Correspondence to: Tsung-Yu Chen, Center for Neuroscience, University of California, 1544 Newton Court, Davis, California 95616. Fax:(530)757-8827 E-mail:tycchen{at}ucdavis.edu.
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Abstract |
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The fast gate of the muscle-type ClC channels (ClC-0 and ClC-1) opens in response to the change of membrane potential (V). This gating process is intimately associated with the binding of external Cl- to the channel pore in a way that the occupancy of Cl- on the binding site increases the channel's open probability (Po). External H+ also enhances the fast-gate opening in these channels, prompting a hypothesis that protonation of the binding site may increase the Cl- binding affinity, and this is possibly the underlying mechanism for the H+ modulation. However, Cl- and H+, modulate the fast-gate Po-V curve in different ways. Varying the external Cl- concentrations ([Cl-]o) shifts the Po-V curve in parallel along the voltage axis, whereas reducing external pH mainly increases the minimal Po of the curve. Furthermore, H+ modulations at saturating and nonsaturating [Cl-]o are similar. Thus, the H+ effect on the fast gating appears not to be a consequence of an increase in the Cl- binding affinity. We previously found that a hyperpolarization-favored opening process is important to determine the fast-gate Po of ClC-0 at very negative voltages. This [Cl-]o-independent mechanism attracted little attention, but it appears to be the opening process that is modulated by external H+.
Key Words: channel gating, ClC-0, ClC-1, pH regulation, Cl- dependence
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INTRODUCTION |
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Ion channels catalyze the transportation of small ions across the cell membrane and, in doing so, maintain normal cellular functions. To achieve functional flexibility, these membrane proteins are regulated by a variety of factors, such as transmembrane voltages, mechanical stretches, extracellular or intracellular ligands, and sometimes the small ions in the aqueous solution (
Molecular cloning work in the last 10 yr has made significant progress toward understanding the identity of various Cl- channels. The principal Cl- channel in the muscle membrane, ClC-1 (56% identity. The similarity between these two channels is also shown in their functional properties. The opening of both types of channels display "double-barrel"like activity (
Consistent with the early findings on the muscle Cl- conductance, varying the external pH also affects the fast gating of ClC-1. With macroscopic current recordings,
As external Cl- appears to control the fast gating of ClC-0 and ClC-1 in the same manner, one would expect that H+ would have a similar modulatory effect on the fast gating of ClC-0. However, previous studies on ClC-0 have not clearly shown an effect of external H+ on the fast gating. 7.4 and
6.0. Another study at the single-channel level reported that although internal pH profoundly affected the fast gating of ClC-0, no effect was discernible when the pH of the trans chamber (corresponding to the external side) was raised from pH 7.0 to 10.0 (
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MATERIALS AND METHODS |
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Expression of ClC-0 and an Inactivation-suppressed Mutant C212S
The present study focuses on the fast gating of ClC-0. We have isolated the fast gating from the inactivation gating by using an inactivation-suppressed mutant C212S. The construction of the cDNA of the wild-type (WT) channel and the site-directed mutagenesis used to create the C212S mutant have been described previously (
Macroscopic Current Recording
Whole oocyte currents were recorded at room temperatures (2023°C) using standard two-electrode voltage clamp techniques. Microelectrodes were pulled by a PP-83 puller (Narashige) and, when filled with 3 M KCl, had resistance of 0.21.0 M. The ground electrodes were connected to the recording chamber via 3-M KCl salt bridges. The recorded current was filtered by the built-in filter of the amplifier (model 725C; Warner Instruments) and was digitized at 1 or 2 kHz using a Digidata 1200 data acquisition board and pClamp6 software (Axon Instruments). The standard solution for the recording of whole oocyte current contained the following (in mM): 98 NaCl, 2 MgSO4, and 5 HEPES. The pH of the solution was titrated by adding NaOH or glutamic acid. To make solutions with a lower Cl- concentration, part of the NaCl was replaced by sodium glutamate. When a higher Cl- concentration such as 300 mM was needed, NaCl was added to the standard solution to obtain the desired concentration. Because the channel is open at the holding potential, a change of [Cl-]o inevitably leads to an alteration of the internal Cl- concentration ([Cl-]i). Based on the measured reversal potentials at a steady-state, we estimated that effective [Cl-]i were
6070,
3648, and
1520 mM when [Cl-]o were 300, 98, and 15 mM, respectively.
To examine the quasi steady-state inactivation curve of the channel (see Fig 1), a protocol modified from a previously described method (
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The fast-gating properties of the channel were evaluated with a voltage protocol described before () was first determined by fitting the current deactivation process to a single-exponential function. The opening rate (
) and closing rate (ß), thus, were calculated according to:
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(1a) |
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(1b) |
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However, this method was limited by the degree of the current deactivation. When the membrane potential is more depolarized than -40 mV, or when the external pH is <5, the current deactivation component is so small that the time constant cannot be reliably estimated. The opening and closing rates, thus, cannot be obtained under these conditions.
Single-channel Recordings
The single-channel behavior of the channel was examined at room temperatures (1923°C) using excised inside-out patch recording techniques (
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(2) |
where f1 and f2 represent the probabilities of the intermediate and the fully open levels, respectively. Intuitively, the opening rate () and closing rate (ß) are as follows:
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(3a) |
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(3b) |
where 0 and
2 are the time constants of the distributions of nonconducting and fully open events, respectively. However, in the very acidic condition (pH = 5.6),
0 was difficult to obtain because Po became so large that the closed events were rare. We exploited the fact that events at the middle level occurred more frequently and also had a longer dwell time than those at the zero current level. The time constant of the dwell-time distribution of the middle level
1, therefore, was determined and the opening and closing rates were calculated according to a method described in
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(3c) |
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(3d) |
Under experimental conditions where both 0 and
1 can be reliably obtained,
and ß were calculated using both these methods (Equation 3aEquation 3bEquation 3cEquation 3d a and 3b versus Equation 3c and Equation 3d) and agreed within 10% (for the comparison of these two methods also see
Data Analysis
Data analysis of the macroscopic current was conducted using the software programs, pClamp6 (Axon Instruments) and Origin 4.0 (Microcal Software, Inc.). Analysis of the single-channel recording trace was performed using a homemade program (140 Hz (-3dB). We did not correct the missed events since the change in opening rate by H+ was much greater than the error that resulted from filtering the recording traces. A previous single-channel recording study using the same digitizing rate and a similar cutoff frequency was shown to have an estimated error for the opening rates of
56% (
Single-channel current amplitudes were determined from all-points amplitude histograms and the time constants of the three current levels were estimated from the dwell-time distribution. All nonconducting events of C212S were included in the analysis because the inactivation of this mutant is suppressed ((0) in each dataset. See Fig 8 legend for a detailed description.
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RESULTS |
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Inactivation of C212S Mutant Is Suppressed in Nonphysiological Conditions
In the attempt to isolate the H+ and Cl- effects on the fast gating of ClC-0, we face a problem due to the inactivation of the channel. In ClC-0, the inactivation mechanism operates at a time scale of tens to hundreds of seconds at room temperatures (
Fast Gating of C212S Is Identical with that of the WT Channel
To ensure that C212S can be used to study the fast gating of ClC-0, we further compared the responses of the fast gating of WT and C212S to different [Cl-]o. Fig 2 A shows macroscopic currents in the presence of 98 and 4 mM [Cl-]o. For the WT channel, the maximal tail current at 4 mM [Cl-]o is smaller than that at 98 mM [Cl-]o. In C212S, the situation is reversed; the maximal tail current at 4 mM [Cl-]o is larger. The inactivation of C212S is also suppressed at low [Cl-]o (data not shown). Thus, the bigger inward tail current at 4 mM than at 98 mM [Cl-]o results from a larger electrochemical driving force that pushes Cl- out of the cell at -100 mV. On the other hand, a smaller tail current at 4 mM [Cl-]o in the WT channel reflects a higher inactivation probability at low [Cl-]o (
To examine the fast gating of the WT and C212S, we systemically varied [Cl-]o and performed a series of experiments similar to those shown in Fig 2 A. The Po-V curves at various [Cl-]o are plotted in Fig 2 B, in which the curve shifts to more depolarized voltages when [Cl-]o is reduced. For the WT channel, the V1/2 of the fitted Po-V curve changes by 2030 mV with a fourfold reduction in [Cl-]o. In C212S, the degree of the shift is not significantly different. These results indicate that WT and C212S have similar [Cl-]o-dependent fast-gating properties. The responsible Cl- binding sites are likely to be intact in the inactivation-suppressed mutant, C212S.
Effects of External pH on the Fast Gating: Macroscopic Current Recordings
The influences of external pH on the fast gating of WT and C212S are compared in Fig 3. At neutral pH, the macroscopic current deactivates according to a single-exponential function in response to membrane hyperpolarization. The deactivated currents at various negative voltages usually cross with each other, a hallmark of ClC-0 current. When the pH is increased from 7.6 to 9.6, the time constant and the steady-state component of the current deactivation are not significantly changed. On the other hand, the proportion of the steady-state component increases significantly as the pH is decreased from 7.6 to 5.6. Consequently, the deactivating currents do not cross with each other in this acidic condition. Fig 4 A shows the Po-V curves derived from the experiments similar to those shown in Fig 3. The effect of H+ is prominent only when the pH is changed from neutral to acidic conditions. Reducing the external pH elevates the open probability of the channel mostly at the hyperpolarized voltages. This behavior is similar to the pH effect on the fast-gate Po-V curve of ClC-1 (, and the closing rate, ß, of the fast gate from the current deactivation time constants and the steady-state open probabilities (see MATERIALS AND METHODS). Fig 4 (B and C) indicates that external H+ profoundly increases the opening rate of the fast gate, whereas the closing rate is almost not affected. The effect of H+ on the opening rate, again, is more prominent as the membranes become more hyperpolarized.
Effects of External pH on the Fast Gating: Single-channel Recordings
To directly observe the channel behavior in response to external H+, we performed single-channel recordings on excised inside-out patches. Representative single-channel recording traces of C212S are shown in Fig 5 A. It is obvious by observing the recording traces or by comparing the all-points amplitude histograms (Fig 5 B) that the channel conductances at various external pH are all the same. The areas of the all-points amplitude histograms or the measured state probabilities of the three current levels (see Fig 5 B legend) also reconfirm the conclusion that the Po of the fast gate is increased mainly as the condition moves from neutral to acidic pH. Dwell-time distributions of the three current levels (Fig 5 C) reveal that the averaged duration of the fully open events is not significantly affected, whereas the time constants of the distributions of the closed and the intermediate current levels are changed upon altering the external pH. Similar results of the H+ modulation were also observed on single WT channels (data not shown). The collected open probabilities, opening, and closing rates of C212S are depicted in Fig 6 (AC, respectively). To a first degree of approximation, the results from the single-channel experiments recapitulate the macroscopic current recordings except a larger minimal Po. This difference between the macroscopic and single-channel recordings is probably due to a different internal Cl- concentration in these two recording conditions (
H+ Modulations at Saturating and Nonsaturating [Cl-]o
The binding site for the [Cl-]o-dependent fast-gate opening has an apparent Cl- binding affinity of 50 mM (
0.2 in Po between 300 and 15 mM [Cl-]o at both pH values is, again, due to an unequal internal Cl- concentration at these two [Cl-]o. Nevertheless, the increase in Po (0.38) in response to a low pH at the saturating [Cl-]o is no less than that (0.30) at the nonsaturating [Cl-]o. Thus, these results do not support the assertion that external H+ affects the fast gating through modulating the [Cl-]o-dependent gating mechanism.
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DISCUSSION |
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We have shown that C212S mutant can be used to study the fast gating of ClC-0. The mutation suppresses the inactivation of ClC-0, but the mutant channel retains normal channel conductance and fast-gating properties, including voltage dependence and modulations by external Cl- (Fig 2) and pH (Fig 3 and Fig 4). Therefore, we are confident that the conclusions drawn with respect to H+ modulation on the fast gating of C212S can be applied to the WT channel.
The effect of external H+ on the fast gating is mainly on the opening rate, whereas the closing rate is insensitive to the pH change. Higher [H+]o increases the opening rate, but the modulation is larger at hyperpolarized voltages. In effect, the Po-V curve shows a larger minimal Po when pH is reduced. This phenomenon is different from the effect obtained by varying [Cl-]o, which shifts the Po-V curve along the voltage axis (Fig 2). With such a difference even at the level of a qualitative description of the Po-V curve, it is already difficult to believe that external H+ modulates the fast gating through protonation of the Cl- binding site. To examine this problem more rigorously, we compared the H+ effect at a nonsaturating (15 mM) and at a nearly saturating [Cl-]o (300 mM). Experimental results show that H+ does not have a smaller modulatory effect in a saturating [Cl-]o condition (Fig 7). This result strongly supports the idea that H+ modulation of the fast gating is unlikely to be due to an increase in the affinity of the Cl- binding site responsible for the [Cl-]o-dependent channel opening.
In a previous single-channel recording study, the fast gate of ClC-0 was found to consist of two opening processes with opposite voltage dependence (
As the effect of external H+ modulation is more prominent at more negative voltages, we suspect that H+ is acting on this [Cl-]o-independent opening process. To explore this possibility, we adopt a five-state scheme to describe the summation of the above two fast-gate opening processes:
This five-state scheme simplifies the described previously six-state model ( determines the opening rate in this [Cl-]o-dependent process. The rate constant
1 represents the rate of the hyperpolarization-favored opening process. The observed opening rate
, thus, is the sum of
1 and
weighted by a factor determined by [Cl-]o and the Cl- dissociation constant K (
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(4) |
where the rate parameters vary with voltage according to Equation 5a and Equation 5b:
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(5a) |
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(5b) |
If H+ modulation is solely acting on 1, we should be able to describe the effect of pH on the fast-gate opening rate by varying the value of
1 but keeping all the other parameters unchanged. In modeling this condition, we applied the values of all parameters shown in Table II of
1(0) = 2.4 s-1 (pH = 7.6),
(0) = 446 s-1, z
1= -0.29, and z
= 0.7. [Cl-]o is 98 mM in this study. Fig 8 A shows that such a model indeed provides a good approximation. When the value of
1(0) is increased presumably by protonation, the observed opening rates are enhanced mostly at hyperpolarized voltages. At depolarized voltages, in contrast, the opening rates at different external pH converge to the same value.
To gain a structural insight into this H+ modulation site, we fitted the observed opening rates to Equation 4 (Fig 8B and Fig C). Curve fitting for data at different pH was performed at the same time with a shared value of (0) for each dataset. The fitted values of
(0), either from the macroscopic current recordings or from the single-channel experiments, vary only within a factor of 2 from that reported in
1(0), indicating that the modulation of external H+ can be explained by an effect on the hyperpolarization-favored opening process. The fitted
1(0)'s in these analyses were plotted in Fig 8 D as a function of external pH. Curve fitting by a logistic function gives a pKa of
5.3 for the protonation site. However, this number can only be viewed as an approximated pKa value of the titratable site because the titration curve is not saturated even at the most acidic pH in which reliable data can be obtained.
The results presented in this study also raise an interesting possibility that internal Cl- may interact with external H+ to modulate the hyperpolarization-activated fast gating. A comparison of the external H+ modulations between the macroscopic and single-channel experiments shows that the effect is bigger with a higher [Cl-]i (compare Fig 4 with Fig 6). This can be better seen in Fig 8 D, in which the fitted 1(0) in single-channel experiments ([Cl-]i = 120 mM) is more than fivefold larger than that obtained from the macroscopic current recordings ([Cl-]i
40 mM) at an external pH of 5.6. An increase of [Cl-]i is known to raise the minimal Po of the fast-gate Po-V curve (
1(0) between macroscopic and single-channel experiments (Fig 8 D). However, the difference in the fitted
1(0) between these two types of experiments becomes larger when the external pH is more acidic. Because [Cl-]i in the whole oocyte recordings are not precisely known, these observations remain only qualitative. It will require a better control of [Cl-]i to explore the exact functional role of internal Cl- in the hyperpolarization-favored fast gating and to examine the possible coupling between the actions of H+ and Cl-.
In summary, external H+ modulates the fast gate of ClC-0 in a way similar to that observed in ClC-1. The effect is to enhance the opening rate, leading to an increase in the fast-gate Po mostly at the hyperpolarized voltages. This modulation of the fast-gate opening, however, is different from that produced by the external Cl-. External Cl- increases the depolarization-favored opening rate, whereas H+ modulates the hyperpolarization-activated fast-gating. These two opening processes, which not only have opposite voltage dependence, but also show characteristic modulations by different external small ions, should be considered together to properly describe the fast-gate openings of the muscle-type ClC channels.
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Footnotes |
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* Abbreviations used in this paper: Po, open probability; WT, wild-type.
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Acknowledgements |
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We thank Miss Yu-Wen Lin for participating in some experiments at the early stage of this study.
This work was partly supported by grant NHRI-GT-EX89B813CS from the National Health Research Institutes in Taiwan.
Submitted: 8 January 2001
Revised: 17 May 2001
Accepted: 18 May 2001
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