Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

Gordon J. Cooper and Peying Fong

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During maturation of oocytes, Cl- conductance (GCl) oscillates and intracellular pH (pHi) increases. Elevating pHi permits the protein synthesis essential to maturation. To examine whether changes in GCl and pHi are coupled, the Cl- channel ClC-0 was heterologously expressed. Overexpressing ClC-0 elevates pHi, decreases intracellular Cl- concentration ([Cl-]i), and reduces volume. Acute acidification with butyrate does not activate acid extrusion in ClC-0-expressing or control oocytes. The ClC-0-induced pHi change increases after overnight incubation at extracellular pH 8.5 but is unaltered after incubation at extracellular pH 6.5. Membrane depolarization did not change pHi. In contrast, hyperpolarization elevates pHi. Thus neither membrane depolarization nor acute activation of acid extrusion accounts for the ClC-0-dependent alkalinization. Overnight incubation in low extracellular Cl- concentration increases pHi and decreases [Cl-]i in control and ClC-0 expressing oocytes, with the effect greater in the latter. Incubation in hypotonic, low extracellular Cl- solutions prevented pHi elevation, although the decrease in [Cl-]i persisted. Taken together, our observations suggest that KCl loss leads to oocyte shrinkage, which transiently activates acid extrusion. In conclusion, expressing ClC-0 in oocytes increases pHi and decreases [Cl-]i. These parameters are coupled via shrinkage activation of proton extrusion. Normal, cyclical changes of oocyte GCl may exert an effect on pHi via shrinkage, thus inducing meiotic maturation.

cell volume; acid extrusion; ion-sensitive microelectrodes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GROWTH-ARRESTED (stage V and VI) amphibian oocytes are a popular model system for the heterologous expression of ion channels and transporters (9, 30). Throughout this dormant period, oocytes continue to exhibit changes in intracellular ionic activities, including those of Ca2+ and Cl- (1, 15). With the onset of maturation, oocytes increase their intracellular pH (pHi). Alkalinization in response to hormonal agents that promote maturation, such as progesterone, is a well-documented phenomenon thought to be permissive for protein synthesis (16). In contrast, there is a paucity of information concerning the possibility of linkage between ionic fluctuations and pHi changes during meiotic maturation.

After the fertilization of mature eggs, the progression of ionic events is well described, with the development of a fertilization potential that coincides with elevated intracellular Ca2+ concentrations ([Ca2+]i) and precedes slow intracellular alkalinization (for review see Ref. 32). This sequence of events has been exhaustively documented for oocytes from organisms ranging from echinoderms to mammals. Cl- currents, including those activated by elevation of [Ca2+]i, have also been described in many oocyte and germ line systems, including frog oocytes (1, 21, 22) and ascidian eggs (2). However, whether and how Cl- conductances (GCl) play a role in the transduction of Ca2+ signals to changes in pHi in meiotic maturation or postfertilization mitotic events are questions that beg further investigation.

Intracellular Ca2+ activities in developmentally arrested Xenopus oocytes are cyclical in nature (15). This periodicity confounds investigations testing whether increased GCl (e.g., via stimulation of an endogenous Ca2+-activated GCl) can mediate pHi changes, as well as the underlying mechanism of any such effect. The present study circumvents this problem and approaches the question by 1) introducing, by means of heterologous expression, a large and tonically active GCl, ClC-0 (24) to the Xenopus oocyte plasma membrane and 2) measuring the effects of this perturbation on the pHi and intracellular Cl- concentration ([Cl-]i). ClC-0, the Cl- channel native to the Torpedo electroplax, is a member of the CLC family of voltage-gated Cl- channels. ClC-0 possesses two distinct gates that display opposite voltage dependence (10, 13, 18, 24). Although in their own right both gates are very intriguing, the important aspect that we exploit here is that, at physiological voltages, both gates have a high probability of opening. Thus, in contrast to cystic fibrosis transmembrane conductance regulator, the gating properties of ClC-0 mean that this channel is likely to be active throughout our experiments. Moreover, this channel expresses strongly and rapidly in Xenopus oocytes. Taken together, these properties make ClC-0 an ideal channel for use in our studies.

The present study demonstrates that oocytes expressing ClC-0 have resting pHi values that are significantly elevated relative to those measured in control, water-injected oocytes. [Cl-]i is lower in ClC-0-expressing oocytes than in controls. Incubation in hypotonic, low-Cl- solutions eliminates the elevation in pHi, although the decrease in [Cl-]i persists. These data suggest a novel mechanism that links increases in membrane GCl to intracellular alkalinization via an increased exit of KCl and, hence, cell shrinkage.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Preparation of cRNA. The plasmid cDNA encoding for the Cl- channel, ClC-0, was a generous gift of Prof. Thomas J. Jentsch (Center for Molecular Neurobiology, Hamburg, Germany). The cDNA had been previously subcloned into the expression vector pTLN (17), which contains the 5' and 3' regions of the Xenopus beta -globin gene. The plasmid was linearized with SnaBI (New England Biolabs, Beverly, MA) and used in cRNA capping and transcription reactions (mMessage mMachine, Ambion, Austin, TX).

Solutions. The composition of experimental solutions is summarized in Table 1. All solutions were prepared using deionized Millipore-filtered water. The OR3 medium contained 6.85 g/l of Leibovitz's L-15 cell culture medium (GIBCO-BRL, Gaithersburg, MA), 10,000 U/ml penicillin G sodium, 10,000 µg/ml streptomycin sulfate (GIBCO-BRL), and 5 mM HEPES (pH adjusted to 7.50 with NaOH or HCl). Solutions were adjusted to the desired pH by addition of NaOH, HCl, or gluconic acid (for low-Cl- solutions). Osmolarities, measured using a vapor pressure osmometer (Wescor, Logan, UT), were 195-200 and 169-172 mosmol/kg for isotonic and hypotonic solutions, respectively.

                              
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Table 1.   Composition of experimental solutions

Isolation and injection of Xenopus oocytes. Mature female Xenopus laevis were anesthetized by immersion in 0.2% ethyl m-amino benzoate (tricaine; Sigma Chemical, St. Louis, MO). A small incision was made in the abdomen, and an ovarian lobe was removed. The animal was allowed to recover from anesthesia at room temperature after closure of the incision with 6-0 silk sutures. For experiments performed at the University of Sheffield, Xenopus were killed using a procedure in accord with current UK legislation and the ovarian lobes were removed. Oocytes were isolated from lobes and enzymatically defolliculated by treatment with 2 mg/ml type IA collagenase (Sigma Chemical) in 0-Ca2+ ND96 and then stored in OR3 medium before they were sorted. Stage V and VI oocytes were selected for injection.

Oocytes were injected with 1 ng of cRNA (50 nl of a 0.02 µg/µl cRNA solution) or an equal volume of water using a microinjector (Drummond Instruments, Broomall, PA). Injected oocytes were incubated overnight at 18°C in OR3 medium or the appropriate version of ND96. Oocytes incubated in OR3 medium and normal ND96 displayed no obvious differences in any parameters measured in this study. The rapid and robust expression of ClC-0 permitted the initiation of experiments 18 h after injection (10).

Measurements of membrane potential, pHi, and [Cl-]i. Conventional voltage electrodes were pulled from borosilicate capillary glass (Warner Instruments, Hamden, CT) and filled with 3 M KCl. Ion-sensitive microelectrodes were prepared as described previously (27). The tips of pH-sensitive electrodes were filled with hydrogen ionophore I-cocktail B (Fluka) before backfilling with a buffer containing 0.04 M KH2PO4, 0.023 M NaOH, and 0.015 M NaCl (pH 7.0). The pH electrodes were calibrated using National Institute of Standards and Technology-traceable pH 6.0 and 8.0 standards (Fisher Scientific, Pittsburgh, PA), with slopes ranging between -55 and -59 mV/pH unit. For Cl--sensing electrodes, the electrode tips were filled with Cl--selective liquid ion-exchanger microelectrode cocktail A (Fluka) and backfilled with 3 M KCl. Cl- electrodes were calibrated using a modification of the calibration method described by Thomas (28). The calibration solutions for Cl- electrodes were prepared by mixing calibration stock solutions A and B (Table 1) to give final Cl- concentrations of 10, 15, 25, 50, 80, and 100 mM. Fresh stocks were mixed on each experimental day. Figure 1A shows the voltage response of a Cl--sensitive electrode to these calibration solutions. The calibration voltages were fitted to the following equation
V<SUB>Cl</SUB> = {m · log([Cl] + corrfact)} + offset
where VCl is the voltage measured by the Cl--sensitive electrode, [Cl-] represents the known Cl- concentration of the standard solution, corrfact adjusts the exponential fit of the data, and offset represents the deviation of the electrode asymptote from 0 mV. Figure 1B shows a fit of the calibration data shown in Fig. 1A. Acceptable slopes were -50 to -59 mV per 10-fold change in [Cl-].


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Fig. 1.   Calibration of Cl--sensitive microelectrodes. A: voltage response of a Cl--sensitive microelectrode to change in Cl- concentration. B: plot of data from A fitted using Eq. 1. Fitted slope for this electrode was -50.9 mV/10-fold change in Cl- concentration.

The membrane potential (Vm) and ion-sensitive electrodes were connected to high-impedance electrometers (World Precision Instruments, Sarasota, FL) interfaced to an IBM-compatible personal computer. A bath calomel served as the reference for the Vm electrode. The voltage due to pH (or Cl-) was determined by electronic subtraction of the Vm and pH (or Cl-) electrode signals (27).

Programs written in-house facilitated the acquisition and initial analysis of data. Further analysis of data utilized SigmaPlot and SigmaStat for Windows programs (Jandel Scientific, Corte Madera, CA).

Measurement of cell water. A previous study has shown that the diffusional water permeability of various cell types, including Xenopus oocytes, is relatively high, with half time for the equilibration of the cell water with the external media <4.5 min (23). To determine whether expression of CLC-0 altered oocyte volume, we measured the amount of water in the oocytes after equilibration with an isotonic external solution containing 3H2O. Oocytes were placed in ND96 containing 3H2O (1 µCi/ml) for 1 h. At the end of this period, the distribution of 3H2O into the oocyte was halted by three rapid washes in ice-cold ND96 solution, the osmolarity of which had been reduced to 150 mM. The oocytes were dissolved in 20% SDS, and 3H was detected by scintillation counting.

Statistics. Unless otherwise specified, values are means ± SE. Paired and unpaired Student's t-tests were used in tests of significance. We have assumed significance at the 5% level.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Expression of ClC-0 results in elevated resting pHi. As noted previously by us (8, 10) and others (13), Xenopus oocytes injected with ClC-0 reproducibly show robust currents with the peculiar gating characteristics of this channel, as well as resting Vm of approximately -30 mV. In contrast, water-injected oocytes from Xenopus and Bufo have been reported to display a wide range of resting Vm values. This variability has been attributed to the hormonal status of the donor animal (20, 29).

Figure 2A illustrates the scatter of paired Vm values in oocytes from nine animals. As expected, the measured Vm values of water-injected oocytes encompassed a wide range: -30 to -84.1 mV (-52.5 ± 1.8 mV, n = 45). In contrast, expression of ClC-0 significantly depolarized Vm to -33.8 ± 0.7 mV (n = 45, P < 0.0001, unpaired t-test). Figure 2B summarizes the data represented in Fig. 2A. The error bars (SD values) show that the scatter among the Vm values measured in ClC-0-expressing oocytes is substantially decreased compared with that of water-injected controls.


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Fig. 2.   Effect of ClC-0 expression on membrane potential (Vm) and intracellular pH (pHi) of oocytes expressing ClC-0. A and C: paired comparison of changes in Vm or pHi in ClC-0-expressing () and control oocytes (open circle ). B and D: summary of data in A and C. Values are means ± SD. * Significantly different from paired control group.

Resting pHi varied for water-injected oocytes from 7.09 to 7.61 (7.30 ± 0.02, n = 44). After expression of ClC-0, the mean pHi increased to 7.49 ± 0.02 (n = 45, P < 0.0001, unpaired t-test). Oocytes paired from nine animals (Fig. 2, C and D) show that expression of ClC-0 elevates resting pHi by ~0.2 pH unit relative to the paired control.

Does expression of ClC-0 facilitate acid extrusion when the oocyte is challenged by an acid load? Oocytes were pulsed for 15 min with a solution containing the weak acid sodium butyrate (Table 1). This maneuver results in an intracellular acidification that is reversed on removal of butyrate (Fig. 3) (7). The presence of an active acid extrusion process would produce a pHi recovery at the end of the challenge period. Similarly, the pHi would overshoot its initial resting level during the recovery period.


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Fig. 3.   Effect of changing pHi and extracellular pH (pHo) on ClC-0-induced alkalinization. A: effect of sodium butyrate on pHi. Oocytes expressing ClC-0 or control water-injected oocytes were exposed to a solution containing 5 mM sodium butyrate for 15 min (horizontal bar). Net butyrate-induced change in pHi, calculated as the difference between resting pHi and steady-state pHi during butyrate application (Delta pHi in ClC-0 trace), did not differ. Rate of change of pHi was calculated over the last 5 min of butyrate exposure, and likewise there was no difference between the groups. Data were obtained from 6 water-injected and 9 ClC-0-expressing oocytes from 5 animals. B: effect of changing pHo on ClC-0-induced alkalinization. After overnight incubation in pH 6.5, 7.5, or 8.5 medium, pHi was measured in control (H2O) and ClC-0-expressing oocytes. Values are means ± SE for oocytes from 2 animals. A paired comparison on oocyte groups indicated that the magnitude of the ClC-0-induced pHi change was larger after incubation at pHo 8.5 than after incubation at pHo 7.5. However, oocytes incubated at pHo 6.5 did not show a difference in the pHi change compared with oocytes incubated at pHo 7.5.

Figure 3 shows the typical response of a water-injected and a ClC-0-expressing oocyte to a butyrate pulse. As shown in these superimposed, continuous measurements of pHi, exposure to butyrate caused the oocytes to acidify steadily and reach a new steady state after ~10 min. The Delta pHi elicited by butyrate did not differ between the two groups. However, in neither case did the pHi recover, as judged by the rate of change of pHi over the last 5 min. These data suggest that acid extrusion is not activated under these conditions for water- or ClC-0-injected oocytes.

Overnight incubation of ClC-0-injected oocytes at pH 8.5 augments the difference in resting pHi. The results described above (see Does expression of ClC-0 facilitate acid extrusion when the oocyte is challenged by an acid load?) imply that once a new steady-state pHi is achieved, acid extrusion mechanisms switch off and are not activated by an acute acid challenge, as is the case for control oocytes. So how does resting pHi (Delta pHi) increase in ClC-0-expressing oocytes? The establishment of a new steady-state pHi must occur during the ~18 h of oocyte incubation after cRNA injection. To assess whether the proton distribution across the membrane influences this mechanism, oocytes were incubated overnight in high- or low-pH solutions. Figure 3B summarizes the effect of incubation in ND96 adjusted to pH 6.5 or 8.5. Compared with control oocytes at extracellular pH (pHo) 7.5, those incubated at pHo 6.5 showed a reduction in pHi from 7.32 ± 0.02 to 7.21 ± 0.04 (n = 2, P = 0.04, 1-tailed t-test). Similar to control oocytes, those expressing ClC-0 and maintained at pHo 6.5 showed pHi values reduced compared with ClC-0-injected oocytes maintained at pHo 7.5. However, the Delta pHi between control and ClC-0 oocytes remained constant.

With incubation at pHo 8.5, water-injected oocytes showed no change in pHi relative to those kept at pHo 7.5. However, after incubation at pHo 8.5, the magnitude of the pHi increase induced by ClC-0 expression (Delta pHi = 0.33 ± 0.01, P = 0.04) exceeded that observed for the pHo 7.5 group (Delta pHi = 0.18 ± 0.02). Incubation at pHo 6.5 had no effect on the magnitude of the ClC-0-induced alkalinization (Delta pHi = 0.16 ± 0.05). In all cases, the Vm of ClC-0- and water-injected oocytes did not differ from that of oocytes maintained in ND96 at pH 7.5.

Membrane depolarization does not cause redistribution of H+ or OH- that can account for the ClC-0-induced shift in resting pHi. As noted above, the Vm of ClC-0-expressing oocytes is substantially depolarized. We entertained the notion that this membrane depolarization could drive H+ exit or OH- entry via another conductive pathway. To test this hypothesis, we chronically altered the Vm of the oocytes by incubating them in high K+ (depolarization) or low K+ (hyperpolarization). Figure 4 summarizes the results of these experiments. Although the predicted effects on Vm were verified by our measurements, pHi changed in the opposite direction. Figure 4A shows that, in water- and ClC-0-injected oocytes, depolarization by overnight incubation in 25 mM K+ caused an acidic shift in pHi, whereas hyperpolarization in 0.1 mM K+ caused an alkalinization. For control oocytes and those expressing ClC-0, pHi and Vm follow a linear relationship. The fitted regression line has a negative slope, clearly opposite in direction to that expected if alkalinization resulted from membrane depolarization. Moreover, as judged from the absolute slope of the regression line, the pHi of ClC-0-expressing oocytes displayed greater sensitivity to changes in Vm (Fig. 4B).


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Fig. 4.   Effect of changing Vm by altering external K+ concentration on ClC-0-induced alkalinization. A: summary of measured pHi after overnight incubation of control (H2O) and ClC-0-expressing oocytes in 0.1, 2, or 25 mM K+. Values are means ± SE. * Significant increase in pHi compared with paired control group. For control and ClC-0-expressing oocytes, Vm after incubation at each extracellular K+ concentration was measured. B: Vm vs. pHi for control (open circle ) or ClC-0-expressing oocytes (). Data are binned into groups corresponding to the appropriate extracellular K+ concentration group, and the regression line is calculated through all the individual points. The regression line for control oocytes fits the following equation: pHi = -0.0034Vm + 7.1283 (n = 31, r = 0.589). The regression line for the ClC-0 group fits the following equation: pHi = -0.0101Vm + 7.0737 (n = 17, r = 0.717).

Lowering the Cl- concentration of the incubation medium increases Delta pHi and decreases [Cl-]i in oocytes expressing ClC-0. The above observations suggest the involvement of nonpassive pathways of H+ extrusion. Xenopus oocytes functionally express a shrinkage-activated Na+/H+ exchanger (NHE) (3, 4, 12). The ClC-0-injected oocytes often appeared shrunken 1 day after injection. However, it was not possible to easily visualize this shrinkage using traditional optical techniques. When visualized from above, the oocytes do not appear smaller, but when turned onto their sides it was clear that the oocytes had settled on the base of the chamber and resembled a punctured football. To determine whether expression of ClC-0 does lead to a reduction in oocyte volume, we calculated the volume of water in the oocytes. The volume of water in the oocytes expressing ClC-0 was reduced by ~30% compared with control oocytes (Fig. 5). In biological systems, shrinkage often is associated with the exit of K+ and Cl-, followed by obligatory water movement. Therefore, we hypothesize that native Cl- permeability is rate limiting in Xenopus oocytes. Expression of ClC-0 removes a limitation on KCl efflux from the oocyte, leading to oocyte shrinkage and activation of NHE. Raising external K+ would decrease KCl efflux, oocyte shrinkage, and, therefore, the pHi rise, whereas lowering K+ would increase these effects. Thus it follows that lowering external Cl- should not only augment the changes in pHi observed in ClC-0-expressing oocytes but should also decrease [Cl-]i. We therefore measured pHi and [Cl-]i in control and ClC-0-expressing oocytes after overnight incubation in low-Cl- media. Figure 6 summarizes the results of these experiments. In oocytes expressing ClC-0, pHi increased with overnight exposure to 53 mM Cl- solution (Fig. 6A) and the [Cl-]i fell (Fig. 6B).


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Fig. 5.   Effect of expressing ClC-0 on oocyte water volume. Control and ClC-0-expressing oocytes were incubated in 3H2O for 1 h. Values are means ± SE of number of observations in parentheses. * Significant reduction in cell water as judged by unpaired t-test.



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Fig. 6.   Effect of low-Cl- media: a summary of the effect of overnight incubation in low-Cl- medium (53.6 mM) on pHi (A) and [Cl-]i (B) in control and ClC-0-expressing oocytes. C and D: summary of effects of overnight incubation in control ND96, a low-Cl- ND96 modification, or a hypotonic, low-Cl- solution on pHi and [Cl-]i in oocytes expressing ClC-0. Values are means ± SE of number of observations in parentheses. * Significantly different from control. dagger  Significantly different from low Cl-.

The effects of low-Cl- incubation on the pHi and [Cl-]i of water-injected oocytes were qualitatively similar. The rise in pHi was significant, whereas the decrease in [Cl-]i was slight.

Incubation in hypotonic solutions uncouples the relationship between [Cl-]i and pHi. At this point, the data show a coupling between pHi and [Cl-]i and suggest a dependency on cell volume. We sought to test further whether the net loss of KCl causes shrinkage and, in turn, alkalinization. If this were the case, offsetting the shrinkage by incubation in hypotonic, low-[Cl-]o solutions ought to abrogate the alkalinization. On the other hand, the decrease in [Cl-]i ought not to be abolished.

We summarize the results of experiments in which ClC-0-injected oocytes were incubated overnight in hypotonic, low-Cl- medium (53.6 mM Cl, ~170 mosmol/kg) in Fig. 6, C and D. Consistent with the experiments described above (see Lowering the Cl- concentration of the incubation medium increases Delta pHi and decreases [Cl-]i in oocytes expressing ClC-0), oocytes expressing ClC-0 and maintained in isotonic, low-Cl- solution show an elevation in pHi exceeding that of oocytes incubated in standard ND96. However, reducing the osmolarity of the low-Cl- incubation medium prevented this increase in pHi (Fig. 6C). In contrast, under hypotonic, low-Cl- conditions, [Cl-]i fell below that measured for oocytes held in isotonic, low-Cl- solution (Fig. 6D). Interestingly, water-injected control oocytes treated similarly did not show such an uncoupling of pHi and [Cl-]i. Although [Cl-]i decreased further (to 21.2 ± 1.86 mM, n = 3 oocytes from 3 batches), pHi remained elevated (7.38 ± 0.02, n = 3 oocytes from 3 batches).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our primary observation demonstrates that the presence of an active GCl in the oocyte membrane initiates an intracellular alkalinization. The extent of alkalinization, ~0.2 pH unit, agrees astonishingly well with that measured by Lee and Steinhardt (16) in Xenopus oocytes undergoing progesterone-induced meiotic maturation. Although, in principle, whether GCl is linked to alkalinization can be addressed in uninjected oocytes, the presence of functionally active endogenous Cl- currents is highly variable. We therefore circumvented this problem by expression of a well-characterized, exogenous GCl, ClC-0. Membrane depolarization, which is a notable feature of ClC-0 expression, also has been linked to oocyte maturation (29). In contrast to the cystic fibrosis transmembrane conductance regulator, the activity of which is dependent on phosphorylation, the unusual gating properties of ClC-0 mean that, for Vm in the physiological range, the probability of channel opening is substantial. Interestingly, previous studies on oocytes expressing members of the CLC family have not considered that these parameters may have been altered. Inasmuch as gating of fast and slow voltage gates of ClC-0 are affected by pHi and [Cl-]i (6, 11,18, 24, 25), it may be necessary to reevaluate the outcomes of previous studies that employed voltage-clamp measurements.

Does ClC-0 increase membrane conductive pathways for H+ equivalents? Because we noted that expression of ClC-0 depolarizes the membrane, we considered whether the channel itself acts as an OH- conductive pathway or, alternatively, whether the depolarization drives H+ efflux through a separate, endogenous conductance. To this end, we studied the effects of chronically altering Vm. Figure 4, which summarizes experiments testing the effects of altering Vm on pHi, argues that the pathway(s) involved in ClC-0-induced alkalinization is not conductive. The linear relationship between pHi and Vm indicates that depolarization acts to acidify, rather than alkalinize, the oocyte. Moreover, compared with a similar plot of control data, the slope of the relationship is greater for the oocytes expressing ClC-0. This further emphasizes that the increase in pHi does not result from conductive H+-equivalent pathways. In addition, these findings also rule out possible contributions by a K+/H+ exchange mechanism or the passage of OH- through ClC-0.

Does expression of ClC-0 upregulate active acid extrusion mechanisms? Xenopus oocytes endogenously express NHE (3, 5). To address whether this mechanism underlies the increase in pHi, we examined the ability of oocytes to recover from an acute acid load. Water- and ClC-0-injected oocytes challenged with sodium butyrate showed no evidence of active H+ extrusion, despite showing similar magnitudes of acidification. These experiments were performed once a new steady-state pHi had been achieved. Thus the possibility remains that NHE upregulation underlies the difference between resting steady-state pHi of the two groups of oocytes. Once a steady state is reached, the exchanger could be inactivated.

One problem to be considered is how to test for the involvement of NHE, a task that poses some challenges. A logical approach would be to utilize specific inhibitors of these transporters and then determine whether the expression of ClC-0 still produces alkalinization. The time scale of the experiments necessitates that block be complete, for any residual activity resulting from incomplete block would be sufficient to bring about the pH change. We attempted to test this hypothesis directly by incubating the oocytes overnight in ND96 containing 1 mM amiloride or in 0-Na+ solutions. Unfortunately, we found that amiloride had unpredictable effects on the oocytes, whereas oocytes maintained in 0-Na+ media were not stable enough to allow prolonged recordings. In principle, more potent inhibitors of Na+/H+ exchange, such as cariporide, could be tested. However, unlike human NHE-1, the NHE that is endogenous to Xenopus oocytes is not blocked by HOE-694, the structurally related congener of cariporide (5). This makes it highly unlikely that cariporide would prove useful in further testing of the validity of our hypothesis. Therefore, although the evidence points toward a mechanism based on activation of native Na+/H+ exchange, it remains unclear how the new steady-state pHi is established.

Does increasing total membrane GCl facilitate KCl efflux? One clue rests in recognizing that the oocyte isoform of NHE is activated by cell shrinkage. Data obtained from experiments in which external K+ was altered suggests that increasing the chemical driving force for K+ movement augments the ClC-0-induced alkalinization. A mechanism consistent with our observations is depicted in Fig. 7. The concerted action of the Na+-K+-ATPase and K+ conductance contributes to a negative resting potential that could drive the exit of Cl-. However, at rest, for the measured Vm of -52.5 mV (Fig. 2B), [Cl-]i is 36.4 mM (Fig. 6B), which is greater than that calculated at equilibrium (~13 mM). We therefore postulate that Cl- efflux is limited by the total membrane GCl in a native oocyte. Expression of an exogenous Cl- channel, ClC-0, removes this limitation, leading to depolarization of Vm to -33.8 mV and lowering of [Cl-]i to ~30 mM, substantial efflux of KCl, and, hence, oocyte shrinkage (Fig. 5). The shrinkage then activates the NHE, resulting in elevated pHi. We predict from this model that imposing an outwardly directed Cl- gradient will additionally alkalinize the cell and lower [Cl-]i beyond levels seen with ClC-0 expression alone. Therefore, we executed the experiments summarized in Fig. 6, A and B. Our data show that water-injected oocytes also show smaller, but qualitatively similar, changes in pHi and [Cl-]i. We therefore must take this to suggest that these effects reflect the contributions of a small, but nonetheless measurable, GCl. Although the resultant data support our hypothesis, these studies do not per se test the effects of shrinkage.


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Fig. 7.   Model summarizing the shrinkage hypothesis. Top: under resting conditions, K+ exit needs to be accompanied by exit of Cl- or another anionic species. As noted in DISCUSSION, for the measured resting Vm of -52.5 mV, [Cl-]i is 36.4 mM, which is above the calculated value for [Cl-]i at equilibrium (~13 mM). This leads us to note that the native Cl- conductance (GCl,native) must be the limiting factor for net KCl efflux. Middle: when GCl,native (right) or a heterologously expressed GCl (GCl,expressed, left) is active, movement of K+ and Cl- is permitted. This leads to the situation shown at bottom, with heterologous expression of ClC-0 depolarizing Vm to -33.8 mV and lowering [Cl-]i to ~30 mM. Thus net KCl efflux causes cell shrinkage, resulting in activation of the Na+/H+ exchanger (NHE) and intracellular alkalinization.

From our model, we also predict that offsetting shrinkage would effectively uncouple the pHi and [Cl-]i changes, attenuating the alkalinization, yet leaving the [Cl-]i effect intact. Our findings, depicted in Fig. 5, C and D, are in line with the hypothesis. Endogenous hypotonicity-activated GCl cannot account for these observations, because 1) collagenase-isolated oocytes lack these conductances (31) and 2) the paired water controls did not show uncoupling of the pHi and [Cl-]i changes. Another prediction from our hypothesis suggests that overnight incubation under hypertonic conditions should drive pHi to an even higher value. However, preliminary experiments in which oocytes were incubated in a series of hypertonic media indicate an apparent saturation of alkalinization, with pHi achieving a plateau at a maximal value of ~7.8. Further increases in tonicity had no additional effect on pHi (unpublished observations). A possible explanation for this is that the oocyte has a limited amount of osmotically active water (~30% of the oocyte volume; unpublished observation). On expression of ClC-0, the oocytes lose this osmotically active water, so changing tonicity cannot effect an additional change in volume.

Implications of the model. The linkage between oscillatory changes in GCl and increases in pHi during developmental processes, such as exit from meiotic arrest and fertilization, has not been extensively investigated. Using unfertilized eggs and embryos of Boltenia villosa, Block and Moody (2) provided evidence for cell cycle-linked oscillations in a voltage-dependent Cl- current. These authors postulated a connection between [Cl-]i and pHi changes, suggesting a link to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> fluxes. However, elucidation of a mechanism coupling these effects was not pursued. We conducted our experiments in the nominal absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, ruling out possible contributions of this species to the observed alkalinization. However, in contrast to NHE, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent transport mechanisms have not been found in Xenopus oocytes (3, 26).

In summary, the findings of our study lead us to postulate a mechanism by which pHi and [Cl-]i are linked via cell volume changes, thereby resulting in the maturation of meiotically arrested oocytes. The conclusions of the present study may extend to other developmental stages, thus underscoring their significance.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the generosity of Dr. Walter F. Boron for providing the space, equipment, and intellectual environment necessary for this study. We also thank Duncan Wong for designing the online data acquisition programs, Emilia M. Hogan and Dr. Emile L. Boulpaep for stimulating discussions, and Dr. Nancy K. Wills for critically reading the manuscript. This work would not have been possible without the collegial cooperation offered by Drs. Stanley White and William B. Guggino.


    FOOTNOTES

G. J. Cooper was the recipient of a Long-Term Fellowship from the International Human Frontiers Science Program Organization. P. Fong was supported by National Institute of Diabetes and Digestive and Kidney Diseases Training Grant DK-07259 during the course of this work.

Present address of G. J. Cooper: Dept. of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK (E-mail: g.j.cooper{at}sheffield.ac.uk).

Address for reprint requests and other correspondence: P. Fong, Dept. of Physiology, The Johns Hopkins University School of Medicine, 725 North Wolfe St., 202C Physiology, Baltimore, MD 21205 (E-mail: pfong{at}jhmi.edu).

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.

First published October 3, 2002;10.1152/ajpcell.00406.2002

Received 4 September 2002; accepted in final form 26 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barish, ME. A transient calcium-dependent chloride current in the immature Xenopus oocyte. J Physiol 342: 309-325, 1983[Abstract].

2.   Block, ML, and Moody WJ. A voltage-dependent chloride current linked to the cell cycle in ascidian embryos. Science 247: 1090-1092, 1990[ISI][Medline].

3.   Burkhardt, BC, Kroll B, and Frömter E. Proton transport mechanism in the cell membrane of Xenopus laevis oocytes. Pflügers Arch 434: 306-312, 1992.

4.   Busch, S. Cloning and sequencing of the cDNA encoding for a Na+/H+ exchanger from Xenopus laevis (X1-NHE). Biochim Biophys Acta 1325: 13-16, 1997[ISI][Medline].

5.   Busch, S, Burckhardt BC, and Siffert W. Expression of the human sodium/proton exchanger NHE-1 in Xenopus laevis oocytes enhances sodium/proton exchange activity and establishes sodium/lithium countertransport. Pflügers Arch 429: 859-869, 1995[ISI][Medline].

6.   Chen, TY, and Miller C. Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel. J Gen Physiol 108: 237-250, 1996[Abstract].

7.   Choi, I, Aalkjaer C, Boulpaep EL, and Boron WF. An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel. Nature 405: 571-575, 2000[ISI][Medline].

8.   Cooper, GJ, and Fong P. Resting intracellular pH is increased by expression of ClC-0, a voltage-gated chloride channel (Abstract). FASEB J 12: A373, 1998.

9.   Dascal, N. The use of Xenopus oocytes for the study of ion channels. CRC Crit Rev Biochem 22: 317-387, 1987[ISI][Medline].

10.   Fong, P, Rehfeldt A, and Jentsch TJ. Determinants of slow gating of ClC-0, the voltage-gated chloride channel of Torpedo marmorata. Am J Physiol Cell Physiol 274: C966-C973, 1998[Abstract/Free Full Text].

11.   Hanke, W, and Miller C. Single chloride channels from Torpedo electroplax. Activation by protons. J Gen Physiol 82: 25-45, 1983[Abstract].

12.   Humphreys, BD, Jiang L, Chernova MN, and Alper SL. Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization. Am J Physiol Cell Physiol 268: C201-C209, 1995[Abstract/Free Full Text].

13.   Jentsch, TJ, Steinmeyer K, and Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348: 510-514, 1990[ISI][Medline].

14.   Krieg, PA, and Melton DA. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12: 7057-7070, 1984[Abstract].

15.   Lechleiter, JD, and Clapham DE. Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes. Cell 69: 283-294, 1992[ISI][Medline].

16.   Lee, SC, and Steinhardt RA. pH changes associated with meiotic maturation in oocytes of Xenopus laevis. Dev Biol 85: 358-369, 1981[ISI][Medline].

17.   Lorenz, C, Pusch M, and Jentsch TJ. Heteromultimeric ClC chloride channels with novel properties. Proc Natl Acad Sci USA 93: 13362-13366, 1996[Abstract/Free Full Text].

18.   Ludewig, U, Jentsch TJ, and Pusch M. Analysis of a protein region involved in permeation and gating of the voltage-gated Torpedo chloride channel ClC-0. J Physiol 498: 691-702, 1997[Abstract].

19.   Ludewig, U, Jentsch TJ, and Pusch M. Inward rectification of ClC-0 chloride channels caused by mutations in several protein regions. J Gen Physiol 110: 165-171, 1997[Abstract/Free Full Text].

20.   Maeda, T. Electrical characteristics and activation potential of Bufo eggs. J Gen Physiol 43: 139-185, 1959[Abstract/Free Full Text].

21.   Parker, I, Gundersen CB, and Miledi R. A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes. Proc R Soc Lond B Biol Sci 223: 279-292, 1985[ISI][Medline].

22.   Peres, A, and Bernardini G. A hyperpolarization-activated chloride current in Xenopus laevis oocytes under voltage clamp. Pflügers Arch 399: 157-159, 1983[ISI][Medline].

23.   Prescott, DM, and Zeuthen E. Comparison of water diffusion and water filtration across cell surfaces. Acta Physiol Scand 28: 77-94, 1953[ISI].

24.   Pusch, M, Ludewig U, Rehfeldt A, and Jentsch TJ. Gating of the voltage-dependent chloride channel ClC-0 by the permeant anion. Nature 373: 527-531, 1995[ISI][Medline].

25.   Pusch, M, Jordt SE, Stein V, and Jentsch TJ. Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol 515: 341-353, 1999[Abstract/Free Full Text].

26.   Sasaki, S, Ishibashi K, Nagai T, and Marumo F. Regulation mechanisms of intracellular pH of Xenopus laevis oocyte. Biochim Biophys Acta 1137: 45-51, 1992[ISI][Medline].

27.   Siebens, AW, and Boron WF. Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubule. J Gen Physiol 90: 799-831, 1987[Abstract].

28.   Thomas, RC. Ion-Sensitive Intracellular Microelectrodes. London: Academic, 1978, p. 92-94.

29.   Wallace, RA, and Steinhardt RA. Maturation of Xenopus oocytes. II. Observations on membrane potential. Dev Biol 57: 305-316, 1977[ISI][Medline].

30.   Weber, WM. Endogenous ion channels in oocytes of Xenopus laevis: recent developments. J Membr Biol 170: 1-12, 1999[ISI][Medline].

31.   Weber, WM. Ion currents of Xenopus laevis oocytes: state of the art. Biochim Biophys Acta 1421: 213-233, 1999[ISI][Medline].

32.   Whitaker, MJ, and Steinhardt RA. Ionic regulation of egg activation. Q Rev Biophys 15: 593-666, 1982[ISI][Medline].


Am J Physiol Cell Physiol 284(2):C331-C338
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