Correspondence to: Tsung-Yu Chen, Department of Physiology, National Yang-Ming University, 155, Section 2, Li-Nung Street, Taipei, Taiwan, 11221. Fax:886-2-2826-4049 E-mail:tychen{at}ym.edu.tw.
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Abstract |
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The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share 5060% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl- less effectively, so the Po-V curve was shifted to a more depolarized potential by
45 mV. The K165C-K165 heterodimer showed double-barrellike channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.
Key Words: chloride channel, ClC-0, 2-aminoethyl methanethiosulfonate, double barrel
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INTRODUCTION |
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ClC chloride channels play important roles in many physiological processes, including the control of cellular excitability, secretion of epithelial cells, and cell volume regulation (5060% amino acid sequence identity and biochemical studies have indicated that each is a homodimer (
In ClC-0, early electrophysiological studies suggested that a functional channel contains two identical pores. This double-barrel model was first proposed based on the single-channel recording trace, in which the active channel displays three current levels D (down, zero-current level), M (middle), and U (upper), likely corresponding to the opening of zero, one, and two pores, respectively. The argument was supported by the equally spaced current levels and a binomial distribution of their probabilities, which were seen across all tested membrane potentials and under diverse ionic conditions (
On the other hand, the pore stoichiometry of ClC-1 was more difficult to address owing to a small single-channel conductance (20-fold higher than was the single-cysteine heterodimer K231C-K231A. Assuming that MTS inhibition arises from pore occlusion, this result was considered as inconsistent with the two-pore model. Together with the inhibition of the various cysteine mutants by externally applied Cu2+-phenanthroline and by internally applied cadmium ions, these results were used to argue that ClC-1 contains only one pore (
This apparent difference of pore stoichiometry between ClC-0 and ClC-1 is important because the biochemical components of these two channels are very similar. The controversy is even more intriguing after the demonstration of double-barrellike single-channel activity in ClC-1 (
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MATERIALS AND METHODS |
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Mutagenesis and Channel Expression
Site-specific mutants were generated using PCR techniques, and all the PCR-generated regions were sequenced to exclude polymerase-induced errors. Wild-type ClC-0 and all mutants were constructed in pBluescript (Stratagene). Tandem dimers were constructed by linking two monomers with four residues (GTTS). Homodimeric channels were expressed by injecting oocytes with RNAs from monomeric constructs. Synthesis of RNAs and injection of Xenopus oocytes were done according to previously described methods (
Macroscopic Current Recordings
Whole oocyte currents were measured using two-electrode voltage-clamp techniques. The standard bath (extracellular) solution was ND96 solution containing (mM): 96 NaCl, 2 KCl, 1 MgCl2, 0.3 CaCl2, 5 HEPES, pH 7.6. MTS reagents were purchased from Toronto Research Chemicals. Stock solutions of 0.1 M were prepared in distilled water and stored at -70°C. Working solutions containing the indicated concentrations of the MTS reagents were made immediately before use. To examine the MTSEA-induced current, the membrane potential of the oocyte was clamped at -30 mV, and the current was monitored with two types of repeated pulsing protocols: (a) a +40-mV voltage step for 50 ms, and (b) a +40-mV voltage step for 50 ms followed by a -150-mV voltage step for 100 ms. The voltage pulses were given at 0.11 Hz and the current was measured at the end of the +40-mV voltage step.
Examination of the fast-gate open probability (Po) from macroscopic oocyte current followed the previously described voltage protocols ( 100.6 mM, no Naglutamate was added). To make 1.6 mM [Cl-] solution, 1 mM KCl from the 4 mM [Cl-] solution was removed and MgCl2 was replaced by MgSO4. The opening rate
of the fast gate was calculated from the equation
= Po/
, in which
was the time constant of the current relaxation at the membrane potential where Po was obtained. Current block by SCN- was examined by pulsing protocol 2, with the indicated concentrations of NaSCN being directly added to the bath ND96 solution. The anomalous mole fraction experiments were also performed with pulsing protocol 2 in solutions containing (mM): X NaCl, (100-X) NaSCN, 1 MgSO4, 5 HEPES, pH 7.6, where NaSCN concentrations were 0, 3, 10, 20, 50, 80, and 100 mM, and the currents in different ionic mixtures were measured at +40 mV. The permeability ratio was determined by measuring the reversal potential shift upon replacing total external Cl- by other anions. All the results in this paper are presented as mean ± SEM.
Single-Channel Recordings
Procedures for obtaining inside-out patches were as previously described (
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To analyze a two-pore channel (see Fig 6A and Fig B), the absolute open probability of the channel (average of two pores) was determined from the probabilities of three equally spaced conductance levels, D (down), M (middle), and U (upper) (Equation 1):
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(1) |
where fM and fU were the measured probabilities at the M and U levels, respectively. When only two levels were present, as in the one-pore channel, Po was determined, after inactivation events were removed, as the probability of the open level.
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To examine the state probabilities of the three conductance levels in the heterodimer (see Fig 6 D), the expected state probabilities of the D, M, and U levels were calculated according to two different models. In one, a binomial distribution was assumed and the predicted probabilities of the three current levels were:
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(2a) |
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(2b) |
and
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(2c) |
where Po was the average open probability of the channel obtained from the Po-V curve of the heterodimer. In the other model, the two pores were assumed to have different fast-gate open probabilities, and the expected probabilities of the three current levels follow a multinomial distribution:
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(3a) |
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(3b) |
and
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(3c) |
where Pa and Pb are the measured open probabilities obtained from the Po-V curves of the K165 and K165C* homodimers, respectively. The expected values, f0, f1, and f2, were then compared with the experimental values, fD, fM, and fU, as shown in Fig 6 D (below).
Comparison of MTSEA Modification Rates
The MTSEA modification rate was examined with pulsing protocol 2 at 0.5 Hz. Because the induced current was measured at +40 mV where the fast gate opens completely (Po 1) in both the homo- and heterodimers, the apparent current induction rate is not influenced by the fast-gate open probability. On the other hand, the current induction rate is affected by the slow-gate open probability of the channel. Fig 9 A (below) depicts two schemes for the MTSEA modifications in the homodimeric and heterodimeric channels. In low concentrations of MTSEA, the reaction schemes can be further reduced to linear models with two (S1 and S2 in the heterodimer) and three (D0, D1 and D2 in the homodimer) states, where S and D represent single and double cysteine mutations and the subscripts denote the number of the open pores. Let
and ß be the slow-gating transition rates,
and µ the pseudo-first-order on and off rate for K165C modification, and PS1, PS2, PD1, and PD2 the slow-gate open probabilities of S1, S2, D1, and D2 states, respectively. When µ is small, the normalized current-induction time courses of the heterodimeric and homodimeric channels are approximated by Equation 4 and Equation 5:
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(4) |
and
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(5) |
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Theoretical curves were thus generated assuming PS1 = PD1 and PS2 = PD2 = 1 (see Fig 9 B, left). The curves were fitted to single-exponential functions and the ratios of the fitted time constants were plotted against PD1/PD2 (see Fig 9 B, right). Experimentally, the modification rates were examined in ND96 containing 110 µM MTSEA (see Fig 9 C) with a bath solution exchange time constant of 3 s. Current amplitudes were measured at the end of the +40-mV voltage step and data points were fitted to single-exponential functions.
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RESULTS |
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Functional Recovery of the Cysteine Mutant with MTSEA Modification
The residue K231 in ClC-1 was suggested to be within the pore (
An example of current induction of K165C by MTSEA is shown in Fig 1 A. Upon the application of MTSEA in the bath solution, ClC-0-like current can be induced. This MTSEA-induced current can be reversed slowly by washing out the modifying reagent, with a current reduction time constant of 30 min at
20°C (Fig 1 B). A reducing reagent, dithiothreitol, speeded up this reversing process (Fig 1 A), indicating that the effect was through a sulfhydryl group. The modification was specific to the introduced cysteine because MTSEA had no effect on wild-type ClC-0 or C212S (data not shown). Another two MTS reagents, 2-(trimethylammonium)ethyl MTS (MTSET) and 2-sulphonatoethyl MTS (MTSES) (
Gating Properties of the MTSEA-modified Homodimer
Modification of cysteine by MTSEA converts the side chain to a structure similar to that of a lysine residue (Fig 1 C), presumably the reason for the reopening of the channel. We thus adopt the name K165C* for the MTSEA-induced functional homodimeric channel with C* representing the MTSEA-modified cysteine. The fast gating of K165C* was similar to that of the wild-type channel containing lysine at the 165 position, with current deactivation at -150 mV (Fig 1 A). However, the Po-V curve of K165C* was right shifted by 45 mV when compared with that of the K165 channel at an external Cl- concentration ([Cl-]o) of 100 mM (Fig 2 A). This curve was further shifted towards a more depolarized membrane potential in response to a low [Cl-]o. The V1/2 of the Po-V curves in both channels had a more negative value at higher [Cl-]o, but the effect was saturated at [Cl-]o > 150300 mM. Furthermore, there was always an
4050-mV difference in V1/2 between two channels at the same [Cl-]o (Fig 2 B). This difference was mostly due to a shift of the opening rate curve of K165C* towards a more depolarized membrane potential (Fig 2 C), an effect similar to that of lowering [Cl-]o on the wild-type channel (
K165 Is Likely to be Located in the Pore Region
As the shift of the Po-V curve was thought to be a pore property, we suspected that K165 may be located in the pore region. To address this possibility more directly, we compared pore properties between K165 and K165C* channels. The blockage by thiocyanate (SCN-), a pore blocker of ClC-0 (5060% of that of the wild-type or the MTSEA-modified pore (also see results below). In addition, the MTSPA-modified pores have the same conductance irrespective of the side chain at residue 165 in the other subunit, an observation favoring two independent pores. The MTSPA-modified cysteine has a side chain slightly longer than the MTSEA-modified side chain (Fig 1 C), a subtle change that influences the rate of Cl- permeation. We have also compared the permeation of various anions in the K165 and K165C* channels. Both channels revealed an anomalous mole fraction effect for mixtures of Cl- and SCN- with a difference in the left arms of the normalized curves (Fig 5), reflecting different SCN- blocking affinities in these two channels. The anion permeability sequence was SCN- > Cl- > Br- > NO3- > I- for both channels (permeability ratios: K165, 1.20 ± 0.04:1:0.80 ± 0.02:0.66 ± 0.02:0.44 ± 0.04, n = 5; K165C*, 1.22 ± 0.08:1:0.95 ± 0.03:0.74 ± 0.02:0.51 ± 0.05, n = 35). The small alteration in ion permeation suggests that the K165C* mutation has not altered too much the global structure of the channel pore.
Fast Gating of Heterodimeric Channels Revealed Double-Barrellike Behaviors
With tandem heterodimers containing K165 in one subunit and K165C in the other, the functional role of K165 in ClC-0 gating can be further characterized. The MTSEA-modified tandem heterodimers revealed a double-barrellike structure, as with K165 or K165C* homodimers (Fig 6 A). The average Po of the two pores in either configurations (K165C*-K165 or K165-K165C*) was close to the mean of those of the K165 and K165C* homodimers (Fig 6 B). However, the Po of each individual pore was different from that of the other pore. For example, the probabilities of the three current levels showed a multinomial distribution when the Po of one pore was equal to the Po of K165 and the other to that of K165C* channels (Fig 6C and Fig D). Furthermore, when recording single MTSEA-modified heterodimeric channels, we frequently observed a transition from three to two current levels, presumably due to the loss of the modifying group. Subsequent examination of such a two-level trace always revealed a Po close to that of the K165 channel (Fig 7). These results together indicate that the two pores of the MTSEA-modified heterodimer have equal conductance and different Po; nevertheless, the principle of independent gating is still preserved.
One-Pore Channel Is Easier to Inactivate
The independence shown above for the fast gating, however, was not observed with respect to inactivation because the latter is influenced by both pores (
Independent Modification of the Two Introduced Cysteine Residues in a Homodimer
Complete modification of a homodimeric K165C channel requires two steps and must go through the low slow-gate open probability, one-pore state. Thus, the current-induction rate in the homodimer cannot be identical with that in the heterodimer. Fig 9 A shows possible schemes for MTSEA reaction with the hetero- and homodimeric channels. We assume that each cysteine residue is modified independently and the reaction rates in the inactivated and noninactivated channels are the same. When the on rate () of MTSEA modification is slower than the slow-gating transition rates (
and ß), but much faster than the off rate (µ), the reaction schemes can, respectively, be reduced to a two- (S1 and S2) and a three- (D0, D1, and D2) state model. When each state X has a slow-gate open probability PX, calculations show that the apparent modification rate would be slower in the homodimer than in the heterodimer as long as PD1 < PD2 (Fig 9 B). Experimentally, we applied 110 µM MTSEA to induce the current with time constants in the range of
20200 s (Fig 9 C, left). The ratios of the time constants for current induction in the homodimer compared with those in the heterodimer ranged from 1.4 to 1.7 (Fig 9 C, right), a result close to the expected ratio if the one-pore channel has a low open probability for the slow gate (B). This result indicates that the two cysteine residues reacted with MTSEA independently and the difference in the apparent modification rate was due to the distinct open probabilities of the slow gate in the one- and two-pore channels.
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DISCUSSION |
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Evidence for a Possible Pore Location of K165
We have found that residue K165 plays important roles in gating and permeation properties of ClC-0. The residue is freely accessible to all external MTS reagents tested. Therefore, K165 in ClC-0 is located at the extracellular side, consistent with both topological models proposed previously (45 mV, an effect similar to that of [Cl-]o reduction. The shift of the fast-gate Po-V curve of ClC-0 upon reducing [Cl-]o has been suggested to be mediated by chloride binding sites in the pore (
MTS Modifications of K165C Support the Double-Barrel Model
The shift of the fast-gate Po-V curve of the K165C* channel also provides a chance to examine the pore stoichiometry in this channel. When analyzing the fast gating of the heterodimers, we found that a multinomial distribution describes gating among the three current levels, with the Po of individual fast gates corresponding to those of the K165 and K165C* pores (Fig 6 D). Furthermore, the channel without MTSEA modification showed only one pore, and the Po was similar to that of the K165 homodimer (Fig 7). These results are all consistent with a picture of two independent fast gates. In addition, both the MTSPA- and MTSEA-modified channels revealed that the conductances of the two pores are independent of each other; that is, irrespective of the side chain of residue 165 in the other subunit (Fig 4 and Fig 6 A). This result further supports the two-pore picture.
Gating Effects of MTS Modification Invalidate the Assumption Used to Compare the MTS Inhibition Rates in ClC-1
One of the experiments supporting the one-pore conclusion in ClC-1 was a comparison of the MTS modification rates between the homo- and heterodimers. In this experiment on ClC-1, a key assumption was that the MTS inhibition of the channel is due to pore occlusion and thus the modification rates of the homo- and heterodimers should be the same if the channel has two pores (45-mV shift in the Po-V curve, it is important to monitor the MTSEA-induced current at a membrane potential where the fast-gate open probabilities of the homo- and heterodimers are the same. We achieved this requirement by monitoring the MTSEA-induced current at +40 mV, where the fast-gate open probabilities of the homo- and heterodimers are both close to unity (Fig 6 B).
A second point to be considered in comparing the apparent MTS modification rates concerns the slow gating of the channel. Our results showed that the channel with only one functional pore was easier to inactivate than the two-pore channel (Fig 8). This is consistent with a previous observation on the single-channel behaviors that the channel inactivates from the middle level and recovers from the inactivation state into the upper level more frequently (
The third caution in the interpretation of MTS modification rates concerns the concentrations of the modifying reagents. The MTS modification schemes (Fig 9 A, top) can be reduced to linear schemes only when the modification rate is much slower than the slow-gating transition rate. In our experiments, we used very low concentrations of MTSEA (110 µM) to fulfill this condition. Under such circumstances, the time courses of current induction were typically 210-fold longer than that of the slow-gating relaxation of the heterodimer. We are therefore confident that the apparent current-induction rates mostly reflect the true MTSEA modification rates on the introduced cysteine residues. On the other hand, if the true modification rate were faster than the slow-gating transition rate, the apparent rate would mostly reflect the gating transition rate of the channel.
It is not known at this stage which of the above reasons contributes most to the different apparent modification rates between the homo- and heterodimers of ClC-1 because characterization of the MTS effects on channel gating has not been performed in this muscle channel. However, the K231C homo- and heterodimers of ClC-1 showed very distinct gating behaviors from those of the wild-type channel (
Unsettled Inconsistency between the Cu2+-Phenanthroline Effect in ClC-1 and the Double-Barrel Conclusion in ClC-0
In ClC-1, the vicinity of K231 in D4 and the end of D5 have been proposed to form the outer vestibule of the pore (
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Footnotes |
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1 Abbreviations used in this paper: MTS, methanethiosulfonate; MTSEA, 2-aminoethyl MTS; MTSES, 2-sulphonatoethyl MTS; MTSET, 2-(trimethylammonium)ethyl MTS; MTSPA, 3-aminopropyl MTS.
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Acknowledgements |
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We thank Dr. M. Maduke and Mr. K.H. Hong from Dr. C. Miller's lab for kindly providing us two mother constructs with convenient restriction enzyme cutting sites to make ClC-0 heterodimers. We also thank Drs. T.-C. Hwang and C. Miller for comments on the manuscript and helpful discussions.
This work was supported by grant NHRI-GT-EX89B813C from the National Health Research Institutes in Taiwan.
Submitted: 3 May 2000
Revised: 2 August 2000
Accepted: 22 August 2000
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References |
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