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Am J Physiol Heart Circ Physiol 290: H1905-H1914, 2006. First published December 9, 2005; doi:10.1152/ajpheart.00965.2005
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Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Shintaro Yamamoto and Tsuguhisa Ehara

Department of Physiology, Saga University Faculty of Medicine, Saga, Japan

Submitted 9 September 2005 ; accepted in final form 2 December 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular acidic pH was found to induce an outwardly rectifying Cl– current (ICl,acid) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN– > Br– > I– > Cl– > F– > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl– channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl– current (ICl,vol) and CFTR Cl– current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.

acidosis; chloride channel; heart

CHLORIDE (Cl–) channels play a role in a variety of cell functions, such as cell cycle, apoptosis, ion homeostasis, cell volume regulation, and muscle tone (for review see Refs. 11, 12, and 20). Several types of Cl– currents have been recorded in native cardiac cells (for review see Refs. 9 and 11). These include Cl– currents activated by intracellular PKA (ICl,PKA), PKC (ICl,PKC), Ca2+ (ICl,Ca), extracellular ATP (ICl,ATP), or cell swelling (ICl,vol) and an inwardly rectifying Cl– current (ICl,ir). The channels responsible for ICl,PKA, ICl,PKC, and ICl,ATP likely share the same molecular basis derived from CFTR gene, and thus these currents can be unified as ICl,CFTR (11). It has been shown that some of these Cl– currents depend on extracellular pH (pHo). Acidic pHo enhances ICl,Ca in rabbit ventricular cells (10) and ICl,ir in rat atrial and ventricular cells (13, 14) and inhibits ICl,CFTR in guinea pig ventricular cells (17). The roles of such pHo-dependent attenuation of Cl– currents in cardiac electrical activity under pathophysiological conditions have been investigated (9–11, 13, 14).

On the other hand, a more directly pHo-dependent regulation has been found in cultured cells. Auzanneau et al. (1) showed that acidic pHo induces a distinct type of Cl– current (ICl,acid) in cultured rat Sertoli cells. This ICl,acid is characterized by activation by severe acidification, outward rectification in current-voltage (I-V) relationships, time-dependent activation during depolarization, sensitivity to DIDS, and independence of intracellular Ca2+. Nobles et al. (19) recorded a similar ICl,acid as well as ICl,vol in several types of cultured cell lines including HEK293 cells. However, they argued that ICl,acid and ICl,vol are different manifestations of the same channel. According to their proposal, acidification alters the properties of the ICl,vol channels, so that acidification alone can activate a ICl,vol whose pharmacological and biophysical characteristics are quite different from those of ICl,vol at normal pHo.

Recently, however, Lambert and Oberwinkler (15) extensively studied the properties of ICl,acid and ICl,vol in HEK293 cells. Their important findings are as follows. The recorded single-channel events, which were likely to underlie the whole cell ICl,acid, showed characteristics different from those of ICl,vol channels recorded in endothelial cells (22). The outward ICl,vol showed a biphasic change on external acidification, first decreasing and then increasing, and this behavior was difficult to explain by a simple transition model like that predicted by Nobles et al. (19). ICl,acid and ICl,vol showed, respectively, time-dependent activation and inactivation during depolarization, and the experiments in which repetitive depolarizing pulses were applied to the cells revealed that the above time-dependent behavior of each current differentially persisted even under acidic hypotonic conditions. On the basis of these findings, Lambert and Oberwinkler (15) concluded that ICl,acid and ICl,vol are caused by different channels.

As described above, ICl,acid has been demonstrated in cultured nonexcitable cells. We report here that acidic pHo activates ICl,acid in freshly isolated mammalian cardiac cells. Our results show that the biophysical and pharmacological properties of cardiac ICl,acid are, in many respects, similar to those of ICl,acid in cultured cells (1, 3, 15). The present study further shows that ICl,acid, unlike ICl,vol and ICl,CFTR, activates independently of the presence of intracellular ATP and that GTP-binding protein is not involved in its activation. In addition, we obtained data that indicate that hypotonic or hypertonic conditions, respectively, are inhibitory or facilitatory for the development of ICl,acid. On the other hand, ICl,CFTR induced by beta-adrenergic stimulation was inhibited by acidification in a pH-dependent manner. Because an acidic environment can develop in local myocardium under pathological conditions such as myocardial ischemia, ICl,acid would play a role in regulation of cardiac electrical activity and cell volume under these pathological conditions. Preliminary accounts of this work have appeared in abstract form (26, 27).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
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Cell preparation. The Committee of Animal Welfare in Saga Medical School approved the use and treatment of all animals used in the experiments described here. The investigation conforms also with the Guiding Principles of the Physiological Society of Japan. Single cardiac myocytes from mouse (18–25 g, C57BL/6J/black inbred, male and female) and guinea pig (250–350 g, male and female) hearts were isolated with an enzymatic dispersion technique as described previously (25, 28, 29). The animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The chest was opened, and the heart was rapidly removed and perfused at 37°C, using a modified Langendorff technique with a fully oxygenated physiological saline solution (PSS; see Solutions and drugs) for 2–5 min, then with a normally Ca2+-free PSS until the heart ceased to beat, and finally with a Ca2+-free solution containing 0.1% collagenase (CLS II, Worthington, Lakewood, NJ) and 1.0% bovine serum albumin for 10–20 min. The collagenase was then washed out of the heart with a high-K+, low-Cl– storage solution (see Solutions and drugs). The digested atria and ventricles were separately dissected to disperse the cells in the high-K+, low-Cl– solution. Isolated myocytes were stored in this medium at 4°C until use. Only rod-shaped atrial and ventricular myocytes with clear cross-striations and no blebs were used for experiments.

Electrophysiological techniques. The tight-seal whole cell patch-clamp technique was used to record membrane currents in isolated cells. Patch pipettes (borosilicate glass electrodes) had a tip resistance of 1–3 M{Omega} when filled with pipette solution. Voltage-clamp recordings were performed with a patch-clamp amplifier (TM-1000; Act ME, Tokyo, Japan), and membrane currents were filtered at 2.5 kHz and sampled at 5 kHz with an analog-to-digital converter (Digidata 1322A) and pCLAMP 9.0 software (Axon Instruments, Foster City, CA). Action potentials (APs) were recorded in current-clamp configuration with a patch-clamp amplifier (CEZ-2300; Nihon Kohden, Tokyo, Japan). Depolarizing pulses of 2-ms duration, sufficient to trigger an AP, were injected at a frequency of 5 Hz. A 3 M KCl-agar bridge was set between the bath and the Ag-AgCl reference electrode to minimize changes in liquid junction potential. Unless otherwise stated, current recordings were made by applying voltage pulses of 400-ms duration to various potentials (between –100 and +120 mV in 20-mV steps) from a holding potential of 0 mV every 2 s. When necessary, current density (current magnitude per membrane capacitive area) was calculated, cell membrane capacitance being measured with pCLAMP 9.0 software. All voltage-clamp recordings were performed at room temperature and AP recordings at 36 ± 1°C.

Solutions and drugs. PSS for cell preparation contained (mM) 126 NaCl, 10 glucose, 4.4 KCl, 5.0 MgCl2, 1.5 CaCl2, 20 taurine, 5.0 creatine, 5.0 sodium pyruvate, 1.0 NaH2PO4, and 10 HEPES, pH 7.4 adjusted with NaOH. The high-K+, low Cl– solution for cell storage contained (mM) 70 potassium glutamate, 20 KCl, 1.0 MgCl2, 10 KH2PO4, 10 taurine, 10 EGTA, 10 glucose, 0.1% albumin, 10 beta-hydroxybutyric acid, and 10 HEPES, pH 7.2 with KOH, 300 mosM with mannitol. All bath and pipette solutions were prepared to minimize cation currents and Ca2+-dependent currents. For mouse ICl,acid and ICl,CFTR recordings, the standard bath solution contained (mM) 127 NaCl, 0.8 MgCl2, 1.0 CaCl2, 5.0 CsCl, 2.0 BaCl2, 0.2 CdCl2, 5.5 glucose, 10 HEPES, 5.0 MES, and 0.01 nicardipine, total extracellular Cl– concentration ([Cl–]o) = 140 mM and pH 4.5–8.5 adjusted with L-aspartic acid or N-methyl-D-glucamine (NMDG), where appropriate. For guinea pig ICl,acid and ICl,CFTR recordings, NaCl was totally replaced with NMDG-Cl to inhibit an acidification-induced cationic current (17). CdCl2 in these solutions was expected to inhibit ICl,ir, if any (13, 14). Low-[Cl–]o bath solutions were prepared by replacing NaCl in the standard bath solution with Na salt of various anions (aspartate–, I–, Br–, F–, and SCN–) on an equimolar basis. Hypotonic and isotonic bath solutions for ICl,vol recording were prepared by reducing NaCl concentration in the standard bath solution to 77 mM, with [Cl–]o = 90 mM and 200 and 320 mosM with mannitol, respectively. Thus both hypotonic and isotonic solutions had a constant ionic strength.

The standard pipette solution contained (mM) 140 NMDG, 140 HCl, 5.0 MgATP, 5.0 EGTA, and 10 HEPES, pH 7.3 adjusted with NMDG and total intracellular Cl– concentration ([Cl–]i) = 140 mM. In some experiments, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; 1.0 mM) was added to this solution or EGTA was replaced with BAPTA on an equimolar basis. The pipette solution with 30 mM [Cl–]i was prepared by replacing 110 mM NMDG-Cl in the above solution with an equimolar amount of NMDG-aspartate. Osmolarity of standard bath and pipette solutions was adjusted to 320 and 290 mosM with mannitol, respectively, for inhibition of swelling-induced currents under control conditions. The pipette solution for ICl,vol recordings was prepared by reducing both NMDG and HCl in the standard pipette solution to 90 mM, pH 7.3 with NMDG and 290 mosM with mannitol. When simultaneous recordings of ICl,acid and ICl,vol were attempted (see Figs. 5–8), the bath and pipette solutions for ICl,vol recordings were used. For AP recordings, the bath solutions contained (mM) 140 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 5.5 glucose, and 5 HEPES, total [Cl–]o = 150 mM, pH 7.4 with NaOH, and 320 mosM with mannitol. The pipette solution for AP recordings contained (mM) 110 potassium glutamate, 30 KCl, 10 NaCl, 5.0 MgATP, 0.1 Tris-GTP, 5.0 EGTA, and 10 HEPES, total [Cl–]i = 40 mM, pH 7.3 with KOH, and 290 mosM with mannitol. Osmolarity was measured with a freezing point depression osmometer (model OM-801; Vogel, Giessen, Germany). Drugs used were glibenclamide (Sigma), DIDS (Sigma), tamoxifen (Sigma), niflumic acid (Sigma), forskolin (Sigma), 8-bromoadenosine 5'-O-(2-thiodiphoshate) (8-BrcAMP; Sigma), and isoprenaline (Daiichi, Tokyo, Japan).


Figure 5
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  		Fig. 5. Inhibition of ICl,acid by hypotonic challenge in ATP-depleted cells. A: time course of changes in whole cell currents during acidification followed by hypotonic challenge. Pipette solution did not contain ATP [intracellular ATP concentration ([ATP]i) = 0], and bath solution was glucose free. Currents were measured at +80 and –80 mV in a cell under symmetrical [Cl–] (90 mM) conditions. Acidic (pH 4.5) solution and acidic hypotonic (Hypo) solution were applied during the time indicated by bars. The osmolarity of the isotonic solution (Iso) was 320 mosM and that of the hypotonic solution was 200 mosM. B: records of membrane currents obtained at the time points indicated by a, b, c, and d in A. Acidification activated ICl,acid (b), which was reduced in acidic hypotonic solution (c). C: mean I-V relationships (n = 4) of ICl,acid (difference current; b – a in B) and ICl,acid as influenced by hypotonicity (c – a). *Significant change at P < 0.05. In D, relative change in ICl,acid at +120 mV produced by hypotonic or hypertonic challenge (ratio of ICl,acid in test solution vs. ICl,acid in control) is plotted against osmolarity. ICl,acid was elicited at pH 4.5. Number of cells examined at each osmolarity is given in parentheses. *,**Significant change at P < 0.05 and 0.01, respectively.

 

Figure 8
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  		Fig. 8. Activation of ICl,vol in the presence of ICl,acid. A: time course of activation of whole cell currents recorded during application of acidic (pH 6.0) solution and subsequent application of acidic hypotonic solution. The osmolarity of the isotonic solution was 320 mosM, and that of the hypotonic solution was 200 mosM. The currents were measured at +80 and –80 mV in a cell under symmetrical [Cl–] (90 mM) conditions. The test solutions were applied during the time indicated by bars. B: records of membrane currents obtained at the time points indicated by a, b, c, and d in A. Acidification induced ICl,acid (b), and the subsequent acidic hypotonic challenge initially decreased (c) and then increased (d) the whole cell currents. C: I-V relationships of the whole cell currents shown in a, b, c, and d in B. D: mean I-V relationship (n = 4) of hypotonicity-induced currents (difference current) corresponding to d – c in B. ICl,acid was activated at pH 6.0.

 
Data analysis. The linear fitting of the reversal potential-log [Cl–]o relationships (see Fig. 2A) was made according to a regression analysis with the least-squares method. Data are expressed as means ± SE; n indicates the number of cells. Statistical comparisons were performed either by one-way or two-way ANOVA with post hoc test (Scheffé's multiple comparison test) for group data as appropriate or by Student's paired t-test when only paired groups were compared. A two-tailed probability of <0.05 was taken to indicate statistical significance.


Figure 2
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  		Fig. 2. Anion sensitivity of acidic pHo-induced currents. A: reversal potential (Vrev)-log extracellular [Cl–] ([Cl–]o) relationship of acidic pHo-induced currents (difference currents) obtained with 30 mM intracellular [Cl–] ([Cl–]i). Data were obtained in 4–6 cells exposed to pH 4.5 at each [Cl–]o. Linear regression analysis revealed Vrev = 58.1 log (34.7/[Cl–]o). B: mean I-V relationships of acidic pHo-induced (difference) current obtained in solutions containing different anion species at pH 4.5. I–-rich (I–) or aspartate-rich (Asp–) solution was made by replacing 127 of 140 mM NaCl in normal-Cl– solution (Cl–) with an equimolar amount of NaI or Na-aspartate, respectively; n = 4 for each solution. C: anion permeability (P) sequence of the acidic pHo-induced current. Experiments similar to those shown in B were performed with various anion species (anion X–) as indicated, and Vrev was determined from the I-V relation of the difference current for each X–-rich solution. The relative P for each anion X– to Cl– (PX/PCl) was calculated with the Goldman-Hodgkin-Katz equation. Number of cells examined in each solution is given in parentheses.

 

   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Figure 1 shows the activation of whole cell currents by acidic pHo observed in a mouse ventricular cell. When pHo was changed from normal (7.4) to 6.5, the whole cell currents at both positive and negative voltages gradually increased and reached an almost steady level in ~5 min (Fig. 1A). The whole cell currents further increased on subsequent pH change to 4.5 and decreased to control level quickly (within 1 min) on resumption of pHo 7.4. The I-V relationships of the acidic pHo-activated current exhibited a clear outward rectification (Fig. 1, Bc and C). During the applied voltage pulses, the current was largely time independent, but a time-dependent slow activation was noted during large positive pulses (e.g., +100 and +120 mV; Fig. 1Bc). Using solutions with various pHo, we obtained pHo-response relationships for acidic pHo-induced (difference) currents at +120, +40, and –80 mV (Fig. 1D). At all voltage steps, the threshold pH value for current activation appeared to be ~pH 7, and the maximum activation was obtained at ~pH 4.5. An analysis with the Hill equation for the currents at +120 mV revealed the pH value for half-maximal activation (EC50) and Hill coefficient (nH) to be pH 5.89 and 1.12, respectively. Such acidic pHo-induced currents were observed in 24 of 26 ventricular cells examined (9 mice). Similar currents were also observed (data not shown) in mouse atrial cells (n = 6) and in atrial (n = 4) and ventricular (n = 10) cells from guinea pig. We could not detect any outwardly rectifying whole cell current in cells dialyzed with an acidic (pH 6.5) pipette solution and perfused at pH 7.4, but exposure of these cells to an acidic (pH 4.5) solution induced a current similar to that described above (data not shown). Although pipette solutions with lower pH values were not examined, the above findings might suggest that the proton-acting site is located outside the membrane.


Figure 1
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  		Fig. 1. Activation of whole cell currents by acidic extracellular pH (pHo) in mouse ventricular myocytes. A: time course of activation of whole cell currents recorded in a cell under symmetrical Cl– concentration ([Cl–]; 140 mM) conditions during application of acidic bathing solution. In this and subsequent similar figures, currents were measured at +80 (bullet) and –80 ({circ}) mV, with the pulse protocol shown in the inset. pH of the bathing solution was changed from 7.4 to 6.5 or 4.5 during the time indicated by bar. B: records of membrane currents obtained at the time points indicated by a, b, and c in A. C: mean steady-state current-voltage (I-V) relationships (n = 24) of whole cell currents obtained at pH 7.4 and pH 4.5. *,**Significant change at P < 0.05 and 0.01, respectively. D: pHo-response relationship of acidic pHo-induced (difference) currents ({Delta}I). Each point represents mean of data obtained in 4 different cells, and curves were obtained by fitting the data points to the Hill equation. Data were obtained at +120, +40, and –80 mV at steady state.

 
The reversal potential (Vrev) of the acidic pHo-induced current depended on [Cl–]o. Figure 2A shows Vrev-log [Cl–]o relationships obtained at three different [Cl–]o with a pipette (internal) solution containing 30 mM Cl–. The regression line had a slope of 58.1 mV per 10-fold change in [Cl–]o, indicating that chloride ions were the main charge carrier of this current. In the preceding experiments (Fig. 1C), Vrev was 3.1 ± 1.9 mV (n = 24), and this value is also close to the Cl– equilibrium potential (ECl = 0 mV) predicted for those symmetrical [Cl–] conditions. Thus we designated the acidic pHo-induced current as ICl,acid. To examine the anion selectivity of ICl,acid, we measured I-V relationships of ICl,acid in low-[Cl–]o solutions containing high concentrations of different anions. Examples are shown in Fig. 2B, in which I-V relationships obtained in Cl–-, I–-, and aspartate-rich solutions are illustrated. Quantitative analysis with the Goldman-Hodgkin-Katz equation revealed that the relative permeability (P) for each anion X– to Cl– (PX/PCl) was SCN– > Br– > I– > Cl– > F– > aspartate (Fig. 2C).

We next compared the effects of several Cl– channel inhibitors (DIDS, glibenclamide, niflumic acid, and tamoxifen) on ICl,acid. To activate ICl,acid in these experiments, we used a bath solution with pH 5.5, because high (50–100 µM) concentrations of glibenclamide were insoluble at pH 4.5. DIDS, glibenclamide, and niflumic acid were soluble at a concentration of 100 µM and tamoxifen at 10 µM. Figure 3A shows an example of such experiments. One hundred micromolar DIDS strongly inhibited the outward ICl,acid, whereas it inhibited the inward ICl,acid only weakly, indicating that DIDS exerted a voltage-dependent inhibition on ICl,acid. Figure 3B summarizes the effect of the inhibitors on ICl,acid. Glibenclamide and niflumic acid moderately inhibited ICl,acid in a voltage-independent manner, whereas ICl,acid was little sensitive to tamoxifen. On the other hand, DIDS (100 µM) and tamoxifen (10 µM) exerted little effect on the background current at pH 7.4 (data not shown), suggesting that any inhibitor-sensitive current like ICl,vol was absent at least at pH 7.4.


Figure 3
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  		Fig. 3. Effect of Cl– channel inhibitors on acid-induced Cl– current (ICl,acid). A: representative I-V relationship of the whole-cell current obtained in control solution (pH 7.4), after application of acidic (pH 4.5) bathing solution, and after further application of 100 µM DIDS. B: comparison of the effects of several Cl– channel blockers on ICl,acid: 100 µM glibenclamide (Glib), 100 µM niflumic acid (NFA), 10 µM tamoxifen (TXF), and 100 µM DIDS were examined. {Delta}Idrug/{Delta}Iacid, which represents the ratio of inhibitor-sensitive current component ({Delta}Idrug) to control {Delta}Iacid, was determined at +100 and –100 mV. ICl,acid was activated at pH 5.5. Data are expressed as means ± SE for no. of cells in parentheses. **Significantly different at P < 0.01.

 
The beta-adrenergic stimulation of ICl,CFTR involves activation of the receptor-coupled GTP-binding protein (G protein), whereas several cardiac membrane currents such as ICl,Ca depend on intracellular Ca2+. We examined whether such mechanisms are involved in the activation of ICl,acid. Figure 4A shows the results of the experiments performed in GDPbetaS (a nonhydrolyzable GDP analog)-loaded cells, while GDPbetaS was expected to block activation of G proteins (6). ICl,acid was activated by acidification as usual in these cells (Fig. 4A), suggesting that G protein is not involved in the activation process of ICl,acid. ICl,acid could also be activated in cells dialyzed with a pipette solution in which the intracellular Ca2+ buffer was changed from EGTA to BAPTA (5 mM; Fig. 4B), the latter being expected to better chelate Ca2+ and to render the intracellular Ca2+ concentration ([Ca2+]i) far lower than a functional concentration. The experiments were done also in cells in which [Ca2+]i was set at 150 nM with a mixture of 5 mM EGTA and 5 mM Ca2+. This also did not affect the activation of ICl,acid (data not shown), and the size of ICl,acid was similar at both these two [Ca2+]i, suggesting that ICl,acid activation is independent of intracellular Ca2+.


Figure 4
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  		Fig. 4. Independence of ICl,acid on intracellular GTP-binding protein (A) and calcium ions (B). In A and B, whole cell currents were recorded at pHo 7.4 (a), pHo 4.5 (b), and pHo 7.4 again (c). Mean I-V relationships (n = 4) of acidic pHo-induced (difference) currents ({Delta}I) are shown in d. Cells were dialyzed with a pipette solution containing 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; A) or 5 mM BAPTA (B).

 
The activation of ICl,CFTR (18) and ICl,vol (23) requires the presence of intracellular ATP. The suppression of the activation of these two currents in ATP-depleted cells was confirmed also in the present study (data not shown). In contrast, ICl,acid could be recorded in the absence of intracellular ATP (see Figs. 5 and 6). Similar results were obtained in 5 mM adenosine 5'-(beta,{gamma}-imido)triphosphate (a nonhydrolyzable ATP analog)-loaded cells (data not shown). These may indicate that ATP hydrolysis, ATP-mediated phosphorylation, or related reactions are not involved in the activation process of ICl,acid.


Figure 6
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  		Fig. 6. Effect of tamoxifen on ICl,acid during hypotonic challenge in ATP-depleted cells. Experiments similar to those shown in Fig. 5A were performed, but tamoxifen (10 µM) was applied to the cell during hypotonic challenge at pH 4.5. A: current recordings obtained in acidic isotonic solution (a), in acidic hypotonic solution (b), and after addition of tamoxifen to the latter solution (c); d shows the difference current (c – b) as "tamoxifen-induced" current. B: mean I-V relation of the tamoxifen-induced current (d in A; n = 4).

 
The biophysical and pharmacological properties of ICl,acid noted above did not appear to be the same as those of other cardiac Cl– currents such as ICl,vol and ICl,CFTR. In the following experiments, we attempted to further characterize ICl,acid compared with the properties of ICl,vol and ICl,CFTR. First, we examined how the tonicity of the bathing solution influenced the activation of ICl,acid in ATP-depleted cells. In these experiments, current measurements were begun at least 10 min after establishment of the whole cell configuration with ATP-free pipette solutions. In the experiment shown in Fig. 5A, the cell was first exposed to acidic (pH 4.5) isotonic (320 mosM) solution, which resulted in a development of ICl,acid as usual (Fig. 5, A and Bb). When the cell was consecutively exposed to acidic (pH 4.5) hypotonic (200 mosM) solution, a reduction of ICl,acid was observed (Fig. 5, A and Bc), and the reduction was reversible (Fig. 5, A and Bd). It should be noted that, as described above, ICl,vol hardly develops in ATP-depleted cells. Even when the acidic hypotonic challenge was prolonged for >10 min in similar experiments, there was little increase in the whole cell currents during this challenge (data not shown). Mean I-V relationships of ICl,acid obtained under isotonic and hypotonic conditions are shown in Fig. 5C. Similar experiments were performed with a less hypotonic solution (250 mosM) or a hypertonic solution (360 mosM). The results of these experiments are summarized in Fig. 5C. Hypotonic conditions inhibited ICl,acid, and the inhibition appeared to be greater the lower the tonicity, whereas hypertonic conditions enhanced ICl,acid.

Tamoxifen is a well-known inhibitor of ICl,vol. We confirmed that this drug greatly attenuated hypotonicity-induced ICl,vol (4 cells), 10 µM tamoxifen reducing ICl,vol by >60% (data not shown). Curiously, this drug was found to increase ICl,acid in ATP-depleted cells under hypotonic conditions. As shown in Fig. 6A, when tamoxifen was applied to the ATP-depleted cell in acidic hypotonic solution, there was an increase in the whole cell currents (Fig. 6, Ab and Ac). The "tamoxifen-induced" current showed a time-dependent activation at positive potentials (Fig. 6Ad), and its I-V relation exhibited outward rectification (Fig. 6B), suggesting that this current represented a fraction of ICl,acid. The mechanism underlying this response is unclear. The hypotonicity-induced cell swelling might be related to this tamoxifen action, but we did not further investigate this point.

We next examined how ICl,acid and ICl,vol developed simultaneously, using cells dialyzed with ATP-containing pipette solution. In the experiment shown in Fig. 7A the cell was first exposed to hypotonic solution, and this resulted in development of the ICl,vol, in which a time-dependent inactivation is barely visible at positive potentials (Fig. 7Bb). The I-V relationship of ICl,vol is shown in Fig. 7D. When pH of the hypotonic solution was changed from 7.4 to 4.5, there was a sizable increase in the whole cell currents (Fig. 7, A and Bc). It is noteworthy that the increased outward currents, like ICl,acid shown above (e.g., Fig. 1Bc), exhibited a time-dependent activation at large positive potentials (Fig. 7Bc). The acid-induced current (difference current) obtained during the hypotonic challenge and its I-V relationships are shown in Fig. 7, Bd and E, respectively.


Figure 7
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  		Fig. 7. Activation of ICl,acid in the presence of volume-regulated Cl– current (ICl,vol) in cells with 5 mM [ATP]i. Cells were dialyzed with pipette solution containing 5 mM ATP. A: time course of activation of whole cell currents recorded during hypotonic challenge followed by acidification. Currents were measured at +80 and –80 mV in a cell under symmetrical [Cl–] (90 mM) conditions. Hypotonic solution and acidic hypotonic (pH 4.5) solution were applied during the time indicated by bars. The osmolarity of the isotonic solution was 320 mosM, and that of the hypotonic solution was 200 mosM. B: records of membrane currents obtained at the time points indicated by a, b, and c in A. Hypotonic challenge induced ICl,vol (b), and the additional acidification further increased the whole cell currents (c). The difference current (c – b) is shown in d. C: records of membrane currents obtained in an experiment similar to that shown in A. The experimental protocol was the same as in A, but here tamoxifen (10 µM) was present throughout. D: mean I-V relationships (n = 4) of ICl,vol (difference current, b – a in B and C) obtained in the presence or absence of tamoxifen. E: mean I-V relationships (n = 4) of acid-induced currents obtained in the presence of ICl,vol (d in B and C) with and without tamoxifen.

 
Similar experiments were also performed in the continuous presence of tamoxifen (10 µM) with the experimental protocol shown in Fig. 7A. The records of membrane currents, I-V relationships of ICl,vol, and acid-induced current (difference current) obtained in these experiments are shown in Fig. 7, C, D, and E, respectively. Tamoxifen inhibited the development of ICl,vol, as expected (Fig. 7D). However, this agent did not inhibit the development of acid-induced current (Fig. 7Cc). The acid-induced current showed a time-dependent activation at large positive potentials (Fig. 7Cd), and its magnitude was comparable to that seen without tamoxifen (Fig. 7E).

Lambert and Oberwinkler (15) observed that when hypotonic solution was switched to acidic hypotonic solution, there was a decrease in the outward ICl,vol before development of ICl,acid. We did not consistently observe such a phenomenon in the present study, and the effect of acidification on ICl,vol in our preparation is unclear. Hence the difference current shown in Fig. 7Bd cannot be regarded as representing the true magnitude of ICl,acid. However, the density of the acid-induced currents (Fig. 7E) is comparable to that of ICl,acid obtained in ATP-depleted cells (Fig. 5C) in which ICl,vol was presumed to be absent. We consider that the acid-induced currents obtained here (Fig. 7, Bd and E) represent largely, if not entirely, ICl,acid and that acidification exerts no profound effect on ICl,vol.

In the above experiments, ICl,acid developed in the presence of ICl,vol. We also observed that ICl,vol developed in the presence of ICl,acid. In the experiment shown in Fig. 8, the cell was first exposed to acidic solution to activate ICl,acid (Fig. 8Bb). When an acidic hypotonic solution was consecutively introduced, the whole cell currents initially decreased (Fig. 8, A and Bc) and increased thereafter, the increase overwhelming the current level caused by acidification alone (Fig. 8, A and Bd). It is conceivable that ICl,vol developed in addition to ICl,acid at this situation. On the other hand, the current decrease observed shortly after acidic hypotonic solution (Fig. 8Bc) was considered to reflect the inhibitory effect of hypotonic condition on ICl,acid, which was noted above (Fig. 5). The I-V relationship of the whole cell currents obtained during the course of the experiment is shown in Fig. 8C and that of hypotonicity-induced currents obtained at acidic pH in Fig. 8D. We conclude that ICl,acid and ICl,vol can develop in an additive manner in cardiac cells.

We next examined the effects of acidification on ICl,CFTR. For this purpose, we used guinea pig ventricular cells, because these cells, unlike mouse cells (5, 16), develop ICl,CFTR in response to beta-adrenergic stimulation (9, 11, 12). In the experiment shown in Fig. 9A, application of 1 µM isoprenaline to the cells induced ICl,CFTR (Fig. 9Ab). Subsequent acidification in the presence of isoprenaline clearly increased the outward currents, and the increased currents showed time-dependent activation at large positive potentials, but the inward currents increased only slightly in this situation (Fig. 9Ac), the whole cell current revealing clear outward rectification. Curiously, withdrawal of isoprenaline from the acidic solution had little effect on the currents at any voltage (Fig. 9, Ac and d). It appears that acidification to pHo 4.5 eliminated ICl,CFTR, while inducing ICl,acid.


Figure 9
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  		Fig. 9. Effects of acidification on membrane currents in the presence of an activator of CFTR Cl– currents (ICl,CFTR) in guinea pig ventricular cells. A: after control records (a) were obtained at pH 7.4, 1 µM isoprenaline was introduced to the bath at pH 7.4, which led to development of ICl,CFTR (b). pHo of the agonist-containing solution was then changed to 4.5, which resulted in a further increase in the whole cell current (c). Finally, isoprenaline was omitted from the bath at pH 4.5 (d). B: results of an experiment similar to that shown in A, but in which 100 µM DIDS was present throughout and an intermediately acidic (pH 6.5) solution with isoprenaline was additionally examined. C: mean I-V relationship (n = 4) of acid-sensitive component of the whole cell current seen in the presence of both isoprenaline and DIDS. Data were obtained by subtracting the currents at pH 4.5 (d) from those at pH 7.4 (b). D: effects of acidic pHo on the whole cell current in the presence of isoprenaline (1 µM) and DIDS (100 µM). y-Axis, density of the whole cell current at +100 mV observed in the presence of both of the two agents (bullet); x-axis, pHo value. The control current level obtained before application of isoprenaline at pH 7.4 is also shown ({circ}). Number of myocytes examined at each pHo is given in parentheses.

 
The effect of acidic pHo on ICl,CFTR was further studied at pH 6.5, 5.5, and 4.5 in the presence of 100 µM DIDS, the latter being expected to suppress ICl,acid. In the presence of DIDS, the lower the pH value of agonist-containing solution the smaller the whole cell current (Fig. 9B), confirming the acid-induced inhibition of ICl,CFTR. The I-V relationship of the current component suppressed by acidification to pHo 4.5 (difference current) is shown in Fig. 9C. Vrev of this component was near 0 mV, and the outward portion of I-V relation was almost linear, in line with the property of ICl,CFTR. The inward portion exhibited a rectification-like bending. This bending was attributed to an insufficient inhibition of ICl,acid by DIDS at negative voltages (Fig. 3). The inward currents at pHo 4.5 (Fig. 9Bd) may have contained a substantial amount of ICl,acid, which would lead to a decrease of the difference currents because the current at pHo 4.5 was subtracted from that at pHo 7.4. A similar inhibitory effect of acidic pHo on ICl,CFTR was observed when ICl,CFTR was activated by application of other PKA activators, 5.5 mM forskolin and 0.5 mM 8-BrcAMP (data not shown).

Figure 9D shows the relationship between density of the outward current at +100 mV and the pHo value. If we consider that the outward whole cell current observed at a given pH in the presence of both isoprenaline and DIDS largely represents ICl,CFTR, the data shown in Fig. 9D can be a measure of the pHo-response relationship for ICl,CFTR. We conclude that ICl,acid is distinct from ICl,CFTR and that acidic pHo depresses the latter, in agreement with earlier results (17). In additional experiments in which mouse ventricular cells were used, application of isoprenaline (1 µM), which did not induce ICl,CFTR in these cells, had no effect on ICl,acid (n = 6, data not shown), suggesting that ICl,acid is independent of the beta-adrenoceptor-PKA system.

Finally, we examined the effect of acidic pHo on the AP in mouse ventricular cells, using more physiological bath and pipette solutions (see MATERIALS AND METHODS). Figure 10 shows a representative result obtained before and after the bath solution was switched from control (pH 7.4) to acidic (pH 6.5) solution. Acidic pHo caused a prolongation of the action potential duration (APD). Quantitatively, APD at –65 mV level (n = 4) was 50.1 ± 3.5 ms in control and 70.0 ± 6.8 ms at pH 6.5, and this was a significant change (P < 0.05). Acidic pHo also caused a significant (P < 0.05) depolarization of the resting membrane. The resting potential (RP) was –83.3 ± 2.1 mV (n = 4) in control and shifted to –74.8 ± 2.7 mV at pH 6.5. If ICl,acid is activated at acidic pHo, ICl,acid should provide an inward current at membrane voltages negative to ECl, and such inward ICl,acid can be one factor contributing to the observed prolongation of APD and depolarization of RP, because most of the plateau potential and RP in mouse cells are below –50 mV (Fig. 10) and hence more negative than the predicted ECl, which was –33 mV under the present conditions. It must be noted, however, that acidification should directly or indirectly affect the properties of other current systems, which would also modulate the AP configuration.


Figure 10
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  		Fig. 10. Effect of acidic pHo on action potential. Action potentials were recorded from a mouse ventricular cell with a conventional whole cell patch clamp at a stimulation rate of 5 Hz. Data for pH 6.5 were obtained 5 min after switching the bath solution from control (pH 7.4) to acidic solution. The bath solutions were Cl– rich and contained 5.4 mM K+, and the pipette solution was K+ rich and contained 40 mM Cl– (see MATERIALS AND METHODS). The predicted Cl– equilibrium potential value was –33 mV.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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ICl,acid observed in the present study showed the following properties. The I-V relation of ICl,acid outwardly rectifies under symmetrical [Cl–] conditions (Fig. 1C). ICl,acid shows a time-dependent activation at large positive potentials (Fig. 1B). It is sensitive to DIDS, glibenclamide, and niflumic acid but insensitive to tamoxifen under isotonic conditions (Fig. 3B) and is independent of intracellular calcium ions, ATP, and G protein (Figs. 4 and 5). The relative permeability sequence for anions appears to be SCN– > Br– > I– > Cl– > F– > aspartate (Fig. 2C). The magnitude of ICl,acid appears to depend on osmotic conditions in ATP-depleted cells (Fig. 5). Tamoxifen increases ICl,acid under hypotonic conditions (Fig. 6), in contrast with its effect on ICl,acid under isotonic conditions. These properties of ICl,acid are totally or partially different from those of other cardiac Cl– currents (ICl,CFTR, ICl,vol, ICl,Ca, and ICl,ir) identified so far. ICl,ir (4, 13, 14) increases at low pHo and on cell swelling, but it inwardly rectifies. ICl,Ca (9, 11, 24) requires an elevation of [Ca2+]i for its activation. ICl,vol (8, 9, 11, 29) exhibits a time-dependent inactivation at positive potentials and is sensitive to both DIDS and tamoxifen. ICl,CFTR (9, 11) shows little rectification under symmetrical [Cl–] conditions and is insensitive to DIDS. With respect to intracellular regulation, activation of ICl,CFTR (18) and ICl,vol (23) has been shown to depend on intracellular ATP. Furthermore, ICl,acid appears to develop independently of ICl,vol (Figs. 7 and 8) or ICl,CFTR (Fig. 9).

Some technical considerations must be made. We determined the pH dependence and Vrev of ICl,acid by using acid-induced currents (difference currents), and it was assumed that the background currents other than ICl,acid were insensitive to acidification. In reality, however, the magnitude of the background currents might change depending on acidity. For example, although DIDS and tamoxifen were ineffective on the background currents at neutral pH, it cannot be ruled out that some inhibitor-sensitive or -insensitive background currents developed at acidic pH. Therefore, our data on the pH dependence and permeability sequence of ICl,acid might contain some inaccuracies, although, as pointed out by Lambert and Oberwinkler (15), the large currents of ICl,acid should minimally be affected by such changes in background currents.

ICl,acid has been recorded in many types of cultured cells derived from nonexcitable cells (1, 3, 15, 19). The current in these cells has common features. It exhibits outward rectification in its I-V relation, time-dependent activation during depolarization, and independence of activation on intracellular Ca2+. The sensitivity of ICl,acid to DIDS (1, 15, 19), niflumic acid (19), and glibenclamide (2) has been demonstrated in some cells. The capability of ICl,acid channels to carry several kinds of anions has also been noted (1, 15, 19). These properties are in agreement with those of cardiac ICl,acid observed in the present study. As with anion selectivity, I– and Br– appear to be more permeable than Cl– in cardiac ICl,acid. Although this feature resembles that for ICl,acid in HEK293 cells (15, 19), a reverse relationship (Cl– > Br– > I–) has been reported for ICl,acid in Sertoli cells (1). The nature of this difference is unknown (see Ref. 15).

Nobles et al. (19) observed a tamoxifen-resistant ICl,acid in cultured cells. However, they considered that this ICl,acid was a manifestation of ICl,vol channels. In their view, the biophysical as well as pharmacological properties of ICl,vol channels are altered by acidic pHo, so that the channels can be activated by acidic pHo under isotonic conditions. This proposal might not be incompatible with the feature of ICl,acid mentioned above. Recently, however, Lambert and Oberwinkler (15) extensively examined ICl,acid and ICl,vol in HEK293 cells. On the basis of their detailed analysis including examinations of the single-channel events, the effects of acidification on ICl,vol, and the effects of train of depolarizing pulses on ICl,vol at acidic pHo (see introduction), they concluded that ICl,acid and ICl,vol are derived from different channels.

In our study, ICl,acid and ICl,vol appeared to develop in an additive fashion in acidic hypotonic solutions, with each current preserving its own voltage-dependent behavior, ICl,acid showing time-dependent activation and ICl,vol time-dependent inactivation at positive voltages (Figs. 7 and 8). We observed an inhibitory effect of hypotonic condition on ICl,acid (Figs. 5 and 8). The mechanism underlying this effect is unclear. Nevertheless, as discussed by Lambert and Oberwinkler (15), it seems difficult to explain the biphasic change of the whole cell current observed during acidic hypotonic challenge (Fig. 8) by simply assuming that ICl,acid channels are acidification-modified ICl,vol channels. In addition, ICl,acid, unlike ICl,vol, can develop independent of intracellular ATP (Figs. 5 and 6), suggesting involvement of different intracellular regulatory mechanisms in the activation of these two currents. The simplest explanation of our findings may be that ICl,acid can develop independently of ICl,vol, in agreement with the conclusion of Lambert and Oberwinkler (15).

The mechanism of activation of ICl,acid by acidic pHo is unclear. Our study suggested that popular intracellular signaling molecules such as G protein, PKA, and ATP were not required for activation of ICl,acid. The pH-response relationship for ICl,acid obtained in our study revealed that EC50 is ~pH 5.9 with a threshold pH value of ~pH 7 and nH of ~1.1 (Fig. 1D). The feature is somewhat different in HEK293 cells, in which EC50 of pH 5.1, threshold of ~pH 5.5, and nH of 3.6 have been reported (15). The difference might indicate a difference in the molecular structure of the ICl,acid channel itself or related regulatory proteins. Further studies are necessary to elucidate this point.

The activation of cardiac ICl,acid at severely acidic pH with a threshold of ~pH 7 and EC50 of pH 5.9 means that the ICl,acid channels can hardly function under physiological conditions. However, local external acidosis is frequently induced by myocardial ischemia and is known to alter properties of several ion channels and transporters (10, 13, 14, 21), which might lead to development of cardiac arrhythmias (21). Yan and Kleber (30) reported that in blood-perfused rabbit papillary muscles, no-flow ischemia for 14 min resulted in an acidification of the extracellular space up to pH 6.64–6.30, depending on PCO2. Essentially similar changes in pHo have been observed in pig heart in vivo after 10-min occlusion of a branch of the coronary artery (7). Thus it is possible that ICl,acid is activated to some extent under such ischemic conditions and that the activated ICl,acid exerts some effects on the electrical activity of cardiac cells.

We observed an acidification-induced prolongation of APD in mouse ventricular cells (Fig. 10), in agreement with the observation in rat ventricular cells (14). However, the effects of acidosis on cardiac AP appear to depend on tissues and animal species. For example, acidosis-induced shortening of APD has been reported in rat atrial (13) and rabbit ventricular (10) cells. Because ICl,acid can flow outwardly or inwardly depending on membrane voltage and ECl, ICl,acid should intrinsically have the potential to prolong and to shorten APD. Although the acidosis-induced changes in cardiac electrical activity must be related to variable ionic mechanisms (10, 13, 14, 21), our study suggests that activation of ICl,acid can be one of the factors contributing to acidosis- or ischemia-induced changes in cardiac AP.

On the other hand, involvement of ICl,vol and ICl,CFTR in regulation of cardiac cell volume has been investigated (25, 28). Because ICl,CFTR is suppressed at acidic pHo (Fig. 9), ICl,acid might play a cell volume-regulatory role in place of ICl,CFTR during myocardial acidosis. As with the pH dependence of ICl,acid, it should be noted that the ICl,acid channels might change their properties depending on unknown cellular signaling events so that they would become more active under less acidic conditions. Obviously, further studies are necessary to elucidate the physiological significance of ICl,acid, including its roles in cardiac function under pathological conditions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was partly supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture (no. 17500276 to T. Ehara and no. 16790138 to S. Yamamoto), the Salt Science Research Foundation (no. 00–02C3 to T. Ehara), the 2004 COE program in Saga University Faculty of Medicine (no. 27, 2004 to S. Yamamoto), and the Young Medical Researcher Grants in Saga University Faculty of Medicine (no. 1104081026 to T. Ehara and S. Yamamoto).


   ACKNOWLEDGMENTS
 
The authors are grateful to Drs. K. Ishihara and T. Shioya for useful discussions and M. Fuchigami and S. Kamohara for secretarial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Yamamoto, Dept. of Physiology, Saga Univ. Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan (e-mail: yamamot3{at}cc.saga-u.ac.jp)

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.


   REFERENCES
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