Identification of the pH sensor and activation by chemical modification of the ClC-2G Clminus channel

Katarina Stroffekova1, Elena Y. Kupert1, Danuta H. Malinowska1, and John Cuppoletti1,2

1 Department of Molecular and Cellular Physiology and 2 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (Po) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pHtrans), with an apparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased Po threefold at pHtrans 7.4, at which the channel normally exhibits low Po. With extracellular pH reduction (protonation) or amidation, increased Po was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.

human and rabbit chloride channels; Xenopus laevis oocytes; cystic fibrosis; acid-activated chloride channel; protein kinase-regulated chloride channel; protein kinase A; gastric secretion; epithelial transport; chloride transport; water-soluble carbodiimide; amidation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE CHLORIDE CHANNEL, ClC-2G, was cloned from the rabbit stomach (16), human intestine (2), and human lung and stomach (23) and has also been shown to be present in a variety of rabbit tissues (7). It is voltage dependent and activated by protein kinase A (PKA; Ref. 16). In the stomach, it is involved in acid secretion and is present in the apical membrane of the gastric parietal cell (3, 16), which is exposed to a highly acidic environment (approximate pH < 1). In this context, acid stability of ClC-2G Cl- channel function is essential. In addition, rabbit and human ClC-2G Cl- channels are activated by extracellular acid (low pHtrans) in the range of pH of the gastric secretions (3, 16, 23).

The ClC-2G Cl- channel is a member of the rapidly expanding ClC family of Cl- channels, which includes at least nine different mammalian members (11). The common features of this channel family are that they are voltage gated and their predicted transmembrane topology is similar (11, 12). The location of the pore of the channel is not known and has been variably suggested to be near the carboxy-terminal (9) or the amino-terminal (5) transmembrane region.

Activation of both the rabbit and the human ClC-2G Cl- channels by low extracellular pH has recently been confirmed by Furukawa et al. (8) and Schwiebert et al. (22), respectively, but the mechanism involved was not investigated. In addition, rat ClC-2 (13), rat ClC-1 (20), and human ClC-1 (4) have also recently been shown to be affected by changes in extracellular pH. Several different mechanisms have been suggested to mediate these effects.

The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel as well as the molecular basis for this acid activation. The half-maximal effect occurred at an extracellular pH of ~5.0, suggesting that the effect on channel activity occurred through neutralization of the negative charge of carboxy groups by protonation. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC)-catalyzed amidation of reactive carboxy groups with glycine methyl ester (GME) (18) was used to test this hypothesis and to probe the topology and accessibility of the potential pH activation site on the extracellular surface of the channel. This treatment activated the channel. To investigate the molecular basis for activation of the ClC-2G Cl- channel by low extracellular pH, a negatively charged potential pH sensor region between predicted transmembrane regions D8 and D9 was examined using site-directed mutagenesis. Analysis of the effect of low extracellular pH on the channel gating was carried out to investigate the mechanism of acid activation of the ClC-2G Cl- channel.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression in Xenopus laevis oocytes and measurement of Cl- channel activity. Rabbit ClC-2G Cl- channel cRNA was made from the wild-type and mutant cDNAs using the mMessage mMachine cRNA kit (Ambion). Xenopus laevis oocytes were prepared and injected with cRNA as described by Malinowska et al. (16). One day after isolation, oocytes were injected with 10 ng cRNA/50 nl H2O per oocyte. After 4-5 days at room temperature, oocyte plasma membrane vesicles expressing the recombinant Cl- channel protein were isolated for electrophysiological studies as previously described (16). The equipment and procedures for vesicle incorporation into the bilayer and single-channel recording were essentially the same as previously described by Cuppoletti et al. (3). Routinely, 100-s channel recordings were obtained at set holding potentials and filtered at 500 Hz. Identification of events and determination of dwell times and amplitudes as well as curve fitting were carried out using the pCLAMP program (version 5.5). Solutions for measuring Cl- channel activity in the bilayer contained 800 mM tetraethylammonium (TEA) chloride, 10 mM EGTA (pH 7.4), 2 mM MgCl2, and 1 mM ATP and were buffered with either 10 mM TEA-HEPES (pH 7.4) or TEA citrate (pH 3.0) as indicated. In titration experiments, concentrated citric acid was added to the solutions on the trans side of the bilayer in concentrations sufficient to give the indicated pH, as determined in separate measurements of pH. PKA catalytic subunit (50 U/ml) was added to the cis side of the bilayer, which corresponds to the cytosolic side of the channel. The electrical reference side of the bilayer was the trans side, which corresponds to the extracellular side of the channel. In most cases, two channels were incorporated into the bilayer. However, occasionally a single channel or three channels were observed. When two or more open-state current levels were observed, which were even multiples of the lowest open-state current level, they were interpreted as reflecting the activity of two or more (as appropriate) channels present in the bilayer. All open probability (Po) values reported have been corrected for the number of channels present. Single-channel Po was calculated using the relationship
<IT>P</IT><SUB>o</SUB> = <LIM><OP>∑</OP><LL><IT>i</IT>=1</LL><UL><IT>N</IT></UL></LIM> <IT>t</IT><SUB>i</SUB>/<IT>TN</IT>
where ti is actual time spent at appropriate level, T is the total time of the recording (100 s), and N is the number of channels present in the membrane (17). At very low Po, the chance of underestimating the number of channels increases, but multiple (generally 3-10) 100-s recordings were carried out in each reported measurement to increase the chances of observing the correct number of channels in the bilayer. The time constants of the Cl- currents in the open states and closed states were measured and reported without correction for the number of channels present. Thus there is some uncertainty in the relationship between these open time constants and the open time constants that might be obtained in records known to contain only a single channel. However, the open time constants were generally very short with respect to closed times. Thus, here, the error in the open time constants is likely to be smaller than the error in the closed time constants. The reported closed time constants are likely to be shorter than the closed time constants obtainable if single channels were incorporated into the bilayer. All of the channel current records presented were confirmed to contain two channels (except Fig. 4A) and to have Po values and open and closed time constants within the ranges reported here in the summarized data. The channel current recordings shown are therefore representative experiments. Statistical significance of the difference between two means was determined using the Student's t-test. The number of different channel recordings under the same conditions (n) and the number of different membranes or reconstitutions of the channel (N) on which current recordings were performed are indicated in parentheses. Channel conductance was calculated as the slope of the linear current-voltage relationship obtained with current measurements in symmetric 800 mM TEACl solutions using a combination of holding potentials in the range ±150, ±100, ±80, ±60, and/or ±40 mV holding potentials.

Site-directed mutagenesis. Mutants of the rabbit ClC-2G cDNA were prepared using the Transformer site-directed mutagenesis kit (Clontech), and the sequences of resulting mutant cDNAs were fully verified. Single independent clones of each mutant were functionally characterized. The following mutants of region E416-E417-L418-E419 (EELE) were prepared: E416Q-E417Q (QQLE), E419Q (EELQ), E419G (EELG), and E416Q-E417Q-E419G (QQLG). In addition, mutant V349E was prepared.

Materials. Palmitoyl-oleoyl-phosphatidylethanolamine and palmitoyl-oleoyl-phosphatidylserine (Avanti Polar Lipids) were dissolved in n-decane. GME and PKA catalytic subunit were from Sigma. DRIREF electrodes were obtained from World Precision Instruments (Sarasota, FL). EDC was obtained from Pierce. Primers for sequencing, mutagenesis, and selection were synthesized at the Univ. of Cincinnati College of Medicine DNA Core Facility.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of pHtrans reduction on rabbit ClC-2G Cl- channel Po and gating. Rabbit (3, 16) and human (22, 23) ClC-2G Cl- channels are activated by low pHtrans. The effect of pHtrans 3.0 on rabbit ClC-2G Cl- channel activity is shown in Fig. 1A. A channel recording at -80 mV is shown. Two channels are present in the bilayer membrane as shown in the recordings (left) and amplitude histograms (right). At pHtrans 7.4, both channels were evident, and, when pHtrans was reduced to 3.0, activity of both channels increased. The concentration dependence of the increase in channel Po (normalized with respect to the number of channels, see MATERIALS AND METHODS) in response to changes in extracellular acidification was then measured. The results are summarized in Fig. 1B. At -80 mV holding potential, Po significantly (P < 0.001) increased from 0.22 ± 0.02 (n = 25, N = 10) to 0.36 ± 0.04 (n = 14, N = 6) when pHtrans was decreased from 7.4 to 6.0. Between pHtrans 6.0 and 4.5, there was no significant change in channel Po. However, lowering pHtrans further from pH 4.5 to 3.0 resulted in an additional significant (P < 0.001) increase in Po to 0.79 ± 0.07 (n = 6, N = 3). Similar results were obtained at +80 mV holding potential (Fig. 1B).


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Fig. 1.   Effect of low extracellular pH (pHtrans) reduction on open probability (Po) of ClC-2G Cl- channel. A: channel current recordings at pHtrans 7.4 and 3.0 of ClC-2G Cl- channel (left) and corresponding amplitude histograms (right). Open (o) and closed (c) states of the channel are indicated. ct, Counts or number of events. In this and most channel recordings in this study (see MATERIALS AND METHODS), 2 channels were present in the bilayer. Channel current recordings were obtained at -80 mV holding potential in symmetrical 800 mM tetraethylammonium (TEA) chloride (pH 7.4) solutions, which also contained 2 mM MgCl2, 1 mM ATP, 10 mM EGTA (pH 7.4), and 10 mM TEA-HEPES (pH 7.4). Protein kinase A catalytic subunit (50 U/ml) was present in the cis compartment. Reduction of pHtrans from 7.4 to 3.0 was achieved by addition of concentrated citric acid (final concentration, 40 mM). B: effect of stepwise reduction of pHtrans on channel Po at ±80 mV holding potentials. Statistical significance of effect was calculated relative to control values at pHtrans 7.4 (hatched bars). * P < 0.001, # P < 0.02. Number of different channel recordings (n) and number of different membranes (N) are indicated in parentheses at each point. C: Hill plot of the data from B at -80 mV (bullet ) and at +80 mV (open circle ). Log (Po/Po max - Po) is the log of the fractional activation of Po, where Po max was 0.79 (Po max is the maximal Po value at -80 mV and pHtrans 3.0). Hill plot is linear (r = 0.88), and half-maximal activation of Po occurs with an apparent acidic dissociation constant of pH 4.95 (arrow). For B and C, data were plotted as means ± SE. In subsequent experiments, unless otherwise indicated, all components of the solutions were as indicated in A.

To determine the half-maximal H+ concentration leading to activation of the channel, the data shown in Fig. 1B at ±80 mV holding potentials were then plotted as a Hill plot of fractional Po as a function of pHtrans (Fig. 1C). The maximal value of Po (0.79) was at -80 mV and pHtrans 3.0. The Hill plot is linear (r = 0.88) with a slope of ~0.25. Half-maximal activation of Po occurred with an apparent acidic dissociation constant (pKa) of pH 4.95 ± 0.27 (n = 99, N = 3), suggesting binding of protons to one or more negatively charged carboxy groups (glutamic acid, aspartic acid) or possible involvement of histidines of the channel protein exposed to the extracellular surface. Channel conductance was not affected by pHtrans reduction (Table 1).

                              
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Table 1.   Conductances of the wild-type and mutant ClC-2G Cl- channels at pHtrans 7.4 and 3.0 

To examine the basis for the increased channel Po after neutralization of the negative charge(s) by lowering pHtrans, open and closed times of single-channel gating were analyzed. Open and closed times were best fit by two exponentials with time constants tau 1 and tau 2. As shown in Fig. 2, significant changes were observed in both open time (Fig. 2A) and closed time (Fig. 2B) constants, tau 1 and tau 2. When pHtrans was lowered sequentially from 7.4 to 6.0, to 4.5, and then to 3.0, open time constants tau 1 and tau 2 significantly increased only at pHtrans 4.5 and 3.0 (P < 0.02 and P < 0.05, respectively). The closed time constants tau 1 and tau 2 significantly decreased at all pHtrans values (P < 0.001 and P < 0.02). Therefore, the increase in channel Po when extracellular pH was lowered results from both an increase in open times and a decrease in closed times of the channel gating. These experiments provide a direct demonstration that pHtrans affects channel gating.


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Fig. 2.   Effect of pHtrans reduction on open time constants (A) and closed time constants (B) of ClC-2G Cl- channel activity at -80 mV holding potential. Open and closed times were fit by 2 exponentials with time constants tau 1 and tau 2. Data were plotted as means ± SE. Statistical significance was calculated relative to the control values of channel at pHtrans 7.4 (hatched bars). * P < 0.001, # P < 0.02, ## P < 0.05.

Neutralization by amidation of the negative charge of carboxy groups facing the extracellular side of the ClC-2G Cl- channel. If neutralization of the negative charge of carboxy groups by protonation (see Fig. 1) is responsible for the change in Cl- channel activity, amidation with GME catalyzed by the water-soluble EDC, both added to the trans side of the bilayer, would result in a similar neutralization of charge and might therefore activate the channel. Channel activation by amidation will only occur if the target amino acids are carboxy groups (and not histidines) and are easily accessible to the bulky water-soluble compounds GME and EDC (18). Figure 3 shows the effect of amidation on ClC-2G Cl- channel activity. A channel recording at pHtrans 7.4 and -80 mV holding potential is shown in Fig. 3A. Two channels were present in the bilayer membrane, and these displayed minimal activity as illustrated by the corresponding amplitude histogram. Addition of 1 mM EDC alone to the solution in the trans compartment of the bilayer had no effect, but, when 10 mM GME was subsequently added to the trans side, channel activity increased, with a further increase when pHtrans was reduced to 3.0. The data are summarized in Fig. 3B. As found with protonation by lowering pHtrans, amidation with EDC and GME at pHtrans 7.4 and at -80 mV holding potential resulted in a significant increase (P < 0.01) in channel Po from 0.16 ± 0.02 (n = 40, N = 19) to 0.55 ± 0.05 (n = 19, N = 13) (Fig. 3B). The Po of the amidated channel was ~75% of the value obtained by reduction of the pHtrans of the untreated channel to pH 3.0. When pHtrans was lowered to 3.0 in the presence of EDC and GME, Po further increased significantly (P < 0.001) to 0.89 ± 0.04 (n = 16, N = 7), which was not significantly different from the Po at pHtrans 3.0 in the absence of EDC and GME. Similar results were obtained at +80 mV holding potential. EDC alone (Fig. 3) and GME alone (data not shown) had no effect on channel Po, ruling out the possibility of intramolecular cross-linking or noncovalent binding as responsible for the effect. Analyses of the open and closed times of channel gating (Table 2) showed that increased channel Po with EDC and GME at pHtrans 7.4 was due to significantly increased open time constants (tau 1 and tau 2) and significantly decreased closed time constants (tau 1 and tau 2) at ±80 mV. However, no additional change in the time constants occurred due to lowered pHtrans following amidation at pHtrans 7.4 at ±80 mV, except for a significant but minor effect on the closed time constants at -80 mV (P < 0.02; P < 0.01, respectively, for tau 1 and tau 2). These findings suggest that neutralization of one or more negatively charged carboxy groups at the extracellular surface of the channel protein is involved in acid activation of the ClC-2G Cl- channel and that histidines are not involved in the activation.


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Fig. 3.   Effect of amidation on the Po of the ClC-2G Cl- channel. A: channel current recordings at -80 mV holding potential in the absence (control) and in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) alone or EDC and glycine methyl ester (GME) at pHtrans 7.4 and 3.0 (left) and corresponding amplitude histograms (right). Channel recordings were started under control conditions at pHtrans 7.4. Two channels were present, and they exhibited low activity. EDC (1 mM) was then added to the trans chamber, and recordings were continued for 15 min. Activity of the channels did not change. GME (10 mM) was then added to the trans chamber, and recordings were continued for 15 min. Channel activity increased. pHtrans was then subsequently lowered to 3.0, and recordings were continued for 10 min. Channel activity further increased. B: summarized data on the effect of EDC-catalyzed amidation on channel Po. Data were plotted as means ± SE. Statistical significance of the effect of protonation (pHtrans 3.0; open bars) was calculated relative to the control value (pHtrans 7.4, hatched bars). Statistical significance for the amidation experiment was calculated by comparing changes in a stepwise manner with the previous condition. Thus Po in the presence of EDC alone was compared with control and Po in the presence of EDC and GME was compared with the Po in the presence of EDC alone. Hatched bars indicate pHtrans 7.4, open bars indicate pHtrans 3.0. * P < 0.001, ** P < 0.01, # P < 0.02.

                              
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Table 2.   Effect of amidation and low pHtrans on ClC-2G Cl- channel open and closed time constants, tau 1 and tau 2

Identification of the pH sensor region of the rabbit ClC-2G Cl- channel. A potential pH sensor region was identified in the loop between transmembrane domains D8 and D9 of the channel. This region has been shown to be extracellular through glycosylation studies (14, 21) and contains a negatively charged region with three glutamic acid residues in close proximity: E416-E417-L418-E419 (EELE). The effects of replacing these negatively charged residues with neutral, isosteric glutamines by site-directed mutagenesis were investigated. QQLE and EELQ ClC-2G Cl- channel mutants were expressed, and single-channel recordings were obtained throughout the voltage range from -80 mV to +80 mV. Figure 4 shows channel recordings and amplitude histograms at -80 mV holding potential obtained using these two mutant channels. In a comparison of mutant channel activity at pHtrans 3.0 and that at pHtrans 7.4, at -80 mV, the QQLE mutant channel retained acid activation. In contrast, the EELQ mutant channel lost its acid activation. The summarized data are shown in Fig. 5A. At -80 mV, the QQLE mutant channel retained significant (P < 0.001) (~6-fold) acid activation. Although the QQLE mutant channel exhibited significantly lower Po at both pHtrans 7.4 (P < 0.001) and 3.0 (P < 0.02) compared with the wild-type channel, its Po at pHtrans 3.0 was nevertheless ~75% of the Po of the wild-type channel at pHtrans 3.0. This value was similar to that of the amidated wild-type channel at pHtrans 7.4. In contrast, the EELQ mutant channel did not exhibit acid activation and Po values at both pHtrans 7.4 and 3.0 at -80 mV were significantly (P < 0.001) decreased compared with the wild-type channel. Similar results were obtained at +80 mV holding potential (Fig. 5B). Corresponding open and closed time constants (tau 1 and tau 2) at ±80 mV for the mutant channels are shown in Table 3. With the QQLE mutant channel, low pHtrans resulted in significantly increased (P < 0.001) open time constants and significantly decreased (P < 0.01; P < 0.02) closed time constants, as observed with the wild-type channel. With the EELQ mutant channel, low pHtrans had no effect on open time constants, whereas a significant but minor effect on closed time constants was obtained at -80 mV (P < 0.05; P < 0.02 for tau 1 and tau 2, respectively), resulting in the channel being virtually closed. The conductance of these two mutant channels remained similar to that of the wild-type channel (Table 1). These results strongly indicate that E419 is of predominant importance to acid activation of the ClC-2G Cl- channel, mediating ~75% of the maximal acid activation effect. E419 provides negative control over channel gating, which is relieved by neutralization.


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Fig. 4.   Cl- channel activity of QQLE (A) and EELQ (B) mutants of ClC-2G at pHtrans 7.4 and 3.0. A: channel current recordings obtained at -80 mV holding potential of the QQLE mutant channel (left) and corresponding amplitude histograms (right). Channel current recordings were first obtained with symmetric pH 7.4 solutions. One channel was present in the membrane, and it was mainly closed. pHtrans was then reduced to 3.0, and recordings were continued. Activity of the channel greatly increased. B: channel current recordings obtained at -80 mV holding potential for the EELQ mutant channel (left) and corresponding amplitude histograms (right). There were 2 channels present in the membrane, and they were mainly closed. pHtrans was then reduced to 3.0, and recordings were continued. Activity of the channels did not change.


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Fig. 5.   Effect of pHtrans reduction on Po of wild-type (W/T) and mutant ClC-2G Cl- channels at -80 mV (A) and +80 mV (B) holding potential. Po values at ±80 mV holding potential of the wild-type (EELE) and mutant channels (QQLE, EELQ, EELG, QQLG, and V349E) at pHtrans 7.4 (hatched bars) and 3.0 (open bars) are shown. Data were plotted as means ± SE. Statistical significance of the difference between Po of the channels at pHtrans 7.4 and pHtrans 3.0 is indicated: * P < 0.001, ** P < 0.01, # P < 0.02, ## P < 0.05. EELE = E416-E417-L418-E419.

                              
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Table 3.   Effect of low pHtrans on wild-type and mutant ClC-2G Cl- channel open and closed time constants, tau 1 and tau 2

In rat ClC-2, the residue corresponding to E419 of rabbit ClC-2G is neutral glycine and has also been reported to exhibit some activation by low extracellular pH (24). Therefore, an EELG mutant ClC-2G Cl- channel was examined. At -80 mV holding potential (Fig. 5A), the EELG mutant channel exhibited a Po of 0.41 ± 0.05 (n = 32, N = 15) at pHtrans 7.4 that was significantly greater (P < 0.001) than that of the wild-type channel [0.22 ± 0.02 (n = 25, N = 10)] under the same conditions. There was no increase in Po when pHtrans was lowered from 7.4 to 3.0 [Po = 0.32 ± 0.03 (n = 25, N = 8)]. This loss of acid activation was expected from studies with the EELQ mutant channel. Similarly, a significantly increased channel Po at pHtrans 7.4 was obtained at +80 mV holding potential (Fig. 5B). However, at +80 mV at pHtrans 3.0, the EELG mutant channel Po was significantly lower (P < 0.001) than at pHtrans 7.4. Thus, in contrast to the large fourfold increase in channel Po at reduced pHtrans (acid activation) observed with the wild-type channel at ±80 mV, the EELG mutant channel was constitutively more active at pHtrans 7.4 at ±80 mV and exhibited no change in Po with lowering of pHtrans at -80 mV. At +80 mV, Po significantly decreased (P < 0.001) with low pHtrans. The EELG mutant channel showed significantly higher activity (Po) than the EELQ mutant channel at ±80 mV and at pHtrans of both 7.4 and 3.0 (P < 0.001; P < 0.01, respectively). The failure of these mutants to show acid activation is reflected in the striking lack of increase in open time constants and lack of decrease in the closed time constants (Table 3). Conductances of the EELG and QQLG mutant channels remained similar to the wild-type channel at both pHtrans 7.4 and 3.0 (Table 1). These results are consistent with the findings that E419 plays a major role in the control of channel gating by pHtrans and suggest that both the structure and the charge at position E419 are important.

Interestingly, when the QQLG mutant channel was examined, acid activation of channel Po was evident at -80 mV but was absent at +80 mV (Fig. 5). In a comparison of the QQLE mutant with the QQLG mutant at -80 mV (Fig. 5A), Po values at pHtrans 7.4 were similar in the two mutants and acid activation was evident in both mutants but was significantly (P < 0.001) smaller in QQLG compared with QQLE. However, in contrast with the QQLE mutant, and similar to the findings with the EELG and EELQ mutants, the QQLG mutant channel showed no effects on the gating mechanism (open and closed time constants, Table 3). The findings that the QQLG mutant channel showed an increase in Po at -80 mV but not at +80 mV holding potential together with the lack of effect on open and closed time constants suggest that the effect of low pHtrans on the QQLG mutant channel at -80 mV is probably due to a screening effect of H+ on the surface potential (10) rather than a change in channel gating. This screening effect would be observed as a shift in the voltage dependence of activation of the channel, which is in agreement with the findings reported for the effect of low extracellular pH on rat ClC-2 (13).

Recently Jordt and Jentsch (13) reported that the single-point mutation V352E in the intracellular linker between transmembrane segments D7 and D8 of rat ClC-2 resulted in constitutive activation and loss of pH sensitivity of the rat ClC-2 Cl- channel. The corresponding rabbit V349E mutant ClC-2G Cl- channel was prepared to test whether this region was also involved in gating, pH sensing, or acid activation of channel activity. The Po values of the V349E mutant channel at pHtrans 7.4 and pHtrans 3.0 are shown in Fig. 5. In apparent contrast to the results reported for the rat V352E mutant ClC-2 Cl- channel (13), at -80 mV holding potential, not only was the rabbit V349E mutant ClC-2G Cl- channel activity very low, and not constitutively activated at pHtrans 7.4, but it retained some acid activation. Channel Po significantly increased (P < 0.01) from 0.03 ± 0.02 (n = 9, N = 4) at pHtrans 7.4 to 0.20 ± 0.05 (n = 20, N = 6), but, importantly, channel gating was unaffected as evidenced by lack of pHtrans 3.0 effect on open and closed time constants (Table 3). In contrast to all the other mutant ClC-2G Cl- channels, the conductance of the V349E mutant channel was significantly reduced at both pHtrans 7.4 (P < 0.02) and 3.0 (P < 0.05) compared with the wild-type channel (Table 1), suggesting that this amino acid residue may reside close to the pore of the channel.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The ClC-2G Cl- channel is voltage and PKA activated (16, 23). ClC-2G Cl- channels are significantly activated by low extracellular pH without affecting the voltage dependence of activation (3, 8, 16, 23). This was shown in studies using native parietal cell apical membranes (3) and using recombinant rabbit (16) and human (23) ClC-2G Cl- channels. The goal of the present study was to investigate the mechanism of acid activation of the ClC-2G Cl- channel and to localize the region involved in this activation at the molecular level.

The concentration-dependent response to change of pHtrans covered the range from pH 7.4 to pH 3.0. Extremes of low pH are physiologically relevant for the ClC-2G Cl- channel in the context of the gastric parietal cell, where it is likely to be involved in gastric acid secretion. Reduction of pH on the extracellular surface of the channel from pH 7.4 to pH 3.0 induced a fourfold increase in ClC-2G Cl- channel Po to a value approaching 0.80. Increased channel Po was due to significantly increased open time constants and significantly decreased closed time constants, which indicates that lowering the pHtrans affects the channel gating mechanism.

Channel Po increased in a concentration-dependent manner with increasing H+ concentration. Channel Po was half of its maximal value at pHtrans 4.95. This value was interpreted to be the apparent pKa of an amino acid involved in pH sensor function. Aspartic acid, glutamic acid, and histidine residues are all likely candidates for this role, with respective pKa values of 3.85, 4.25, and 6.0 for each amino acid in free solution (25). Although the value obtained is closest to the value for glutamic acid, the pKa of amino acids is known to be influenced by the environment. The hydrophobicity of the environment of the amino acid, as well as the presence of other charged amino acids in its vicinity, could alter its pKa. In addition, the nature of the solution bathing the amino acid could have an effect on the pKa, depending on how closely the microenvironment around the residue reflects that of the bulk solution.

It was therefore necessary to determine whether the amino acid responsible for acid activation was exposed to the extracellular surface of the channel and to distinguish between carboxylic amino acids and histidine. If the increased Po resulted from protonation and thus neutralization of one or several charged amino acids, then neutralization by amidation should lead to the same effect, namely, an increase in Po. Amidation by EDC occurs under very mild conditions and GME forms a neutral adduct. Both EDC and GME are hydrophilic and relatively bulky. If amidation from the outer surface affects channel activity, this would imply accessibility of the residue or residues to the solution bathing the extracellular surface of the channel. Amidation with EDC and GME at pHtrans 7.4 led to a threefold increase in channel Po that approximated the Po obtained with the QQLE mutant channel at pHtrans 3.0. This Po is ~75% of that obtained for the wild-type channel under the same conditions. When pHtrans was reduced to pH 3.0, the Po of the amidated ClC-2G channel increased to that of the untreated channel at pHtrans 3.0. This additional increase in Po at low pH could be due to acid-induced exposure of new amino acids to the reagents after the initial modification of a single, highly reactive amino acid and/or the known increase in the amidation reaction rate at low pH (18) or protonation of other sites, which are not initially accessible to the reagents. Noncovalent binding of either EDC or GME could affect channel function, and, in the absence of GME, formation of linkages between carboxy and amino groups of the protein could occur. However, neither EDC alone (Fig. 3) nor GME alone (not shown) affected ClC-2G Cl- channel Po or open or closed time constants (Table 2). These findings suggest that the pH sensor is likely to be a carboxylic acid and not histidine, since histidine is not reactive with EDC (18).

Because these large water-soluble compounds were added only to the trans chamber (corresponding to the outside of the channel), the susceptible amino acid(s) must be exposed at the extracellular surface of the channel and to the medium. Amino acid charge neutralization, whether by protonation or by formation of a neutral product through amidation, affected the Po and the closed and open time constants. There are numerous carboxylic amino acid residues in the ClC-2G channel protein (16). Although the primary reaction catalyzed by EDC is with carboxylic amino acids under these mild conditions, the amidation results with EDC must also be interpreted within the framework of knowledge that EDC can react with amino acids other than carboxylic acids and that EDC can gain access to amino acids that may be partially buried within the structure of proteins (18).

All members of the ClC Cl- channel family have a similar predicted topology of 10-12 transmembrane domains with both amino and carboxy termini localized to the cytoplasmic or intracellular side (21). With the assumption that the pH sensor contains carboxylic amino acid residues facing the extracellular environment of the channel, a region of the protein that contains a cluster of negatively charged amino acids and is known to be extracellular was selected for investigation as a possible pH sensor. Although the entire topology has not yet been experimentally verified, the loop between the D8 and D9 transmembrane domains has been shown by glycosylation experiments and protease protection assays (14, 19, 21) to be extracellular. In the rabbit ClC-2G Cl- channel, this loop contains a cluster of negatively charged glutamic acid residues E416, E417, and E419 (EELE). Therefore, to investigate the molecular basis of acid activation of the rabbit ClC-2G Cl- channel, these glutamic acid residues were modified and channel function was studied. The effects of pHtrans on channel Po, open and closed time constants, and voltage dependence of activation were measured.

The results with the EELQ and QQLE mutant channels suggest that E419 plays the key or predominant role in acid-activated gating of the rabbit ClC-2G Cl- channel. The reduction of negative control of the EELG mutant channel, along with loss of acid activation, demonstrates that the structure of the region containing EELE as well as charge neutralization is important to acid activation of channel gating. The voltage dependence of acid activation of the QQLG mutant channel illustrates that acid activation of the ClC-2G channel can occur through other mechanisms, perhaps through changes in surface potential (10) that do not affect channel gating.

Recently, it has been shown by whole cell current studies of rat ClC-2 and several other members of the ClC Cl- channel family, that changes in extracellular pH result in increases in the Cl- current. In rat (20) and human ClC-1 (4), lowering extracellular pH from 7.0 to 5.5 increased the Cl- current in the hyperpolarized range of the membrane potential (~1.3- to 1.6-fold at -80 mV), whereas there was no effect in the depolarized range and there was no effect on the time constants of the Cl- currents. These authors concluded that low extracellular pH affects the voltage dependence of activation of these channels. In studies of rat ClC-2 (13), the time constants of the Cl- currents were not analyzed. However, because acid activation did not occur at positive membrane potentials, the authors concluded that low extracellular pH affected the voltage-dependent activation of these channels.

The effect of extracellular acidification of rabbit ClC-2G has also been studied by measurement of whole cell Cl- currents in Xenopus oocytes (8). Lowering extracellular pH from 7.6 to 3.6 resulted in an increase in the Cl- current in both negative and positive ranges of membrane potential. Similar findings were obtained with whole cell current measurements of human ClC-2G Cl- channels overexpressed in IB3 cells (22), indicating that acid activation is independent of voltage. Although the mechanism of activation was not investigated, the results obtained with rabbit and human ClC-2G using whole cell current measurements are in full agreement with those at the single-channel level as shown in the present study but are in contrast to the findings with rat ClC-2 and ClC-1.

In whole cell recordings, rat ClC-2 (13) and rabbit (8) and human (22) ClC-2G Cl- currents displayed a slowly activating process and no deactivation, but gating models were not suggested in these studies. ClC-0, which has both fast and slow gating modes, has been described with a six-state Markovian model with a minimum of six time constants (1, 15). For ClC-1, which has slow, fast, and ultrafast transitions, a five-state model with six time constants has been proposed (4). To date, none of the members of the ClC Cl- channel family has shown simple two-state gating (open-closed) with only two time constants, tau open and tau closed. In the present study, two channels were generally present, and the "closed" and "open" times may differ from open and closed times that might be obtained with single-channel studies (see MATERIALS AND METHODS) or after "correction" (17). However, the open and closed times obtained were best fit by two exponentials with time constants tau 1 (fast) and tau 2 (slow), suggesting that the gating mechanism of the ClC-2G Cl- channel could be described by at least a three-state Markovian model with fast and slow gating modes and a minimum of four time constants. Similar behavior at the single-channel level has been described for the cardiac Ca2+ release channel (26) and the amiloride-sensitive Na+ channel (6). A model for the ClC-2G Cl- channel would require a detailed analysis of the lifetime distribution of each state and fitting by computer simulation, which is beyond the scope of the present studies.

The region EELE in the extracellular loop between transmembrane domains D8 and D9 is conserved in both rabbit and human ClC-2G Cl- channels (16, 23), whereas, in the rat ClC-2 Cl- channel, this region contains the sequence EDLG. Different amino acid composition between the rat ClC-2 and the human and rabbit ClC-2G Cl- channels, especially in position 419, may be responsible for the different mechanisms in responses to extracellular pH changes.

In rat ClC-2, a point mutation of residue V352E led to a constitutively open channel and loss of pH sensitivity (13). Channel function of the corresponding V349E mutant of the rabbit ClC-2G channel was examined, and significant differences were found. In direct contrast with the findings with rat ClC-2 (13), the V349E mutant of the rabbit ClC-2G Cl- channel did not lead to a constitutively open Cl- channel with loss of pH sensitivity. Instead, the rabbit V349E channel mutant had not only a significant level of pH sensitivity but also had a reduced channel Po compared with the wild type at pHtrans 7.4 and 3.0. In addition, this was the only mutant that showed altered (reduced) conductance compared with the wild-type channel, suggesting that the mutation may be affecting the folding of the protein in the vicinity of the channel pore. These results may reflect the differences in amino acid sequence between the ClC-2 and ClC-2G Cl- channels and the different methodologies used to study channel function.

In summary, the EELE419 sequence within the extracellular D8-D9 loop region in the rabbit ClC-2G Cl- channel is involved in activation of the channel by low pHtrans. Analyses of the channel open and closed time constants indicate that this region exerts negative control of channel gating. Charge neutralization and the structure of this region are both important to the control of channel gating. Of these amino acids, E419 plays the major role.

    ACKNOWLEDGEMENTS

We thank Lisa M. Knapp for her help with the initial experiments performed and Dr. J. W. Hanrahan for helpful discussions regarding analysis of multichannel records.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43816, DK-43377, and DK-58399 and the Cystic Fibrosis Foundation Award CUPPOL96PO to J. Cuppoletti and D. H. Malinowska.

Address for reprint requests: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, PO Box 670576, Cincinnati, OH 45267-0576.

Received 30 June 1997; accepted in final form 10 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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