Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell)

Muriel Nobles, Christopher F. Higgins, and Alessandro Sardini

Medical Research Council, Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, London W12 0NN, United Kingdom

Submitted 25 November 2002 ; accepted in final form 15 July 2004


    ABSTRACT
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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A Cl current activated by extracellular acidification, ICl(pHac), has been characterized in various mammalian cell types. Many of the properties of ICl(pHac) are similar to those of the cell swelling-activated Cl current ICl(swell): ion selectivity (I > Br > Cl > F), pharmacology [ICl(pHac) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 1,9-dideoxyforskolin (DDFSK), diphenylamine-2-carboxylic acid (DPC), and niflumic acid], lack of dependence on intra- or extracellular Ca2+, and presence in all cell types tested. ICl(pHac) differs from ICl(swell) in three aspects: 1) its rate of activation and inactivation is very much more rapid, currents reaching a maximum in seconds rather than minutes; 2) it exhibits a slow voltage-dependent activation in contrast to the fast voltage-dependent activation and time- and voltage-dependent inactivation observed for ICl(swell); and 3) it shows a more pronounced outward rectification. Despite these differences, study of the transition between the two currents strongly suggests that ICl(swell) and ICl(pHac) are related and that extracellular acidification reflects a novel stimulus for activating ICl(swell) that, additionally, alters the biophysical properties of the channel.

cell swelling-activated chloride current; patch clamp; pH


A CHLORIDE CURRENT ACTIVATED by cell swelling (ICl(swell)) has been characterized in many different cell types (for review see Refs. 26 and 37), and it is thought to play a role in regulatory cell volume decrease (RVD) in response to hypotonicity. ICl(swell) exhibits outward rectification, fast voltage-dependent activation, and time- and voltage-dependent inactivation at potentials more positive than +40 mV. The relative anion selectivity for the swelling-activated Cl channel is SCN > I > Br > Cl > F > gluconate; this sequence is referred to as "Eisenman sequence I" and indicates a weak interaction between the channel pore and the permeation anion. A number of different substances, including the stilbene derivative 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (9, 35), the anti-inflammatory drug niflumic acid (20), the antiestrogen drug tamoxifen (43), diphenylamine-2-carboxylic acid (DPC) (22), and 1,9-dideoxyforskolin (DDFSK) (9), inhibit ICl(swell). Furthermore ICl(swell) is insensitive to changes in calcium concentrations (33). Despite much effort and several candidates, P-glycoprotein (P-gp) (42), pICln (30) and ClC-3 (12), none has stood the test of time, and the molecular identity of the channel responsible for ICl(swell) and its mechanism of activation remain unknown.

Extracellular acidification has been reported to both activate and inhibit anion channel activities in different cell types. Extracellular acidification to pH 5–6 inhibits ClC-5 channels (25) and the small-conductance Cl channel from kidney distal tubule (32), yet it activates the ClC-2 (4, 19, 38), ClC-7 (10) and ClC-0 (8) channels. Furthermore eriC, a bacterial ClC-type Cl channel, has been shown to be activated by acidic pH (17). This supports the idea that pH modulation of ClC channels is maintained throughout evolution. Effects of extracellular acidification on ICl(swell) have already been described (27, 31, 44).

In this study we characterize a Cl current activated by extracellular acidification (ICl(pHac)) present in a variety of mammalian cell types. The current shares many characteristics with ICl(swell). Devised experiments looking at the transition between currents activated by extracellular acidification and hypotonicity show that the time course of the change in the biophysical characteristics of the two currents is too fast to be accounted for by two different channels. Finally, ICl(swell) shows in this study a clear pH dependence. Our data suggest that acidic extracellular pH provides an alternative pathway that activates the channel responsible for ICl(swell) and alters its biophysical properties. This has implications for the signal transduction pathways that mediate channel opening and the physiological role of the swelling-activated Cl channel.


    EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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Cell culture. Unless otherwise stated, experiments were carried out with HEK-293 (transformed human embryonic kidney) cells. Cells were grown in Dulbecco's modified Eagle's medium with Glutamax (DMEM; GIBCO Life Technologies), 10% fetal bovine serum (FBS; Helena Laboratories) and 1% penicillin-streptomycin (P/S; GIBCO Life Technologies). PC-12 (transformed rat pheochromocytoma) cells were grown in DMEM, 6% FBS, 6% horse serum, and 1% P/S; Caco-2 (transformed human colon) cells in DMEM, 20% FBS, and 1% P/S; and HeLa (transformed human adenocarcinoma) and CHO (transformed hamster ovary cells) cells in DMEM with Glutamax, 10% FBS, and 1% P/S. HEK-293, PC-12, and Caco-2 cells were grown on bare plastic flasks. Human bronchial epithelial cells (16HBE14o-; Ref. 14) and human tracheal epithelial (HTE) cells (Ref. 47) were grown in DMEM (GIBCO Life Technologies) with 10% fetal bovine serum (GIBCO Life Technologies) and 1% gentamicin (Sigma) in flasks coated with a solution containing 1 mg of human fibronectin (Stratech), 0.33 mg Vitrogen (Imperial Laboratories), and 10 mg bovine serum albumin (ICN Flow) in 100 ml of MEM with Earle's salts (GIBCO Life Technologies). The NIH3T3-MDR1 cell line, a derivative of the mouse NIH 3T3 fibroblast cell line permanently transfected with the human multidrug resistant gene (MDR1) coding for P-gp (34), was grown in DMEM with Glutamax, 10% FBS, 1% P/S, and 1 µg/ml colchicine.

Patch-clamp recording. Cl currents were measured in the whole cell recording mode of the patch-clamp technique as described previously (46).

Cells were plated in 35-mm plastic dishes coated with fibronectin-based solution (see above) and mounted on the stage of an inverted Leica DMIL microscope. Whole cell currents were recorded with an Axon 200A amplifier. Cells were clamped at 0 mV and pulsed for 500 ms from –80 mV to +120 mV in 40-mV steps. For current showing time- and voltage-dependent inactivation maximum currents were measured at the beginning of the 500-ms voltage pulse, whereas for current showing voltage-dependent activation maximum currents were measured at the end of the voltage pulse. Current inactivation was measured by fitting a single exponential to the traces recorded at + 120 mV and comparing the time constant {tau}.

Occasionally, whole cell current-voltage (I-V) curves were obtained by applying a ramp of voltage from –120 mV to +120 mV over a 1-s period. The Strathclyde Electrophysiological Software written by J. Dempster (University of Strathclyde, Glasgow, UK) was used for pulse generation, data acquisition (DigiData 1200 interface Axon Instrument), and subsequent analysis.

Whole cell anion currents were measured with an isotonic extracellular (bathing) solution containing (in mM) 140 N-methyl-D-glucamine chloride (NMDGCl), 0.5 MgCl2, 1.3 CaCl2, and 10 HEPES titrated with Trizma-base solution to the indicated pH. The osmolarity was corrected to 310 mosM with mannitol. The extracellular hypotonic solution, used to elicit swelling-activated currents, had the same composition as the isotonic solution except that it contained 100 mM NMDGCl and that osmolarity was adjusted with mannitol to 220 mosM. Extracellular solutions with Cl substitution were obtained by replacing 140 mM NMDGCl with the respective anion salts (NaI, NaF, or NaBr) at 140 mM, osmolarity was adjusted with mannitol to 310 mosM, and pH was buffered at 4.5. For swelling-activated currents, 100 mM NMDGCl was replaced with the respective anion salts (NaI, NaF, or NaBr) at 100 mM, osmolarity was adjusted with mannitol to 220 mosM, and pH was buffered at 7.4. The intracellular (pipette) solution was (in mM) 140 NMDGCl, 1.2 MgCl2, 1 EGTA, and 10 HEPES, titrated to pH 7.4 with Trizma-base and osmolarity adjusted to 280 mosM with mannitol. To investigate the pH dependence of the Cl currents, the pH of the extracellular solution was adjusted to between pH 4.5 and 7.4 with Trizma-base. In some experiments, as indicated, 10 mM HEPES was replaced with 10 mM MES (Sigma).

Cells were perfused with a gravity-fed bath perfusion system. Drugs and experimental solutions were applied by the same route. The perfusion system was positioned close to the recorded cell to achieve a fast solution exchange.

Inhibitors. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), DDFSK, DIDS, niflumic acid, and tamoxifen were all purchased from Sigma. Stock solutions of niflumic acid and tamoxifen were prepared in ethanol, and DDFSK was diluted in DMSO. DPC was purchased from Fluka, and stock solutions were prepared in DMSO. The solvents at the final dilution were tested alone and showed no effect.

Cell volume measurement. HEK-293 cells, grown on glass coverslips coated with poly-L-lysine, were loaded with 2.5 µM calcein-AM (Molecular Probes) for 10 min at room temperature and washed in isotonic solution for ~30 min before each experiment. Calcein-AM is nonfluorescent and cell membrane permeant. After diffusion into the cells it is converted by intracellular esterases into calcein, which is fluorescent and membrane impermeant. Calcein-loaded cells were imaged with a Leica SP confocal microscope using an oil immersion x63, 1.32 NA plan Apochromat objective lens in a perfusion chamber containing (in mM) 140 NMDGCl, 0.5 MgCl2, 1.3 CaCl2, and 10 HEPES titrated with Trizma-base solution to the indicated pH. Osmolarity was corrected to 310 mosM with mannitol. The extracellular hypotonic solution had the same composition as the isotonic solution except that it contained 100 mM NMDGCl and osmolarity was adjusted with mannitol to 220 mosM. Calcein was excited by the 488-nm line of an argon laser, and fluorescence was collected with a 499- to 601-nm band pass emission filter. Images were acquired every 20 s, and the average intensity fluorescence signal collected from an area within the cell was analyzed. To calibrate the fluorescence signal, brief exposures to 15% hypotonic and 15% hypertonic solutions were used. The signal was then analyzed and converted in volume measurement (1).

Intracellular pH measurements. To measure intracellular pH (pHi) we used 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes). CMFDA is an analog of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and shows a pH-dependent spectral shift in more acidic pHi than BCECF because of its acidic dissociation constant (pKa) being ~6.4 instead of 7.0 as for BCECF. This fluorophore has been shown to be suitable for pHi measurements (36). Cells were excited alternatively at 495 and 440 nm, and pHi was assessed from the ratio of the respective emitted fluorescent signals at 520 nm. HEK-293 cells were loaded with 5 µM CMFDA for 45 min at 37°C and rinsed for 60 min at 37°C with the extracellular buffer containing (in mM) 140 NaCl, 2.5 KCl, 0.5 MgCl2, 1.2 CaCl2, 10 HEPES, and 5 glucose (310 mosM). The solution was buffered at pH 7.4 or 4.7 with NaOH. Hypotonic experiments were done with an extracellular solution in which 140 mM NaCl was replaced by 100 mM NaCl (220 mosM). pHi calibration was done by exposing cells to a solution containing (in mM) 140 KCl, 10 NaCl, 2 MgCl2, 0.2 CaCl2, 0.5 EGTA, and 10 HEPES with the H+ ionophore nigericin (13 µM; Sigma) titrated to different values of pH, ranging from 5.5 to 9.2, by Trizma-base. The calibration curve was linear for pH values within the range from 6.6 to 7.4 (coefficient of correlation 0.999, slope 2.52).

Fluorescence measurements were performed by using as a light source a polychrome II (T.I.L.L. Photonics) connected to an inverted Zeiss microscope through a x40 oil-immersion objective. Images were analyzed with Openlab (Improvision, Coventry, UK). Measurements were then processed with Sigma Plot software (version 7).

Statistics. Data are expressed as means ± SE (n = number of cells). Statistical analyses were performed by nonpaired t-test; statistical significance was accepted for P < 0.05.


    RESULTS
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 EXPERIMENTAL PROCEDURES
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Extracellular acidification activates a Cl current. When HEK-293 cells were challenged with an isotonic extracellular solution at pH 4.5, a large current developed within 5–10 s (Fig. 1A). The mean maximum current, measured at +120 mV after 1-min exposure to a solution at pH 4.5, was 91.96 ± 10 pA/pF (n = 29). The current disappeared within 1 min after switching back to pH 7.4. Under control conditions (pH 7.4), current measured at +120 mV was negligible (6.91 ± 1.44 pA/pF; n = 41). This current activated by extracellular acidification showed strong outward rectification at voltages greater than +50 mV (Fig. 1B). Because NMDGCl was used in the intracellular and extracellular solutions, and the reversal potential (Erev) was +2.6 ± 2.1 mV in agreement with the predicted Erev of 0 mV, the current activated by extracellular acidification must have been a Cl current, and was designated ICl(pHac).



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Fig. 1. Electrophysiological characteristics of extracellular acidification-activated Cl current (ICl(pHac)) in HEK-293 cells. A: representative Cl currents in isosmotic solutions buffered at pH 7.4 (left) and pH 4.5 (middle) and after returning to pH 7.4 (right). Cells were stimulated for 500 ms with square voltage pulses from –80 mV to +120 mV in 40-mV voltage steps from a 0 mV holding potential. t, Time. B: current-voltage (I-V) relationships of Cl current in HEK-293 cells in presence of the following conditions: isosmotic solution (Iso) at pH 4.5 (n = 12), hyposmotic solution (Hypo) at pH 7.4 (n = 9), and hyposmotic solution at pH 4.5 (n = 5). There is no significant difference in the amplitude of the current measured at +120 mV.

 
A similar current activated by extracellular acidification was recorded in several different cell lines, including CHO, HeLa, PC-12, Caco-2, HBE, and HTE cells. In all cells tested the currents had similar time- and voltage dependence, although the magnitude of the currents varied.

pH profile for activation of ICl(pHac). HEPES, with a pKa of 7.5, has optimal buffering capacity in the pH range 6.8–8.2. To exclude the possibility that the current recorded at pH 4.5–5.0 was due to the poor pH buffering capacity of HEPES, we also used MES (pKa 6.1), which has good buffering capacity in a lower pH range. No significant difference in ICl(pHac) was observed whatever buffer was used. pH activation of the Cl current was observed at or below pH 5.5, but not at pH 6.2, and maximum current was obtained at pH 5.0 (Fig. 2).



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Fig. 2. ICl(pHac) is activated below pH 5.5. Mean Cl currents in HEK-293 cells at +120 mV were elicited at different extracellular pH values with either HEPES- or MES-buffered solutions. Currents were measured at pH 7.4 (HEPES; n = 9), pH 6.2 (HEPES; n = 5), pH 5.5 (MES; n = 4), pH 5.0 (MES; n = 5), and pH 4.5 (HEPES; n = 9).

 
Ion selectivity of current activated by extracellular acidification. Relative anion selectivity for Cl, I, Br, and F was measured for ICl(pHac) (Fig. 3A) from the respective values of Erev. According to the extracellular and intracellular solutions used, the predicted Erev for Cl is 0 mV. In Cl-containing solution, Erev was +2.6 ± 2.1 mV (n = 10), not significantly different from that predicted. Erev was not changed significantly when Cl was replaced by Br (–0.4 ± 1.3 mV; n = 10). However, replacement of Cl with I shifted Erev to more negative values (–28.8 ± 4.5 mV; n = 9), and Erev was shifted to more positive values (+10.2 ± 2 mV; n = 9) when Cl was replaced by F. Thus the selectivity sequence for the channel is I > Br ≥ Cl > F. A similar selectivity sequence was obtained for ICl(swell) (Fig. 3B). To expose the same cell to the different halides, measurements of ICl(swell) were done at the onset of the current. Because ICl(swell) develops with a much slower kinetics than ICl(pHac) (see Fig. 4), this accounts for the difference of current magnitudes of ICl(swell) (Fig. 3B) and ICl(pHac) (Fig. 3A).



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Fig. 3. Anion permeability sequences of ICl(pHac) (A) and cell swelling-activated Cl current (ICl(swell); B). Anion currents were activated at pH 4.5 or after challenge with a hypotonic solution and measured in the presence of different halides, as indicated, with a ramp protocol from –120 mV to +120 mV lasting 1 s. Note that the currents activated by hyposmotic shock are small because measurements were done as soon as the current started to develop. The traces are representative of 9 independent experiments. The anion selectivity sequence was I > Br > Cl > F.

 


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Fig. 4. Time activation and inactivation of ICl(swell) and ICl(pHac). Cells were clamped at 0 mV and pulsed for 500 ms from –80 mV to +120 mV. A: ICl(swell), activated by a hyposmotic solution at pH 7.4. B: ICl(pHac), activated by an isosmotic solution at pH 4.5. Curves represent an average of 6 independent experiments.

 
The maximum currents (Imax) at –120 and +120 mV, and the ratio between Imax at +120 mV and –120 mV, of ICl(swell) and ICl(pHac) are shown in Table 1. For ICl(pHac), there was a striking loss of outward rectification, due to an increase in the size of the inward current, when either I or F replaced Cl. For ICl(swell), a loss in outward rectification due to a decrease in outward current was observed when F replaced Cl.


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Table 1. Anion selectivity for ICl(pHac) and ICl(swell)

 
Pharmacology and calcium dependence of ICl(pHac). The effect of various Cl channel blockers on ICl(pHac) was tested (Tables 2 and 3). Significant inhibition (P < 0.05) was obtained with DIDS, niflumic acid, and DDFSK. There was no significant effect of 5 µM tamoxifen (Table 2), a concentration known to block the swelling-activated Cl channel (43). Preincubation with tamoxifen for 10–15 min before challenge with the low-pH solution also failed to inhibit ICl(pHac). Higher concentrations of tamoxifen (>5 µM) were toxic to HEK-293 cells. In parallel experiments at pH 7.4, ICl(swell) was completely inhibited by 5 µM tamoxifen, showing that the compound is active. Tamoxifen is also an inhibitor of transport mediated by the multidrug resistance ATPase P-gp (6). To exclude the possibility that tamoxifen is inactivated at acidic pH, we showed that P-gp transport activity in NIH3T3-MDR1 cells was inhibited by 10 µM tamoxifen prepared in a solution buffered at pH 4.5 (data not shown). The failure of tamoxifen to inhibit ICl(pHac) is addressed below.


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Table 2. Pharmacology of ICl(pHac)

 

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Table 3. Effects of DIDS

 
DPC was recently shown to inhibit a Cl current activated by extracellular acidification in Sertoli cells (3) as well as ICl(swell) (22). In the presence of 500 µM DPC, the mean maximum current, measured at +120 mV, for ICl(swell) was 13.5 ± 0.17 pA/pF (n = 3) vs. a control of 114 ± 27.84 pA/pF (n = 13) and for ICl(pHac) was 6.6 ± 1.5 pA/pF (n = 3) vs. a control of 91.96 ± 10 pA/pF (n = 29). Therefore, a large inhibition of both types of Cl currents induced by extracellular acidification and cell swelling was observed. The level of inhibition of ICl(pHac) was similar to that described on application of 500 µM DPC in Sertoli cells (3). In contrast, the level of inhibition of ICl(swell) apparently differed from the value of an IC50 of 350 µM reported for a small intestinal human epithelial cell line (22) and an IC50 of 200 µM for cultured chick heart cells (48). These differences are presumably attributable to the different cell types used in the mentioned studies.

ICl(swell) is insensitive to chelation of intracellular calcium (29); therefore, the dependence of ICl(pHac) on calcium ions was also tested. The intracellular solution was calcium free and included 1 mM EGTA. There was no significant effect of removing calcium from the extracellular solution (n = 4, data not shown). To buffer any possible calcium release from intracellular stores, 2 mM BAPTA was added to the patch pipette. There was no significant effect of BAPTA on ICl(pHac) (n = 6, data not shown). From these experiments we can exclude the involvement of calcium ions in the activation of ICl(pHac).

Rate of activation and inactivation of ICl(pHac) and ICl(swell). The activation of Cl currents by cell swelling and extracellular acidification in HEK-293 cells occurs at different rates (Fig. 4). Cl currents activated by cell swelling (hypotonicity) took 4–6 min to reach a maximum and ~10 min to diminish to background level on return to isotonicity (Fig. 4A). On extracellular acidification, the Cl currents reached a maximum within 10–15 s, and they disappeared within 15 s after return of the extracellular pH to 7.4 (Fig. 4B). The slower rate of activation of ICl(swell) is consistent with many previously published studies (43).

Characteristics of ICl(swell) at different extracellular pH values. The data above show that ICl(pHac) has many similarities to ICl(swell), namely, outward rectification, selectivity, and sensitivity to pharmacological inhibitors, and is calcium independent. We therefore hypothesized that ICl(swell) and ICl(pHac) might be a manifestation of the same channel activated by different stimuli. However, specific biophysical properties, rate of activation, voltage-dependent activation, and time- and voltage-dependent inactivation, of the two currents differ. Thus if ICl(swell) and ICl(pHac) are due to the same channel, its biophysical properties after activation must also be pH dependent.

We therefore asked whether extracellular protons affect the properties of ICl(swell). At pH 7.4, ICl(swell) showed time- and voltage-dependent inactivation at voltages greater than +40 mV (Fig. 5A), as expected (mean {tau} = 0.628 s at + 120 mV; n = 5). However, when cells were challenged with a hyposmotic solution at pH 6.5 (a pH at which ICl(pHac) is not activated; Fig. 2) time- and voltage-dependent inactivation was reduced (Fig. 5B; mean {tau} = 1.200 s at + 120 mV, n = 4). A significant difference between the maximum and the plateau current measured was only obtained in the presence of a hyposmotic solution buffered at pH 7.4 (Fig. 5D). At pH 4.5, ICl(swell) showed slow voltage-dependent activation rather than time- and voltage-dependent inactivation (Fig. 5C). The decrease in extracellular pH also appeared to decrease the inward current measured at –80 mV (Fig. 5D), although these changes were not statistically significant. These data suggest that the biophysical properties of ICl(swell) are sensitive to extracellular pH.



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Fig. 5. Characteristics of ICl(swell) at different extracellular pH values. Representative traces of an experiment in which ICl(swell) was activated for 2 min with a 30% hyposmotic solution at pH 7.4 (n = 5; A), pH 6.5 (n = 4; B), or pH 4.5 (n = 4; C) are shown. The value of {tau} in A and B refers to the time constant of a single exponential fitted to the trace recorded at + 120 mV. Each experiment is from a separate cell. Note that because currents were measured 2 min after challenge with the experimental solution, ICl(swell) will not be maximally activated. D: summary of the experiments described in A-C. Currents measured at –80 and +120 mV are shown. For currents measured at +120 mV, the maximum current refers to the current measured at the beginning of the 500-ms voltage pulse whereas the mean current refers to that measured at the end of the voltage pulse.

 
To obtain further evidence that ICl(swell) and ICl(pHac) are the manifestation of the same channel we studied the additivity of the currents. If the two currents are manifestations of different channels, the activation would be expected to be additive and the maximum current elicited by each stimulus to differ. HEK-293 cells were challenged with an isosmotic solution at pH 4.5, a hyposmotic solution at pH 7.4, or an hyposmotic solution at pH 4.5 (Fig. 1B). There was no significant difference in the mean size of currents measured at +120 mV in any of the three different conditions.

pHi measurements. We have addressed the possibility that extracellular acidification may lead to changes in pHi and consequently affect the swelling-activated channel. pHi measurements were conducted in HEK-293 cells loaded with the fluorescent dye CMFDA (see EXPERIMENTAL PROCEDURES). A 2-min exposure to extracellular acidification (pH 4.5) led to a decrease of 0.21 ± 0.02 pHi units (n = 3), whereas a 2-min challenge with a 30% hypotonic solution led to a decrease of 0.2 ± 0.09 pHi units (n = 4). The intracellular acidification following extracellular acidification is unlikely to inhibit ICl(swell) because a similar reduction in pHi is obtained with a hypotonic challenge. Moreover, this pHi decrease is far from the value of pH 6.0 that has been shown to abolish ICl(swell) (31).

Sequential stimulation of cells by hyposmotic shocks at different extracellular pH values. To more directly demonstrate that ICl(pHac) is a manifestation of the same channel as ICl(swell), cells were sequentially exposed to different conditions. First, ICl(swell) was activated by a hyposmotic stimulus at pH 7.4 and, as soon as the current reached a maximum (after 4–6 min), the cell was challenged with a hyposmotic solution buffered at pH 4.5. A typical trace is shown in Fig. 6A. Transition from pH 7.4 to pH 4.5 led to a rapid change in the biophysical characteristics (time- and voltage-dependent inactivation) of the current toward the characteristics of ICl(pHac). Because 1 min after the switch to pH 4.5 was insufficient for ICl(swell) to inactivate (Fig. 4), the change in current biophysical characteristics can only be accounted for by a change in the properties of ICl(swell) and not by inactivation of the swelling-activated channel and activation of another Cl channel. Histograms in Fig. 6B summarize the experiment shown in Fig. 6A. There was no significant difference in the maximum amplitude of the currents recorded at +120 mV when cells were sequentially challenged with hyposmotic solutions at pH 7.4 and 4.5.



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Fig. 6. Change in biophysical characteristics of ICl(swell) on transition from pH 7.4 to pH 4.5. A: cells were first challenged with an hyposmotic solution buffered at pH 7.4 for 4 min, followed by a hyposmotic solution buffered at pH 4.5. Currents were measured 1 min after the shift to pH 4.5. B: histograms showing maximum currents measured at +120 mV summarize experiments in A. No significant additive effect of reduction of osmolarity and extracellular acidification is observed. Representative traces of 6 independent experiments for each condition are shown.

 
Currents in isosmotic or hyposmotic solutions buffered at pH 4.5 were indistinguishable (Figs. 1A and 5C). It should also be noted that the shape of the currents in hyposmotic solution buffered at pH 4.5 differed depending on whether the cells were previously exposed to a hyposmotic solution at pH 7.4 (Fig. 6A) or not (Fig. 5C). Figure 5C shows a typical current from a cell bathed in an isosmotic solution at pH 7.4 and immediately challenged with an hyposmotic solution at pH 4.5. Its characteristics should be compared with those in Fig. 6A, where the cell was exposed for 4 min to a hyposmotic solution at pH 7.4 before the exposure to hyposmotic solution at low pH.

We then did a transition in hyposmotic solution from pH 4.5 to 7.4 (Fig. 7A). Within 30 s of shifting the cells from pH 4.5 to 7.4 the biophysical characteristics changed from the typical ICl(pHac) profile to that of ICl(swell). Because activation of ICl(swell) takes many minutes (Fig. 4A), a delay of 30 s is not sufficient to allow ICl(swell) to develop. Therefore, if the channel responsible for ICl(swell) was inhibited by extracellular acidification (hyposmotic solution, pH 4.5), it would have taken longer for the current to activate during the transition from pH 4.5 to pH 7.4. The phenomenon observed during the transition in hyposmotic solution from pH 4.5 to 7.4 (Fig. 7A) cannot be explained by closure of a channel activated by extracellular acidification and activation of another channel activated by hypotonicity, although it can be explained by activation of a single type of channel under both conditions whose biophysical properties are modified by extracellular pH. The channel responsible for ICl(swell) was therefore, in the experiment shown in Fig. 7A, activated by the hyposmotic solution buffered at pH 4.5. The absence of additivity of the currents, shown by the histogram of the mean maximum currents at +120 mV (Fig. 7B), is a further proof that only one type of channel supports the currents observed in hyposmotic solution at pH 4.5 and in hyposmotic solution at pH 7.4.



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Fig. 7. Change in biophysical and pharmacological characteristics of ICl(swell) on transition from pH 4.5 to pH 7.4. A: cells were challenged for 1 min (by which time the currents have reached the maximal value) with a hyposmotic solution at pH 4.5 and then immediately exposed to a hyposmotic solution buffered at pH 7.4. Currents were recorded 30 and 60 s after the shift in condition, a time too short for activation of ICl(swell). B: histograms showing maximum currents measured at +120 mV summarize experiments in A. No additive effects of extracellular acidification and reduction of osmolarity (ICl(swell)) is observed at any time. C: cells were first challenged for 1 min (until current stabilized to a maximal value) with a hyposmotic solution at pH 4.5 in the presence of 5 µM tamoxifen and then immediately exposed to a hyposmotic solution buffered at pH 7.4 (in the sustained presence of tamoxifen). D: histograms showing maximum currents measured at +120 mV summarize experiments in C. Tamoxifen did not affect the currents measured in acidic conditions, yet currents became sensitive to tamoxifen on transition to pH 7.4. Representative traces of 5 independent experiments are shown.

 
We noted above the failure of tamoxifen to inhibit ICl(pHac), a result apparently inconsistent with identity of ICl(swell) (which is tamoxifen sensitive) and ICl(pHac). To explore this further, we examined the transition between the two currents in the presence of tamoxifen (Fig. 7C). Tamoxifen did not inhibit the Cl currents at acidic pH, yet immediately (<30 s) after the shift from pH 4.5 to pH 7.4 the current was almost completely inhibited, showing that the sensitivity of the channel to tamoxifen is pH dependent. This suggests that tamoxifen interacts with residues on the channel that are protonated at acidic extracellular pH. Together, these experiments strongly suggest that the currents activated by extracellular acidification or hypotonicity are manifestations of the same channel but that the characteristics of this channel, including its kinetics of activation and sensitivity to tamoxifen, are modified by extracellular pH.

Cell volume during extracellular acidification. We asked whether activation of ICl(pHac) by extracellular acidification might be due to change in cell volume caused by acidic pH rather than directly by acidification itself. Cell volume was measured in cells loaded with the fluorescent dye calcein, where the fluorescence signal is proportional to the concentration of calcein and hence to cell volume (41). We observed RVD in extracellular hyposmotic solutions buffered at acidic pH (data not shown). Cell volume was not altered by exposure to acidic pH (Fig. 8A), and, furthermore, pH did not influence the increase in cell volume induced by hypotonic solution (Fig. 8B). Thus the Cl currents activated at acidic pH cannot be due to induction of cell swelling by acidic pH.



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Fig. 8. Extracellular acidification does not affect cell size or cell swelling. Cells were loaded with calcein in an isosmotic solution at pH 7.4 and then challenged with the indicated solutions. Fluorescence intensity, measured for individual cells, was converted in relative volume. The mean ± SE change in relative cell volume (Vt/Vo) is shown. Vo and Vt are the cell volume at time 0 and t, respectively. A: exposure to acidic pH does not, in itself, affect cell volume (n = 39). B: rapid changes in cell volume in response to hypotonic solutions with recovery on return to isotonic solution. There was no difference in the magnitude of the cell swelling whether the hypotonic solution was buffered at pH 7.4 or 4.5 (n = 18).

 

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In this study we describe a Cl current, ICl(pHac), activated by extracellular acidification, showing outward rectification with a selectivity sequence I > Br ≥ Cl > F, and blocked by the Cl channel inhibitors DIDS, DPC, niflumic acid, and DDFSK. Activation of ICl(pHac) was rapid, with a maximum current achieved at pH 5.0 within 10–15 s of exposure to acidic pH.

Many of the characteristics of ICl(pHac), including ion selectivity, pharmacology, and lack of dependence on Ca2+, are similar to those of the well-characterized swelling-activated Cl current ICl(swell), suggesting that ICl(pHac) could be a manifestation of the same channel responsible for ICl(swell). Although ICl(pHac) and ICl(swell) have outwardly rectifying I-V relationships, ICl(pHac) shows a steeper outward rectification due to smaller inward currents. In contrast to ICl(swell), ICl(pHac) showed slow voltage-dependent activation and was tamoxifen insensitive whereas (as reported previously) ICl(swell) showed fast voltage-dependent activation and time- and voltage-dependent inactivation and was tamoxifen sensitive. Furthermore, ICl(swell) shows a clear pH dependence: change in extracellular pH during hypotonic challenge led to loss of time- and voltage-dependent inactivation. Thus if the two currents are manifestations of the same channel, its biophysical properties must be pH sensitive. To test this we studied the transition between the two currents. The change in current characteristics (voltage-dependent activation and time- and voltage-dependent inactivation, tamoxifen sensitivity) was too rapid (30 s) to be accounted for by inactivation of one channel and activation of another (because ICl(swell) takes several minutes to fully activate or inactivate) and could only be explained by a change in biophysical characteristics of a single channel type in response to changes in extracellular pH. Inactivation of the channel responsible for ICl(swell) by extracellular acidification is also unlikely because Sabirov et al. (31) showed that extracellular acidification leads to a significant increase in the single-channel current amplitude.

The maximum current elicited by either stimulus was the same in any cell type, and the combination of both stimuli (decrease in extracellular osmolarity and pH) did not increase the maximum current (absence of additivity). Together, these results suggest that ICl(pHac) could be a manifestation of the same channel as ICl(swell).

ICl(swell) is known to be activated by a number of maneuvers in nonswollen cells, e.g., activation by guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) and low ionic strength (7, 11, 28, 45). Because these maneuvers shift the set point of ICl(swell), osmotic shrinkage is known to be effective in blocking Cl currents activated by the nonswelling maneuvers. ICl(pHac) was not affected by hyperosmotic solution (380 mosM, data not shown), suggesting that if only one type of channel supports ICl(pHac) as well as ICl(swell), the pH- and swelling-sensing domains must be different.

Furthermore, patch excision and depolarization may elicit outwardly rectifying, depolarization-induced Cl channels (ORCC or ORDIC), whose molecular identity is yet unknown. ORCC shows marked outward rectification, sensitivity to general Cl channel blockers, anion selectivity Eisenman sequence I, and voltage- and time-dependent activation at positive potentials (16), characteristics shared with ICl(pHac). ORCC channels have been shown to be distinct from the swelling-activated Cl channels (21). We can clearly elicit ICl(pHac) without patch excision, making ORCC unlikely to be responsible for ICl(pHac). Our data also show a clear pH modulation of ICl(swell) when cells were challenged by a hyposmotic solution buffered at acidic pH. Indeed, the voltage- and time-dependence at positive potentials changed gradually by progressive extracellular acidification from pH 7.4 to pH 6.5 and pH 4.5, supporting the hypothesis of a single type channel for both types of currents.

Activation of ICl(swell) by extracellular acidification could be an essential screening tool for the identification of the protein responsible for ICl(swell) because it would provide an alternative to the conventional stimulus of cell swelling. In rat parotid acinar cells, extracellular acidosis was indeed reported to potentiate swelling-activated Cl currents (2). Interestingly, our data show that the rate of current activation and inactivation by pH is much more rapid than that induced by cell swelling, even though the maximum currents elicited by the two stimuli do not differ. This suggests that pH does not simply act through an effect on cell volume. This was confirmed by demonstrating that extracellular acidification does not lead to cell swelling or influence cell swelling during hypotonic stimulus. Thus the mechanism by which extracellular acidification activates Cl secretion through the channel also responsible for ICl(swell) must involve, at least in part, a different signal transduction pathway. It has been suggested that cell swelling may activate ICl(swell) through production of a second messenger, accounting for the relatively slow rate of activation (for review, see Refs. 15 and 40). The activation by extracellular acidification, which is much more rapid, may circumvent the requirement for second messenger synthesis. Because it has proven very difficult to elucidate the pathway involved in activation of ICl(swell), these findings suggest an alternative approach. Interestingly, the multidrug resistance P-gp is known to increase the rate of activation of ICl(swell), yet the mechanism has remained entirely obscure (5, 24, 41). Because it is well established that P-gp mediates a decrease in extracellular pH (23, 39), it is possible that P-gp increases the rate of activation of ICl(swell) indirectly, through an effect on local extracellular pH.

Another explanation for the effect of extracellular acidification would be the concomitant inhibition of the channel responsible for ICl(swell) by intracellular acidification and activation of a different type of channel responsible for ICl(pHac). The small degree of intracellular acidification detected on a short exposure (2 min) to an extracellular acidic solution (pH 4.5) is unlikely to inhibit ICl(swell) because only a reduction of pHi to pH 6.0 has been shown to abolish ICl(swell) activity (31). It is also unlikely to be the stimulus for ICl(pHac) because it occurs with a much slower kinetics. Indeed, the activation of ICl(pHac) on extracellular acidification reaches a steady state within few seconds (Fig. 4), suggesting that the effect is due to external pH affecting channel gating as has been suggested for ClC-2 (19). A similar decrease in pHi was obtained on challenge with hypotonic solution, in agreement with previously published work (18); indeed, moderate intracellular acidification increases the single-channel conductance of the channel supporting ICl(swell) (31).

In addition to activation of the channel, extracellular acidification also induces a change in the biophysical properties of the channel. At neutral pH ICl(swell) shows fast voltage-dependent activation and time- and voltage-dependent inactivation and is tamoxifen sensitive, yet after extracellular acidification slow voltage activation is seen and the channel is no longer blocked by tamoxifen.

Modulation of the activity of anion channels through changes in pH is an established phenomenon. Sabirov et al. (31) showed that protonation of an extracellular site by exposure to acidic extracellular solution increases the single-channel amplitude of the cell swelling-regulated anion channel. External H+ modulates the fast gate of ClC-0 (8), and mutation of Glu166 of ClC-0 to Ala, Gln, or Val has an effect on the fast gate similar to that of exposure of the wild-type channel to acidic extracellular solution (13), suggesting that the deprotonated form of the glutamate chain closes the wild-type channel. ClC-2 has been shown to be activated by extracellular acidification primarily by affecting its gating (19). Furthermore, by a combination of site-directed mutagenesis and amidation of carboxyl groups, Stroffekova et al. (38) identified in ClC-2 isolated from rabbit gastric mucosa a negatively charged extracellular region, EELE, with a role of pH sensor.

Until the molecular identity of the channel responsible for ICl(swell) is determined, and the mechanism of tamoxifen action is known, the mechanism of modulation by extracellular H+ is likely to remain obscure, although it is not unreasonable to suppose that lowering of the extracellular pH may change the protonation state of amino acid groups in an extracellular domain of the channel and therefore alter its behavior.


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This work was supported by the Medical Research Council, UK.


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Ian McFadzean (King's College, London) for helpful comments on this work.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Nobles, University College London, Dept. of Medicine, Clinical Pharmacology, 5 University St., London WC1E 6JJ, UK (E-mail: m.nobles{at}ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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