Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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
-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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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
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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.
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RESULTS |
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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).
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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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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 (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
pHi between control and ClC-0 oocytes remained constant.
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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Lowering the Cl concentration of
the incubation medium increases
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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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.
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DISCUSSION |
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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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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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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