Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

Tetsushi Furukawa1, Takehiko Ogura1, Yoshifumi Katayama1, and Masayasu Hiraoka2

Departments of 1 Autonomic Physiology and 2 Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101, Japan

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of ×40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- >=  Br- >>  I- >=  F-. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at -160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2Delta NT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2Delta NT-injected oocytes (-5.9 ± 0.4 µA), followed by ClC-2-injected oocytes (-4.3 ± 0.6 µA), and smallest in water-injected oocytes (-0.2 ± 0.2 µA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2Delta NT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopus oocytes appear to act for volume regulation when exposed to a hyposmolar environment.

chloride channel; voltage clamp; cell swelling; ClC supergene family

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE UNDERSTANDING OF MOLECULAR aspects of voltage-dependent Cl- channels has advanced remarkably since cloning of the Cl- channel gene of Torpedo marmorata (ClC-0) by the hybrid-depletion approach (7). Since then, by homology screening or polymerase chain reaction (PCR)-based screening, ten members of related Cl- channels have been cloned from various tissues in different species (8-10). This Cl- channel supergene family is now designated as the ClC family. Despite a growing body of molecular data, understandings of endogenous counterparts and physiological roles of the ClC family are limited to certain members of the family. For instance, ClC-1 is responsible for a major Cl- conductance in skeletal muscle (19) and its abnormalities result in the muscle disorder congenital myotonia (11, 18, 26). ClC-2 has unique features because external hyposmolarity activates rat ClC-2 currents in Xenopus oocytes, and the ClC-2 channel has been suggested to act in some way in volume regulation under hyposmolar conditions (5, 12, 20). The ClC-2 channel has also been suggested to stabilize the relationship between the membrane potential and the Cl- equilibrium potential in neurons (15-17).

A rabbit homologue of ClC-2 was cloned and was designated as ClC-2G because this channel was first isolated from a rabbit gastric cDNA library (13). Rat ClC-2 currents were examined using the two-electrode voltage clamp of oocyte membrane (5, 12, 20), whereas rabbit ClC-2G currents were characterized by voltage clamp of a lipid bilayer, in which oocyte membrane injected with ClC-2G cRNA was incorporated (13). Despite a high sequence homology, ion permeability differed between rat ClC-2 currents and rabbit ClC-2G currents. Rat ClC-2 currents had a sequence of halide anion permeability of Cl- >=  Br- > I-, whereas rabbit ClC-2G had a sequence of I- > Cl-. In addition, some important characteristics found in rat ClC-2 currents were not fully examined in rabbit ClC-2 currents or vice versa. Rat ClC-2 currents were activated by external hyposmolarity, but effects of external hyposmolarity on rabbit ClC-2 currents were not examined. Rabbit ClC-2G currents were activated by application of the catalytic subunit of protein kinase A (PKA), but the effects of PKA on rat ClC-2 were not completely clear. ClC-2G currents were activated by strong external acidification (pH 3.0) and were suggested to be essential for HCl secretion by the gastric parietal cells, but effects of external acidification on rat ClC-2 currents were studied only in moderate acidic conditions (pH 6.0) (12).

Previously, we isolated a clone that is identical to ClC-2G from the rabbit heart cDNA library (ClC-2alpha ) (3). During the screening of the rabbit heart cDNA library, we also isolated a truncated form of ClC-2alpha and we proposed the presence of an alternative splicing form (ClC-2beta ). However, the new 5' sequence [35 base pairs (bp)] is highly homologous to sequences at the very ends of many sequenced cDNAs in different species, such as rice, and contains repeats of palindromic sequence (12). Thus the truncated clone of rabbit ClC-2 (ClC-2beta ) is likely to be an artifact of library construction but not a product of alternative splicing (3). In that study, expressed currents were not carefully separated from endogenous Cl- currents of Xenopus oocytes, and thus rabbit ClC-2G (ClC-2alpha ) currents were not fully characterized.

To assess whether rabbit ClC-2G (ClC-2alpha ) is a tissue-different variant due to alternative splicing of rat ClC-2 or just a counterpart of rat ClC-2, it is imperative to fully characterize rabbit ClC-2 currents and compare them with those of rat ClC-2 currents. Thus, in the present study, we further characterized rabbit ClC-2G (ClC-2alpha ) currents using two-electrode whole cell voltage clamp as used for characterization of rat ClC-2 currents (5, 12, 20). Our data suggest that rabbit ClC-2G (ClC-2alpha ) currents had similar characteristics to rat ClC-2 currents and ClC-2G (ClC-2alpha ) appeared to be a counterpart of rat ClC-2 in rabbit. Thus, in the present study, we use rabbit ClC-2 in place of rabbit ClC-2G (ClC-2alpha ). Second, although it is well documented that rat ClC-2 currents are activated by external hyposmolarity (5, 12, 20), their possible role for cell physiology has not been fully clarified. Thus we examined whether expression of ClC-2 in oocytes affected the volume regulation in response to external osmolarity changes.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Molecular biology. ClC-2 cDNA obtained from rabbit heart (3) was subcloned into pSPORT I (GIBCO BRL, Rockville, MD). As reported for other members of the ClC supergene family, including rat ClC-2, Cl- currents could not be expressed in Xenopus oocytes from the original, putative full-length cDNA for rabbit (19, 20, 22). We had previously reported that outwardly rectifying Cl- currents were expressed by injection of cRNA transcribed from the original, putative full-length cDNA for rabbit ClC-2 (3). However, this current is likely to be an endogenous current present in oocytes but not an expressed current of rabbit ClC-2. Thus DNA of the Torpedo ClC-0 5' untranslated region (83 bp) was synthesized and added to the first ATG of rabbit ClC-2 by recombinant PCR methods (6). The sequence of the new chimeric clone was verified by the dideoxynucleotide chain termination method using a 373A DNA sequencing system (Perkin Elmer, Rockville, MD). In an erratum to the previous study (3), we reported expression of inwardly rectifying Cl- currents by injection of rabbit ClC-2 cRNA (3). Although not mentioned in this erratum (3), in those experiments, DNA of the Torpedo ClC-0 5' untranslated region (83 bp) had been added to the first ATG of rabbit ClC-2. In vitro methyl-capped complementary RNA (cRNA) was made using T7 RNA polymerase (Stratagene, La Jolla, CA).

Oocyte handling and electrophysiology. Xenopus oocyte preparation and handling were carried out as described previously (24). In brief, oocytes were removed from Xenopus laevis (Hamamatsu Seibutsu, Hamamatsu, Japan) under anesthesia, washed in Ca2+-free OR-2 containing (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 5 tris(hydroxymethyl)aminomethane (Tris) (pH 7.6 with HCl). Stage V and VI oocytes were defolliculated by treatment with 2 mg/ml collagenase (type IA; Worthington, Freehold, NJ) in Ca2+-free OR-2 for ~30-60 min, washed extensively with Ca2+-free OR-2 containing no collagenase, and injected with ~30-50 ng of cRNA dissolved in 30 nl of diethylpyrocarbonate-treated distilled water. Injected oocytes were incubated for ~4-6 days at ~12-18°C in modified Barth's solution containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 15 Tris, 0.3 Ca(NO3)2, 0.4 CaCl2, and 0.8 MgSO4 and 100 µg/ml sodium penicillin and 100 µg/ml streptomycin sulfate (pH 7.6 by HCl).

Membrane currents were recorded from oocytes with a two-electrode voltage clamp using an amplifier (TEV-200; Dagan, Minneapolis, MN) at a room temperature of ~24-26°C. Current-injecting and potential-measuring electrodes had resistances of ~0.5-2.0 and ~1.0-3.0 MOmega , respectively, when filled with 3 M KCl. The bath solution was connected to the ground via a low-resistance agarose bridge containing 2% agarose in 3 M KCl. A second reference electrode was used to avoid polarization errors. Junction potentials resulting from solution changes were <2.5 mV in each experiment and were not corrected. Current measurements were low-pass filtered at 0.5 kHz. Data acquisition and analysis were done on an 80386-based microcomputer using pCLAMP software and TL-1 analog-to-digital converter (Axon Instruments, Foster City, CA). Oocytes were perfused continuously with various external solutions. Each experiment began with perfusing a modified ND96 solution with a mean osmolarity of 228 ± 11 mosM [solution 1 (isosmolar solution) in Table 1]. Oocytes were kept in current-clamp mode for at least 5 min before switching to voltage-clamp mode. Only oocytes exhibiting a resting membrane potential negative to -30 mV were used. Oocytes were voltage clamped at a holding potential of -30 or -10 mV in some experiments, and 2-, 4-, or 5-s voltage steps were applied from -160 to +60 mV in 20-mV increments or from -120 to +60 mV in 10-mV increments. Extracellular hyposmolarity was established by perfusing a hyposmolar solution (109 ± 7 mosM), in which 96 mM mannitol was omitted from the control isosmolar solution, leaving other components the same (solution 2 in Table 1), which was a slightly different method from others. In a study by Gründer et al. (5), 0.5× ND96 + 100 mM sucrose was used as an isosmolar solution and hyposmolarity was achieved by omission of 100 mM sucrose. We also used sucrose to balance external osmolarity in a few experiments, but we obtained basically no different findings between sucrose and mannitol used as an agent to balance osmolarity. Osmolarity of the solution was measured using a vapor pressure osmometer (model 5500; Wescor, Salt Lake City, UT). To determine the ion permeability, we measured the reversal potential (Erev) in various external solutions. To determine the permeability for anions, 48 mM NaCl was replaced with equimolar sodium aspartate (solution 3), sodium glutamate (solution 4), or sodium gluconate (solution 5). To determine the permeability for cations, 48 mM NaCl was replaced with equimolar choline chloride (solution 9) or KCl (solution 10). To determine the permeability for halide anions, 48 mM NaCl was replaced with equimolar NaF (solution 8), NaBr (solution 6), or NaI (solution 7). To examine effects of extreme external acidification, 5 mM HEPES in external solution was replaced with equimolar propionic acid for pH 4.6 (solution 11) or with citric acid for pH 3.6 (solution 12), and pH was adjusted with NaOH.

                              
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Table 1.   Compositions of external solutions

4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Sigma, St. Louis, MO), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; Sigma), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; Tokyo Kasei, Tokyo, Japan) were directly dissolved in external test solutions just before experiments. Anthracene-9-carboxylic acid (9-AC; Sigma) and diphenylamine-2-carboxylic acid (DPC; Wako Pure Chemical, Osaka, Japan) were prepared as 1 M stock solutions, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; Research Biochemical International, Natick, MA) as a 100 or 500 mM stock solution, and forskolin (Wako Pure Chemical) as a 10 mM stock solution in dimethyl sulfoxide (DMSO). They were stored at -20°C until needed and were diluted in the external solution at final concentrations described in the text. The final concentration of DMSO in each solution was <= 0.1%, which had no effect on the membrane currents of Xenopus oocytes.

Photographs of oocytes were taken using a camera (model AFM; Nikon, Ofuna, Japan) attached to the microscope with a magnification of ×40. Photographs of oocytes were copied with a magnification of ×2, and oocyte diameters were measured with a pair of dividers with a final magnification of ×80. To check the precision and accuracy of measurement of oocyte diameter, two researchers (Furukawa and Ogura) measured oocyte diameter independently and each researcher repeated measurements three times. Variations of measurement in each researcher and variations of measurement between researchers never exceeded 0.02 mm. Thus we consider the difference in oocyte diameter as significant if the difference is >0.02 mm and had a P value of <0.05.

All values are presented with n (number of measurements in one oocyte) and N (number of measurements in different oocytes). Measurements were repeated three times (n = 3) in the same oocyte if possible. When the difference in measured values was <3 mV for Erev, 5% of measured values for current amplitude, and 0.02 mm for oocyte diameter, measurement in that oocyte was considered to be stable and accurate and included for further analysis. For the experiments in which external osmolarity or pH was changed, current amplitude changed quickly from time to time and repeatedly measured values varied substantially. Thus, for these experiments, measurements were done singly (n = 1) and stability of data was judged from continuous current recordings at a single voltage. Measured values in different oocytes (N) were averaged and are shown as means ± SE. One-way analysis of variance followed by a Student's t-test was used to test for significance (P < 0.05).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Current-voltage relationships and ion permeability. In oocytes injected with rabbit ClC-2 cRNA, slowly activating inward currents at negative potentials and small outward currents at positive potentials were recorded (Fig. 1Aa). For oocytes injected with distilled water, very small outward currents but not inward currents were recorded (Fig. 1Ab), and, therefore, slowly activating inward currents at negative potentials appeared to be expressed currents due to injection of rabbit ClC-2 cRNA and outward currents appeared to be endogenous currents of oocytes. The amplitude of outward currents varied between different batches of oocytes, and we arbitrarily decided to avoid oocytes in which current amplitude at +60 mV was >0.4 µA. Swelling-activated outward Cl- currents and hyperpolarization-activated inward Cl- currents are endogenously present in oocytes (1, 21). The former type of current was decreased in amplitude after defolliculation and was almost undetectable at 2 days after defolliculation (1). The latter type of current shifted its voltage dependence to the negative potential after defolliculation, and potential for activation threshold was more negative than -160 mV at 4 days after defolliculation (21). Thus we performed current measurements at least 4 days after defolliculation. The current-voltage (I-V) curves constructed at the end of 5-s step pulses showed marked inward rectification (Fig. 1B). Because ClC-2 currents showed only a minor, if any, inactivation, to determine ion selectivity through the rabbit ClC-2 channel, quasi-steady-state I-V curves were constructed at the end of 5-s step pulses in various ionic solutions and the Erev values were measured. Permeability for anions through rabbit ClC-2 channel was studied by replacing 48 mM Cl- with equimolar aspartate, glutamate, or gluconate (Fig. 2A). The Erev in Cl- solution (solution 1 in Table 1) was -15.1 ± 1.3 mV (n = 3, N = 10), which was shifted to +31.7 ± 4.2 mV (n = 3, N = 8) in aspartate solution (solution 3), +33.0 ± 4.9 mV (n = 3, N = 9) in glutamate solution (solution 4), and +35.8 ± 5.0 mV (n = 3, N = 10) in gluconate solution (solution 5).


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Fig. 1.   Current-voltage (I-V) relationships of rabbit ClC-2 currents. A: representative membrane currents recorded from an oocyte injected with cRNA of rabbit ClC-2 (a) and that injected with distilled water (b). Membrane was held at -30 mV, step pulses for 5 s to various potentials between -160 and +60 mV in 20-mV intervals were applied, and recorded currents were superimposed. B: I-V curves for membrane currents of oocytes injected with rabbit ClC-2 cRNA and those injected with distilled water. n, Number of measurements in 1 oocyte; N, number of measurements in different oocytes.


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Fig. 2.   Ion permeability through rabbit ClC-2 channels. Aa: representative superimposed currents of oocytes injected with rabbit ClC-2 in control external Cl- solution (left), in aspartate solution (middle), and in glutamate solution (right). Membrane potential was held at -30 mV, and step pulses for 4 s were applied to various potentials between -120 and +60 mV in 10-mV intervals. To minimize current flowing at holding potential and resulting changes in intracellular ionic environment, holding potential was changed to -10 mV for glutamate and aspartate solutions. Ab: I-V curves for rabbit ClC-2 currents in control external Cl- solution, in aspartate solution, in glutamate solution, and in gluconate solution. Ba: rabbit ClC-2 currents in control external solution containing 48 mM Na+ and 2 mM K+ (left), in Na+-free solution containing 0 mM Na+ and 2 mM K+ (middle), and in high-K+ solution containing 0 mM Na+ and 50 mM K+ (right). Concentration of Cl- in these 3 test solutions was identical (55.6 mM). Membrane potential was held at -30 mV, and step pulses for 4 s were applied to various potentials between -120 and +60 mV in 10-mV intervals. Bb: I-V curves of rabbit ClC-2 currents in control solution containing 48 mM Na+ and 2 mM K+ (48Na, 2K), in solution containing 0 mM Na+ and 2 mM K+ (0Na, 2K), and in solution containing 0 mM Na+ and 50 mM K+ (0Na, 50K).

To examine whether cations are also permeable through this channel, extracellular Na+ concentration and K+ concentration were changed, and the Erev was measured. When extracellular Na+ was decreased from 48 to 0 mM by replacing with equimolar choline or when extracellular K+ was increased from 2 to 50 mM by replacing choline with K+, the Erev was not significantly changed (Fig. 2B). The Erev was -13.2 ± 3.2 mV (n = 3, N = 9) in the control solution (solution 1 in Table 1), -14.1 ± 5.0 mV (n = 3, N=9) in the Na+-free solution (solution 9), and -10.7 ± 2.9 mV (n = 3, N = 9) in the high-K+ solution (solution 10). Thus the rabbit ClC-2 channel has a permeability that is highly selective for anions over cations.

We next examined permeability of halide anions through this channel by replacing 48 mM extracellular Cl- with equimolar Br-, I-, or F- (Table 1 and Fig. 3). Even in the same solution, the Erev varied between different batches of oocytes. To avoid influence of this variation on data analysis, the data were used when the Erev could be measured for all four solutions from the same oocyte. In this way, the order of permeability sequence could be assessed accurately. The Erev was -19.5 ± 2.2 mV (n = 3, N = 10) in Cl- solution (solution 1), -11.0 ± 2.6 mV (n = 3, N = 10) in Br- solution (solution 6), +12.0 ± 2.9 mV (n = 3, N = 10) in I- solution (solution 7), and +15.8 ± 5.1 mV (n = 3, N = 10) in F- solution (solution 8). Thus the sequence of halide anion permeability was Cl- >=  Br- >>  I- >=  F-. This selectivity sequence is similar to that of rat ClC-2 currents and corresponds to Eisenman sequence 4 (23).


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Fig. 3.   Permeability of halide anions through rabbit ClC-2 channels. A: rabbit ClC-2 currents in external Cl- solution (a), in Br- solution (b), in I- solution (c), and in F- solution (d). Stimulation protocol used was same as in Fig. 2A. B: I-V curves in Cl- solution, in Br- solution, in I- solution, and in F- solution.

Sensitivity to Cl- channel blockers. To understand sensitivity of ClC-2 currents to various Cl- channel blockers, we tested DIDS, SITS, DNDS, 9-AC, DPC, and NPPB. Rabbit ClC-2 currents were insensitive to stilbene disulfonates. Amounts of 1 mM DIDS, 1 mM SITS, or 10 mM DNDS did not inhibit inward currents [only data for 10 mM DNDS are presented (Fig. 4)]. Application of 10 mM DNDS slightly shifted the I-V curves in a depolarizing direction (Fig. 4Ba). In contrast, 9-AC, DPC, and NPPB inhibited rabbit ClC-2 currents (Fig. 4, Ab and Bb-d). Remaining current amplitudes at -160 mV after drug application were expressed as a fraction of the current level before drug application and are shown in Fig. 4C. It was significantly smaller for 0.5 mM NPPB (0.49 ± 0.13) than for 1 mM DPC (0.72 ± 0.14; P < 0.01 vs. NPPB) or 1 mM 9-AC (0.69 ± 0.07; P < 0.01 vs. NPPB) (P < 0.05). There was no significant difference between 1 mM 9-AC and 1 mM DPC. Thus an order of inhibitory potency was NPPB > DPC = 9-AC. The remaining current fraction for 10 mM DNDS assessed at -160 mV was >1 (1.23 ± 0.17). This was mainly due to a positive shift of voltage dependence by DNDS. Figure 4D displays a dose-response curve for NPPB. The data were fitted by a least-squares analysis using SigmaPlot software (Jandel Scientific, Corte Madera, CA) according to the Hill equation in the following form
<IT>I</IT> = 1/{1 + ([NPPB]/<IT>k</IT>)<SUP><IT>n</IT></SUP>} (1)
where I is current amplitude in the presence of NPPB expressed as a fraction to the current amplitude in the absence of NPPB, k is the NPPB concentration ([NPPB]) causing half-maximal inhibition, and n is the Hill coefficient. The obtained value for k was 0.98 ± 0.03 mM and that for n was 0.97 ± 0.03. 


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Fig. 4.   Effects of various Cl- channel blockers. Aa: rabbit ClC-2 current recorded in the absence (left) or presence (right) of 10 mM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Ab: rabbit ClC-2 currents recorded in the absence (left) or presence (right) of 1 mM diphenylamine-2-carboxylic acid (DPC). Stimulation protocol used was same as for Fig. 1. B: I-V curves of rabbit ClC-2 currents in the absence (filled symbols) or presence (open symbols) of 10 mM DNDS (a), 1 mM anthracene-9-carboxylic acid (9-AC; b), 1 mM DPC (c), or 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; d). * P < 0.05 for current amplitude between absence and presence of each drug. C: residual current amplitude at -160 mV in the presence of drugs expressed as a fraction of current amplitude in the absence of drugs. * P < 0.05 between drugs. D: dose-response relationships for NPPB-induced inhibition of ClC-2 currents. Ordinate is current amplitude expressed as a fraction of control current level, and abscissa is a concentration of NPPB. A continuous line was obtained by fitting Hill equation (Eq. 1) to the data.

Effects of external acidification. We examined the effects of perfusion with external solution of pH 4.6 and 3.6 on rabbit ClC-2 currents. In Fig. 5A, membrane was held at -30 mV and 2-s hyperpolarizing pulses to -160 mV were applied every 15 s. When external solution was switched to pH 4.6, current amplitude quickly increased. Thereafter, current amplitude decreased slowly and reached the steady-state level at ~5 min. When external pH was returned to pH 7.6, current amplitude increased gradually and returned to the initial level at ~10 min of washout. In a different series of experiments, step pulses to various membrane potentials were applied in the control state at pH 7.6 (Fig. 5, Ba and C), at 30 s (Fig. 5, Bb and C), or at 5 min (Fig. 5, Bc and C) after a start of perfusion with solution of pH 4.6. Roughly 1 min was required to complete one series of the step-pulse protocol, and, as one can see in Fig. 5A, current amplitude changed substantially within 1 min. Thus the effects of pH at the end of the step-pulse protocol (e.g., +60 mV) might be different from those at its beginning (e.g., -160 mV). Despite this limitation, it can be clearly seen that current amplitude was increased at 30 s after start of perfusion with a solution of pH 4.6 and was decreased at 5 min. At 30 s after start of perfusion with solution of pH 4.6, the increase in current amplitude was observed both in inward and outward currents. Thus this initial increase in current amplitude did not result from a shift of voltage dependency due to surface charge screening by protons. In the external solution at pH <4.0, oocyte membrane became unstable and step pulses to extremely negative or positive potentials could not be applied. Thus we applied 2-s step pulses to -120 and +40 mV alternatively every 15 s and external pH was changed to 3.6 for 4 min and returned to 7.6 (Fig. 5D). Both inward currents at -120 mV and outward currents at +40 mV were initially increased by external pH 3.6 and thereafter decreased. These changes were observed in all four experiments performed.


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Fig. 5.   Effects of low pH in the external solution. A: representative recording of rabbit ClC-2 current when pH of external solution was changed from 7.6 to 4.6 and returned to 7.6. Membrane was held at -30 mV, and 2-s hyperpolarizing pulses to -160 mV were applied every 15 s. In this and Figs. 6, 7, and 9, inward currents are shown downward and outward currents are shown upward. Top trace: current recording at -30-mV holding potential. Bottom trace: current recording at -160-mV hyperpolarizing pulses. Times when recording chamber was perfused with external solution of pH 7.6 or 4.6 are indicated by bars on top of current recording. B: superimposed ClC-2 currents in the control solution of pH 7.6 (a), at 30 s after start of perfusion with solution of pH 4.6 (b), and at 5 min after start of perfusion with solution of pH 4.6 (c). Stimulation protocol used was the same as in Fig. 1. C: I-V relationships in pH 7.6 solution, at 30 s after start of perfusion with solution of pH 4.6, and at 5 min after start of perfusion with solution of pH 4.6. D: representative recording of rabbit ClC-2 current when pH of external solution was changed from 7.6 to 3.6 and returned to 7.6. Membrane was held at -30 mV, and 2-s hyperpolarizing pulses to -120 or +40 mV were applied alternately every 15 s. Top trace: current recording at +40-mV depolarizing pulses. Middle trace: current recording at -30-mV holding potential. Bottom trace: current recording at -120-mV hyperpolarizing pulses. Times when recording chamber was perfused with external solution of pH 7.6 or pH 3.6 are indicated by bars on top of current recording.

Effect of forskolin. Channel activity of rabbit ClC-2 (ClC-2G) was enhanced by application of the catalytic subunit of PKA to the cytosolic side of the lipid bilayer (13), but effects of PKA on rat ClC-2 were not examined. In a previous study, we reported that external application of forskolin activated rabbit ClC-2 currents (3). However, in those studies, currents enhanced by forskolin were outward Cl- currents that are now believed to be endogenous currents. Thus we again examined effects of forskolin to increase adenosine 3',5'-cyclic monophosphate (cAMP) and activity of endogenous PKA on rabbit ClC-2 (Fig. 6). External application of 10 µM forskolin did not affect ClC-2 current amplitude.


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Fig. 6.   Effects of forskolin. A: representative recording of rabbit ClC-2 current when an oocyte was perfused with solution containing 10 µM forskolin. Membrane was held at -30 mV, and 2-s hyperpolarizing pulses to -120 mV were applied every 5 s. Top trace: current recording at -30-mV holding potential. Bottom trace: current recording at -120-mV hyperpolarizing pulses. At arrows marked with a or b, step pulses for 2 s to various potentials between -160 and +60 mV in 20-mV intervals were applied from a holding potential of -30 mV. Step-pulse protocols were repeated 3 times to check the stability of recordings. B: superimposed ClC-2 currents in the control state (a) and in the presence of 10 µM forskolin (b). Stimulation protocol used was the same as for Fig. 1. C: I-V relationships in the control solution and in the presence of 10 µM forskolin.

Rabbit ClC-2 currents are sensitive to cell swelling. Changing the superfusate from isosmolar solution (228 mosM) to hyposmolar solution (109 mosM) resulted in an ~1-10% increase in oocyte diameter, which was dependent on both the types of cRNA injected and the stimulation protocol used. Similar to rat ClC-2 currents, the rabbit ClC-2 currents were small in isosmolar solution, but current amplitude was markedly increased by superfusion with hyposmolar solution (Fig. 7, Ab-Cb). Reperfusion with isosmolar solution completely or in some cases partially decreased current amplitude. In oocytes injected with distilled water, superfusion with hyposmolar solution did not significantly enhance membrane currents (Fig. 7, Aa-Ca).


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Fig. 7.   Effects of external hyposmolarity on rabbit ClC-2 currents. A: representative continuous recordings of rabbit ClC-2 currents for oocytes injected with distilled water (a) and rabbit ClC-2 cRNA (b) when external solution was changed from isosmolar solution (228 mosM) to hyposmolar solution (109 mosM) and changed back to isosmolar solution. Membrane potential was held at -30 mV, and 2-s hyperpolarizing pulses to -160 mV were applied every 5 s. Top traces: current recordings at a -30-mV holding potential. Bottom traces: current recordings at -160-mV hyperpolarizing pulses. Times when oocytes were externally superfused with hyposmolar solution are indicated by bars. At points marked by arrows, 2-s step pulses were applied to various potentials between -160 and +60 mV in 20-mV increments. B: superimposed membrane currents recorded from oocytes injected with distilled water (a) and rabbit ClC-2 cRNA (b) recorded in the control state (left), in hyposmolar solution (middle), and during washout (right). Stimulation protocol used was the same as in Fig. 1. C: I-V curves for water-injected oocytes (a) and rabbit ClC-2 currents (b) in the control state, in hyposmolar solution, or during washout.

The enhancement of ClC-2 current amplitude in response to external hyposmolarity was a slow phenomenon (see Fig. 7Ab), which seems to be compatible with the hypothesis that slow cellular changes, probably oocyte swelling, may be responsible for current enhancement (5). If this hypothesis is correct, ClC-2 currents and oocyte swelling should change with a similar time course in response to external osmotic changes, as indeed they did. Representative photographs of oocytes at several time points are displayed in Fig. 8Ab. Oocyte diameter was increased in hyposmolar solution and was decreased to the control level during washout of hyposmolar solution. Figure 8Bb depicts the time course of changes in current amplitude at a -160-mV hyperpolarizing pulse and in oocyte diameter. In oocytes injected with ClC-2 cRNA, increases in current amplitude became apparent at ~10 min of superfusion with hyposmolar solution and current amplitude increased more slowly after ~25 min (Fig. 8Bb). When external solution was changed back to isosmolar solution, current amplitude decreased relatively quickly and returned to the control level at ~20 min of washout (Fig. 8Bb). These patterns were also observed from the increase in oocyte diameter during hyposmolar perfusion and decrease in oocyte diameter during washout (Fig. 8Bb). The correlation between time course of changes in current amplitude and time course of changes in oocyte diameter supports the hypothesis that the changes in oocyte volume rather than the changes in extracellular osmolarity per se are directly linked to current activation.


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Fig. 8.   Effects of external hyposmolarity on oocyte diameter. A: representative pictures of oocytes in the control state (left), at 30 min after superfusion with hyposmolar solution (middle), and at 30 min after washout (right) for oocytes injected with distilled water (a), ClC-2 cRNA (b), and ClC-2Delta NT cRNA (c). Here and in Fig. 9, photographs of oocytes are aligned so that 1 electrode is impaled from the left side and the other from the right side. Diameter of oocytes was measured from areas where electrodes were not impaled, namely, as a distance between top and bottom edge of oocytes. B: time courses of changes in current amplitude and oocyte diameter for oocytes injected with distilled water (a), with ClC-2 cRNA (b), or with ClC-2Delta NT cRNA (c). Membrane was held at -30 mV, and 2-s hyperpolarizing pulses to -160 mV were applied every 5 s. Current amplitudes at end of -160-mV hyperpolarizing pulse and oocyte diameter were measured every 5 min and were plotted. Data for oocyte diameter were expressed as percent increase compared with the control value and plotted.

To assess whether expression of ClC-2 currents affects volume regulation of oocytes, we monitored changes in current amplitude and diameter in oocytes injected with distilled water (Fig. 8, Aa and Bb), ClC-2 cRNA (Fig. 8, Ab and Bb), and ClC-2Delta NT cRNA (Fig. 8, Ac and Bc). ClC-2Delta NT is a clone obtained during the course of screening, in which 76 amino acids were artificially deleted from the NH2 terminus (3). Because the NH2 terminus of rat ClC-2 was demonstrated to be a critical region for response to cell swelling and it expressed open channels (5), it was worth examining volume changes in oocytes injected with ClC-2Delta NT cRNA in response to external hyposmolarity (Fig. 8, Ac and Bc). In the control state, there was no significant difference in diameter between oocytes injected with distilled water and those injected with ClC-2 cRNA: 1.13 ± 0.02 mm (n = 3, N = 12) and 1.14 ± 0.01 mm (n = 3, N = 27), respectively. The diameter of oocytes injected with ClC-2Delta NT cRNA was significantly smaller (1.01 ± 0.02 mm; n = 3 and N = 11) than in oocytes injected with distilled water and with ClC-2 cRNA (P < 0.01), which may be explained by activation of inward current even in the isosmolar solution in oocytes injected with ClC-2Delta NT cRNA. Current amplitude in water-injected oocytes was not changed (-0.2 ± 0.2 µA in the control, -0.2 ± 0.2 µA at 30 min of hyposmolarity, and -0.2 ± 0.2 µA at 30 min of washout), but oocyte diameter was increased by external hyposmolarity and decreased by washout (Fig. 8, Aa and Ba). Current amplitude in oocytes injected with ClC-2Delta NT cRNA was the largest in the isosmolar solution (-5.8 ± 0.4 µA). Although current amplitude was not affected by external hyposmolarity, it remained large (-5.9 ± 0.4 µA at 30 min of hyposmolarity). However, oocyte diameter was only slightly increased by external hyposmolarity (Fig. 8, Ac and Bc). The maximum percent increase in oocyte diameter attained in the hyposmolar solution was smallest for oocytes injected with ClC-2Delta NT (1.1 ± 0.2%; n = 1 and N = 5), followed by oocytes injected with ClC-2 (5.3 ± 0.5%; n = 1 and N = 12), and was largest for oocytes injected with distilled water (9.0 ± 0.2%; n = 1 and N = 7). Thus oocyte swelling by hyposmolarity was smallest in oocytes with the largest inward Cl- currents, suggesting that expression of ClC-2 currents markedly influenced regulation of oocyte volume.

We next tested whether the frequency of activation of ClC-2 currents affected volume regulation of oocytes injected with ClC-2. In Fig. 9A, representative time courses of changes in ClC-2 current amplitude are displayed for when step pulses to -120 mV were applied every 15 s and thereafter at every 5 s. External hyposmolarity increased current amplitude at -120-mV hyperpolarizing pulses. The hyposmolarity-induced increase in current amplitude was greater when stimulated every 15 s than every 5 s. Figure 9B shows representative photographs of oocytes whose membrane was hyperpolarized to -120 mV every 15 s (Fig. 9, Ba and Ca) and every 5 s (Fig. Bb and Cb). Oocyte diameter at 30 min of hyposmolarity was greater with stimulation at 1/15 s than with stimulation at 1/5 s. Increases in current amplitude (Fig. 9Ca) and oocyte diameter (Fig. 9Cb) by external hyposmolarity were compared between two stimulation frequencies. Increases in current amplitude (Fig. 9Ca) and oocyte diameter (Fig. 9Cb) were significantly greater by stimulation at 1/15 s than at 1/5 s. Maximum current amplitude at -120-mV hyperpolarizing pulses was -4.4 ± 0.2 µA (n = 3, N=5) at 1/15 s and -2.9 ± 0.3 µA (n = 3, N = 5) at 1/5 s (P < 0.01). The maximum increase in oocyte diameter was 7.8 ± 1.1% (n = 3, N = 5) at 1/15 s and 6.2 ± 0.3% at 1/5 s (n = 3, N = 5) (P < 0.05).


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Fig. 9.   Effects of frequency of current activation on current enhancement and oocyte swelling. A: representative continuous recordings of ClC-2 currents induced by hyperpolarizing pulses to -120 mV at every 15 s (left half of trace), followed by at every 5 s (right half of trace). Times when oocytes were externally superfused with hyposmolar solution are indicated by bars on top of current recordings. B: representative pictures of oocytes in the control state (left), at 30 min after hyposmolar perfusion (middle), and at 30 min after washout (right) for stimulation rates of 1/15 s (a) and 1/5 s (b). C: time courses of changes in current amplitude (a) and oocyte diameter (b) by stimulation at 1/15 and 1/5 s. * P < 0.05 between stimulation at 1/15 and 1/5 s.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Utilizing the Xenopus oocyte heterologous expression system and two-electrode voltage-clamp technique, we have characterized two important features of rabbit ClC-2. First, expressed currents of rabbit ClC-2 had similar electrophysiological features to rat ClC-2 currents, in terms of I-V relationships, ion selectivity, and sensitivity to Cl- channel blockers. Rabbit ClC-2 currents were activated by external hyposmolarity in a manner similar to rat ClC-2 currents. Second, expression of rabbit ClC-2 regulated the swelling of oocytes in response to external hyposmolarity. This regulatory action was dependent on activation of ClC-2 currents.

The protein encoded by rabbit ClC-2 had, overall, 93% similarity and 82% identity to rat ClC-2 (13, 20). [We had reported that identity of overall sequence of amino acid between rat ClC-2 and rabbit ClC-2 was 81% in a previous study. This is because we used ClC-2Delta NT (ClC-2beta ) for rabbit ClC-2. Identity between rat ClC-2 sequence and full-length sequence of rabbit ClC-2 is 82% (3).] Despite the high amino acid homology, reported ion permeability is different between rat ClC-2 currents (5, 12, 20) and rabbit ClC-2 currents (13). In addition, several important electrophysiological characteristics had not been clarified between rat ClC-2 currents and rabbit ClC-2 currents. The sequence of halide anion permeability was Cl- >=  Br- > I- for rat ClC-2 currents (20) and I- > Cl- for rabbit ClC-2G currents (13). Rat ClC-2 currents were inwardly rectifying (20), whereas rabbit ClC-2 currents had linear I-V relationships of single-channel conductance and a higher open probability at a positive potential (+80 mV) compared with a negative potential (-80 mV) (13). However, voltage dependence of whole cell currents of rabbit ClC-2 had not been examined. Rat ClC-2 currents were sensitive to carboxylate derivatives and were resistant to stilbene disulfonates (11). Effects of Cl- channel blockers on rabbit ClC-2G currents have not been examined. These electrophysiological characteristics are fundamental for channel function and must be clarified to determine whether rabbit ClC-2G is a tissue-different variant of rat ClC-2 due to alternative splicing or is just its counterpart. In the present study, the I-V relationships, ion permeability, and sensitivity to channel blockers of rabbit ClC-2 currents were similar to those of rat ClC-2 currents. Thus, when characteristics of expressed currents were examined in the same Xenopus oocyte expression system, rat ClC-2 and rabbit ClC-2 exhibited similar electrophysiological features.

Similarities between rat ClC-2 and rabbit ClC-2 are not restricted to fundamental electrophysiological features. Rat ClC-2 currents were sensitive to external osmolarity changes (5), but effects of external osmolarity on rabbit ClC-2 currents had not been examined yet. We showed that rabbit ClC-2 currents expressed in Xenopus oocytes were activated by external hyposmolarity, as were rat ClC-2 currents. Furthermore, rat ClC-2 and rabbit ClC-2 were similar in terms of tissue distribution. The rat ClC-2 transcript was present ubiquitously in almost all tissues (20). Data from reverse transcription-PCR experiments in our previous study (3) showed that rabbit ClC-2 transcript was present in every tissue we tested. [In the previous study, we also reported the presence of a transcript for ClC-2Delta NT (ClC-2beta ) in heart and brain. However, we could not reproduce these data by repeated experiments using different batches of poly(A)+ RNA and a different set of PCR primers, and thus this reverse transcription-PCR product may be nonspecific products or products due to some experimental errors (3).] Together, these data suggest that rabbit ClC-2 is a counterpart of rat ClC-2 in rabbit and not a tissue-different variant.

Two intriguing modulations of rabbit ClC-2 were reported by Malinowska et al. (13). First, rabbit ClC-2 currents were enhanced by application of the catalytic subunit of PKA to the cytosolic side of membrane (13). A consensus sequence for PKA phosphorylation is present in the COOH-terminal cytoplasmic stretch of rabbit ClC-2, but not in rat ClC-2, which was suggested to be functionally important (rat ClC-2 has PKA sites in different positions) (13, 20). We also reported that rabbit ClC-2 currents were enhanced by external application of forskolin; however, the currents modulated by forskolin were outward currents and are now believed to be endogenous currents of oocytes (3). [This is also the case for different magnitudes of current inhibition by 9-AC in the previous study (~60%; Ref. 3) and in the present study (~31%).] In the present study, we found no effects of forskolin on inwardly rectifying rabbit ClC-2 currents. Expressed currents of the human homologue of ClC-2 that has a consensus PKA site on the COOH-terminal cytoplasmic stretch were not affected by manipulations to increase intracellular cAMP levels. One possible explanation for this discrepancy is different experimental condition. Malinowska et al. (13) applied the PKA catalytic subunit directly to the cytoplasmic side of the membrane. Others (3, 12) applied forskolin or increased intracellular cAMP, in which the local concentration of PKA achieved in close vicinity to the channels might not be as high as the level achieved by direct application of the PKA catalytic subunit. Another possibility is that Malinowska et al. (13) and others studied different classes of channels because a sequence of ion permeability shows stark contrast (Cl- < I- vs. Cl- > I-). In human T84 cells, a native Cl- current resembles ClC-2, and it was reported that this current was inhibited by cAMP (2). It may be possible that different expression systems may potentially cause this discrepancy (the presence of PKA modulation of inward Cl- in human T84 epithelial cell and its absence of ClC-2 current expressed in Xenopus oocytes). High local concentrations of active PKA may be required for modulation of ClC-2 by way of some anchoring protein, which is indeed the case for cardiac L-type Ca2+ channels (4). The Xenopus oocyte may not express the anchoring protein of PKA and may not be an appropriate expression system to test modulation by PKA because enhancement of L-type cardiac Ca2+ channel by PKA could be reproduced in some mammalian cell lines (e.g., Chinese hamster ovary cells) (25) but not in the Xenopus oocyte expression system (14). Thus the scenario that ClC-2 currents would be modulated by PKA in native tissues, which could not be reconstituted in Xenopus oocyte heterologous expression system, is still possible. Further studies are required to clarify this point.

Another important modulator of rabbit ClC-2 reported was external acidification. When rabbit ClC-2 currents were studied by voltage clamp of oocyte membrane incorporated into the lipid bilayer, current amplitude was increased by extreme external acidification (pH 3.0) (13). Thus it was suggested that rabbit ClC-2 (ClC-2G) was active at low pH, which is essential for HCl secretion by the gastric parietal cells. Recently, it was reported that rat ClC-2 currents were also enhanced by moderate external acidification (pH 6.0), but effects of extreme acidification on rat ClC-2 currents were not reported (12). In the present study, we could lower pH of external solution to 3.6 with a decent stability of current recordings. This difference may be due to different experimental conditions. In the system of voltage clamp of lipid bilayer, buffering of pH both in intra- and extracellular solution is more rigid. However, in oocytes, low pH in extracellular solutions may cause intracellular acidification, which may damage oocytes and cause instability of current recording. Within the range that we could study, external acidification initially enhanced and thereafter inhibited rabbit ClC-2 currents. Because current enhancement by external acidification was observed at the moment when low-pH solution reached the recording chamber, this action appeared to be a direct action of external protons. On the other hand, inhibitory effects of low pH were attained gradually and reached steady-state levels after ~5 min of perfusion with low-pH solution and ~10 min was required to wash out this inhibitory effect. Thus this inhibitory effect is likely to be due to some secondary changes triggered by external acidification, such as allosteric changes of channel proteins or intracellular acidification. Further experiments are needed to assess this intriguing finding. The latter possibility could especially be tested in a system in which intracellular and extracellular pH could be controlled more rigidly (e.g., inside-out patch experiment, lipid bilayer voltage clamp).

The time course of changes in ClC-2 current amplitude correlated with the time course of changes in oocyte diameter. The increase in current amplitude was initially fast but became slower after 20 min of superfusion with hyposmolar solution. A similar pattern was observed in the time course of oocyte swelling. These findings support the hypothesis, although indirectly, that changes in cell volume rather than external osmolarity per se are linked to modulation of ClC-2 currents. Oocyte swelling evoked by external hyposmolarity was smallest in oocytes in which expressed current was largest, whereas oocyte swelling was largest in those oocytes in which current amplitude was smallest. Thus ClC-2 currents appear to compensate oocyte swelling in hyposmolar solution. Volume regulatory action appears to be dependent on activity of ClC-2 currents. The more frequently stimulation was applied to activate ClC-2 currents, the less oocyte swelling was attained. These findings are consistent with the idea that efflux of Cl- through activated ClC-2 channels reduces intracellular osmolytes in association with obligatory loss of water, resulting in reduction in oocyte volume. We repeated measurement of oocyte diameter three times by two different observers. Error range of measurement in the same observer and that between two observers was 0.02 mm. A difference, then, >0.02 mm with a P value <0.05 was considered to be significant. Nevertheless, we have to admit that our method of measurement of oocyte diameter still lacks precision and accuracy. Measurement of oocyte diameter with more accurate space resolution is needed to confirm these intriguing conclusions.

In summary, our data indicate that rabbit ClC-2 appears to be a counterpart of rat ClC-2, but not a tissue-different variant, because rabbit ClC-2 currents have fundamentally similar electrophysiological characteristics to those of rat ClC-2 currents. Rabbit ClC-2 currents were enhanced by extracellular hyposmolarity in a similar way as rat ClC-2 currents. For this finding, the rat ClC-2 channel was suggested to be important for volume regulation in a condition such as tissue edema. Our finding that the expression of ClC-2 diminished oocyte swelling in response to external hyposmolarity that is dependent on activation of ClC-2 currents appears to further confirm the implication of ClC-2 for volume regulation.

    NOTE ADDED IN PROOF

After acceptance of this paper, ClC-3 was reported to code a volume-activated Cl- channel in the heart and probably other tissues (D. Duan, C. Winter, S. Cowley, J. R. Hume, and B. Horowitz. Nature 390: 417-421, 1997).

    ACKNOWLEDGEMENTS

We thank Dr. C. D. McCaig (University of Aberdeen) for critical reading and for correcting the English and N. Sakaguchi for secretarial assistance.

    FOOTNOTES

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas of Cardiac Development and Gene Regulation from the Ministry of Education, Science, and Culture, Japan; by a grant from the Japan Cardiovascular Research Foundation; by a Bayer Cardiovascular Disease Research Scholarship; and by a grant from the Molecular Cardiology Study Group, Japan.

Address for reprint requests: T. Furukawa, Dept. of Autonomic Physiology, Medical Research Institute, Tokyo Medical and Dental Univ., 2-3-10, Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan.

Received 3 July 1997; accepted in final form 30 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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