1 currents elicited with the protocol shown in A. C, currents of mutant C277S. D, double logarithmic representation of the trace at -120 mV from B. Single (continuous line) and double (dashed line) exponential fits are also plotted. Parameters obtained with the single exponential fit are: τf = 19.7 ms, while the double exponential fit yielded τf = 12.3 ms, τs = 85.8 ms and Cf/Cs = 3.2. In the inset the same trace and fits are plotted in a linear representation. E, double logarithmic representation of the trace at -120 mV from C, single (continuous line) and double (dashed line) exponential fits are also plotted. Parameters obtained with the single exponential fit are: τf = 13.3 ms, while the double exponential fit yielded τf = 11.8 ms, τs = 32.9 ms and Cf/Cs = 8.7. In the inset the same trace and fits are plotted in a linear representation.

Fast gate time constants of mutant C277S

At positive voltages the gating relaxations of both WT ClC-1 and C277S are too fast to be directly followed (see Fig. 1A and B of Accardi & Pusch, 2000). We employed envelope protocols to investigate the voltage dependence of the time constants (see Methods; Accardi & Pusch, 2000). Figure 2A shows typical currents recorded with an envelope protocol at +100 mV. The flat trace corresponds to the case where tP = 0μs. Even a brief, 10 μs, pulse to +100 mV is sufficient to induce a relatively large jump in the initial current. Increasing the time spent at +100 mV increases the initial current recorded at -140 mV. In Fig. 2B the initial values of the currents recorded at -140 are plotted as a function of the time spent at +100. The dashed line represents a single exponential fit while the continuous line represents a double exponential fit. As can be seen the two fits are almost superimposable indicating that the slow component of gating is also almost absent at positive voltages. If longer prepulse durations were employed a slower component of very small amplitude became apparent. At all investigated voltages the time constant of the residual slow component was of the same order of magnitude as that found for the slow gating process of WT ClC-1 (Accardi & Pusch, 2000) (data not shown). A precise determination of the slow time constant was, however, impeded by its small amplitude.

Figure 2. Voltage dependence of the time constants of mutant C277S.

Figure 2

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A, fast gating process time constant of mutant C277S obtained with envelope protocols. A 200 ms pulse to -140 mV is followed by a short pulse of varying duration to +100 mV (increasing in 10 μs steps). The patch is then hyperpolarised to -140 mV for 10 ms. The value of the initial current recorded upon repolarization back to -140 mV is evaluated with a single exponential fit of the trace at -140 mV. Only 1 trace out of 4 is plotted for clarity's sake. B, initial values of the current at -140 mV plotted as a function of the time spent at +100 mV, tp. Dashed line (hardly visible because almost superimposed on the data points) is a double exponential fit, and the continuous line is a single exponential fit. C, voltage dependence of the time constant of the fast gate of WT ClC-1 (○) and of mutant C277S (•). Error bars represent s.e.m. (n≥ 3 for all voltages). WT values taken from Accardi & Pusch (2000). The time constants of the WT and mutant channel were not different (P > 0.20) at all voltages apart from -100, -40 and +20 mV where P > 0.05. At -60 and +60 mV the test indicated that the two were significantly different (P < 0.05), but the relatively small number of experiments at these voltages prevents a meaningful statistical evaluation.

As can be seen from Fig. 2C the fast gate time constants of mutant C277S (filled circles) are almost identical to those of the fast gate of the wild-type channel (open symbols) at all voltages.

Separation of fast and slow gating processes open probabilities

As mentioned above, the kinetics of the residual slow gating component is in the millisecond range (for V > +60 mV) while the fast process is at least 40 times faster, similar to the situation found for the WT (data not shown). This large kinetic difference allows the separation of the fast and slow gate open probability (see Methods; Accardi & Pusch, 2000). Figure 3A shows typical tail currents of the mutant recorded when, after a 200 ms pulse to various potentials, Vp, the voltage is immediately stepped to -140 mV. In Fig. 3B the currents are shown from the same patch when the voltage was stepped to +200 mV for 200 μs before the repolarisation to -140 mV. Since the fast time constant at +200 mV is smaller than 20 μs, the fast gating process saturates during the 200 μs prepulse. The dependence of the initial current at the -140 mV repolarising pulse on the prepulse voltage, Vp, thus reflects the voltage dependence of a residual slow gating process (Fig. 3C).

Figure 3. Separation of fast and slow gating process open probabilities of mutant C277S.

Figure 3

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A, a 200 ms long pulse of increasing voltage from -140 to +100 mV in 20 mV steps is followed by a 20 ms repolarization to -140 mV. The initial part of the 200 ms prepulse is not shown. B, currents recorded when 200 ms pulses are followed by a short 200 μs pulse to +200 mV, prior to the 20 ms repolarization to -140 mV. The initial part of the 200 ms pulse is not shown. Dashed line represents zero current. C, open probabilities for the slow gate (SG) of WT ClC-1 (▵) and mutant C277S (▴). Continuous lines are the fits of the open probabilities with eqn (1). The values obtained from the mean of at least 7 different experiments are: V½(SG) = -48 ± 4 mV; P0 (SG) = 0.84 ± 0.01; z (SG) = 1.0 ± 0.1. Error bars represent s.e.m. (n = 7). WT values taken from Accardi & Pusch (2000). The residual open probability of the slow gating process in the WT and mutant channel are significantly different (P < 10−8). D, open probabilities for the fast gate (FG) of WT ClC-1 (○) and mutant C277S (•). Continuous lines are the fits of the open probabilities with eqn (1). The values obtained from the mean of at least 6 different experiments are: V½(FG) = -73 ± 3 mV; P0 (FG) = 0.08 ± 0.02; z (FG) = 1.1 ± 0.1. Error bars represent s.e.m. (n = 7). WT values are taken from Accardi & Pusch (2000).

The quantity Δ = (Imax - Imin)/Imax, where Imax is the maximal current (for VP =+100 mV) and Imin is the minimal current (for VP = -140 mV), is an estimate of the weight of the residual slow component. A mean value for Δ of 0.14 ± 0.01 (n = 10) was obtained. This value is much smaller compared with that obtained for WT (Δ = 0.37 ± 0.02; n = 6) indicating that the mutation C277S drastically reduces the weight of the slow component. Several experimental artefacts would tend to increase the value of Δ, e.g. chloride accumulation/ depletion effects, or improper voltage control of the patch. Therefore we think that the mean value of Δ = 0.14 represents an upper limit of the residual slow component.

To derive the voltage dependence of the open probability of the fast and slow component separately we assumed that the residual current observed in the mutant with the protocols described in Fig. 3B corresponds to the slow component of the WT channel. As can be seen in Fig. 3C the residual current can be described with eqn (1) (filled triangles). The half-activation potential of the curve is very similar to that of the WT channel (open triangles), V12S (WT) = -51 mV while V12S (C277S) = (-48 ± 4) mV. The residual open probability is increased from 0.65 in the wild type, to 0.84 ± 0.01. The ratio of the total open probability and the slow component's open probability yields the fast gating process open probability (Accardi & Pusch, 2000) (Fig. 3D). Mutation C277S (filled circles) lowers P0f , the minimal open probability at negative voltages, to 0.08 ± 0.02, compared with the value for WT (open circles) of 0.22. The half-activation potential is virtually unchanged (V12f (C277S) = (-73 ± 3) mV; V12f (WT) = -79 mV).

Effect of lowering the internal pH and the external chloride concentration

To analyse further the properties of mutation C277S we investigated the effect of lowering the internal pH (pHi) or the external chloride concentration ([Cl−]o). Lowering pHi to 6.5 increased the residual open probability to 0.21 ± 0.03, and shifted the half-activation potential, V½, leftward by 23 mV to -92 ± 2 mV (data not shown). These changes are very similar to those induced on the fast gate of WT ClC-1, where the residual open probability was increased threefold and the half-activation potential was shifted towards more negative voltages by about 22 mV (Accardi & Pusch, 2000).

Reducing [Cl−]o to 20 mm slightly lowered the residual open probability to 0.04 ± 0.01, as compared with 0.06 ± 0.01 for the standard conditions. The half-activation potential was shifted towards positive voltages by about 22 mV (data not shown). Both of these changes are very similar to those induced by lowering [Cl−]o on the WT channel, a 27 mV shift towards more positive potentials and a slightly changed residual open probability (Accardi & Pusch, 2000).

Block of WT ClC-1 and C277S mutant by S(-)-CPB

The S(-) enantiomer of the 2-(p-chlorophenoxy) propionic acid (CPP) inhibits ClC-1 (Aromataris et al. 1999) acting from the intracellular side (Pusch et al. 2000). We tested the S(-) enantiomer of the butyric acid derivative, CPB, both on WT and mutant C277S. Figure 4 shows currents of mutant C277S in control (Fig. 4A) and in the presence of 1 mm CPB (Fig. 4B). With 1 mm CPB the current deactivation becomes faster overall, but there is also additionally a slow component, not seen in control, which is introduced with a time constant around 20 ms (see Fig. 4B, inset). The fast component has a time constant between 1 and 5 ms, compared with the fast time constant of 10 ms in control. At positive voltages there is a relief from block, evidenced by the slow activating kinetics with a time constant of about 10 ms at +80 mV. The degree of steady-state inhibition decreases with increasing voltage. Figure 4C shows the initial currents recorded at the beginning of the fixed tail potential of -100 mV without (circles) and with 1 mm CPB (squares). CPB strongly decreases the residual open probability at negative voltages, while at positive voltages the relief from inhibition becomes almost complete. The effect of 1 mm CPB can be described by a shift of about 90 mV towards more positive voltages of the apparent activation curve. A very similar shift (80 mV) was induced also on the WT channel (data not shown). The ratio of the current in the presence of different CPB concentrations (ranging from 0.1 to 1 mm) and in control conditions could be well described with a hyperbola of the form:

graphic file with name tjp0534-0745-m2.jpg (2)

where I (c) is the current recorded in presence of CPB at concentration c, and Kd is the apparent inhibition constant. Figure 4D shows the voltage dependence of Kd both for WT (open symbols) and mutant C277S (filled symbols), derived from analysing either the steady-state currents (squares) or the initial current recorded at -100 mV (circles) using eqn (2). The Kd is similar for the WT and mutant channel, regardless of whether it was evaluated from the tails or from the steady-state currents. At negative voltages it is relatively small (≈50 μm) and almost constant, while for V >-40 mV it increases exponentially with similar slopes.

Figure 4. Block of mutant C277S by S(-)-CPB.

Figure 4

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The stimulation protocol in A and B is similar to that described in Fig. 1A (but the tail voltage is -100 mV instead of -140 mV). A, control. B, current traces recorded in presence of 1 mm S(-)-CPB. Inset: slow deactivating component at -140 mV; the time constant of this component is τvs = 22.3 ms. C, initial tail currents at -100 mV are plotted as a function of the prepulse voltage in control conditions (•) and with 1 mm S(-)-CPB (▪). The continuous line is a fit with eqn (1) with the following parameters: V½(control) = -68 mV; P0 (control) = 0.04; z (control) = 1.2 and V½(CPB 1 mm) = 23 mV; P0 (CPB 1 mm) = 0.003; z (CPB 1 mm) = 0.94. D, comparison of the Kd for S(-)-CPB obtained from tail currents (○, •) and from steady-state current (□, ▪) for WT ClC-1 (○, □) and mutant C277S (•, ▪). The Kd was obtained from eqn (2). Error bars represent s.e.m. (n = 4 both for WT and C277S mutant). The Kd for WT and mutant are not different (P > 0.21 at all voltages except +20 and +40 mV where P > 0.05).

DISCUSSION

Here we present evidence that cysteine 277 is strongly involved in the slow gating process of ClC-1 much as the equivalent amino acid, C212, is involved in the slow gate of ClC-0. Mutation C277S strongly reduced the slow component of macroscopic gating relaxations at all voltages investigated (from -160 to +200 mV) while it had only a small effect on the fast gating process.

This result demonstrates unequivocally that the slow and fast gating relaxations of ClC-1, respectively, reflect the gating of the common pore gate and of the protopore gates of the double-barrelled channel, and thus confirms previous suggestions of such an identification (Saviane et al. 1999; Accardi & Pusch, 2000).

This identification does not imply that the fast and slow gating processes are identical in ClC-0 and ClC-1, as there are some important differences that need to be mentioned. First of all, the voltage dependence of the slow gate is opposite (although several point mutations reverse the voltage of the slow gate of ClC-0 (Ludewig et al. 1996)). Furthermore, the kinetics are extremely different (seconds to minutes for ClC-0 and milliseconds in ClC-1). Another important difference is the fact that while mutation C212S affects only the slow gate in ClC-0 leaving unaltered the fast gate the corresponding mutation in ClC-1 slightly changes the fast gating, indicating that the coupling between the fast and slow process is different and stronger in ClC-1 than ClC-0.

Thus, in a manner similar to the C212S mutant of ClC-0, mutant C277S can be treated as the ClC-1 channel lacking the slow gate. For example, various experimental manipulations (low pHi, low [Cl−]o) affect the open probability of the mutant in a very similar manner to the fast gate of the wild-type channel.

Mechanism of block of ClC-1 by S(-)-CPB

Aromataris et al. (1999) have proposed that CPP acts on ClC-1 as a gating modifier, shifting the activation towards positive potentials. We have recently investigated the mechanism of block of ClC-0 by CPB and concluded that CPB acts on ClC-0 by inhibiting each protopore independently (Pusch et al. 2001). We found that CPB preferentially binds to closed ClC-0 channels, introducing a slow gating component at negative voltages. The affinity of the open state is much smaller such that almost no direct open channel block occurs (Pusch et al. 2000, 2001).

Here we found that the overall affinity of CPB for ClC-1 is much larger than that for ClC-0 (roughly 10-fold larger). Its voltage dependence is, however, quite similar suggesting that the underlying mechanism of block is similar. In contrast to what we have observed for ClC-0, CPB mainly accelerates the fast component of deactivation at negative voltages besides introducing a slow component (see Fig. 4B, inset; see also Aromataris et al. 1999 and Pusch et al. 2000). This behaviour is most easily explained by a substantial binding of CPB to the open state at negative voltages. Mutation C277S has practically no effect on the block by CPB, in accordance with the view that CBP acts on the individual protopores.

Apart from demonstrating that the ‘slow process’ of ClC-1 corresponds to the ‘slow gate’ of the double-barrelled channel, the ClC-1 mutant C277S offers a conveniently simplified system to study biophysical or pharmacological properties of ClC-1 without interference from the slow gate.

Acknowledgments

We thank Professor Diana Conte Camerino for kindly providing the S(-) enantiomer of the 2-(p-chlorophenoxy) butyric acid. This work was partially supported by Telethon Italy, grant 1079 and by Italian CNR Progetto Strategico Biosensori.

References

  1. Accardi A, Pusch M. Fast and slow gating relaxations in the muscle chloride channel ClC-1. Journal of General Physiology. 2000;116:433–444. doi: 10.1085/jgp.116.3.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aromataris EC, Astill DStJ, Rychkov GY, Bryant SH, Bretag AH, Roberts ML. Modulations of the gating of ClC-1 by S(-) 2-(4-chlorophenoxy) propionic acid. British Journal of Pharmacology. 1999;126:1375–1382. doi: 10.1038/sj.bjp.0702459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv. 1981;391:85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  4. Jentsch TJ, Steinmeyer K, Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature. 1990;348:510–514. doi: 10.1038/348510a0. [DOI] [PubMed] [Google Scholar]
  5. Koch MC, Steinmeyer K, Lorenz C, Ricker K, Wolf F, Otto M, Zoll B, Lehmann-Horn F, Grzeschik K-H, Jentsch TJ. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science. 1992;257:797–800. doi: 10.1126/science.1379744. [DOI] [PubMed] [Google Scholar]
  6. Lin YW, Lin CW, Chen TY. Elimination of the slow gating of ClC-0 chloride channel by a point mutation. Journal of General Physiology. 1999;114:1–12. doi: 10.1085/jgp.114.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ludewig U, Pusch M, Jentsch TJ. Two physically distinct pores in the dimeric ClC-0 channel. Nature. 1996;383:340–343. doi: 10.1038/383340a0. [DOI] [PubMed] [Google Scholar]
  8. Maduke M, Miller C, Mindell JA. A decade of ClC chloride channels: structure, mechanism, and many unsettled questions. Annual Review Biophysics and Biomolecular Structure. 2000;29:411–438. doi: 10.1146/annurev.biophys.29.1.411. [DOI] [PubMed] [Google Scholar]
  9. Middleton RE, Pheasant DJ, Miller C. Homodimeric structure of a ClC-type channel. Nature. 1996;383:337–340. doi: 10.1038/383337a0. [DOI] [PubMed] [Google Scholar]
  10. Miller C. Open-state substructure of single chloride channels from Torpedo electroplax. Philosophical Transactions of the Royal Society B. 1982;299:401–411. doi: 10.1098/rstb.1982.0140. [DOI] [PubMed] [Google Scholar]
  11. Mindell JA, Maduke M, Miller C, Grigorieff N. Projection structure of a ClC-type chloride channel at 6. 5 Å resolution. Nature. 2001;409:219–223. doi: 10.1038/35051631. [DOI] [PubMed] [Google Scholar]
  12. Pusch M, Accardi A, Liantonio A, Ferrera L, De Luca A, Conte Camerino D, Conti F. Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(4-chlorophenoxy) butyric acid (CPB) Journal of General Physiology. 2001 doi: 10.1085/jgp.118.1.45. (in the Press) [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pusch M, Liantonio A, Bertorello L, Accardi A, DE Luca A, Pierno S, Tortorella V, Conte Camerino D. Pharmacological characterization of chloride channels belonging to the ClC family by the use of clofibric acid derivatives. Molecular Pharmacology. 2000;58:498–507. doi: 10.1124/mol.58.3.498. [DOI] [PubMed] [Google Scholar]
  14. Pusch M, Ludewig U, Rehfeldt A, Jentsch TJ. Gating of the voltage-dependent chloride channel ClC-0 by the permeant anion. Nature. 1995;373:527–531. doi: 10.1038/373527a0. [DOI] [PubMed] [Google Scholar]
  15. Rychkov GY, Pusch M, Astill DStJ, Roberts ML, Jentsch TJ, Bretag AH. Concentration and pH dependence of skeletal muscle chloride channel ClC-1. Journal of Physiology. 1996;497:423–435. doi: 10.1113/jphysiol.1996.sp021778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Saviane C, Conti F, Pusch M. The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia. Journal of General Physiology. 1999;113:457–467. doi: 10.1085/jgp.113.3.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Steinmeyer K, Ortland C, Jentsch TJ. Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel. Nature. 1991;354:301–304. doi: 10.1038/354301a0. [DOI] [PubMed] [Google Scholar]
  18. Wollnik B, Kubisch C, Steinmeyer K, Pusch M. Identification of functionally important regions of the muscular chloride channel ClC-1 by analysis of recessive and dominant myotonic mutations. Human Molecular Genetics. 1997;6:805–811. doi: 10.1093/hmg/6.5.805. [DOI] [PubMed] [Google Scholar]

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