Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (Cell Physiol. 43): C543-548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ~40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane's lipid, or 4) increasing the expression of a native "gas channel." The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2 permeability is unaffected by treatment with PCMBS. These data rule out options 2-4 listed above. Thus our results suggest that CO2 passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.
p-chloromercuriphenylsulfonic acid; intracellular pH; carbon dioxide
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INTRODUCTION |
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FROM THE TURN of this century, a dogma of biology has been that small molecules such as H2O, CO2, O2, and NH3 cross cell membranes by passing between membrane lipids. However, beginning in 1935 (11) and continuing in 1953 (14, 18), evidence began to point to the existence of "pores" that can mediate fluxes of H2O, at least in certain cell membranes. The cloning of the H2O channel aquaporin (AQP) 1 (19) and the demonstration that expressing AQP1 in Xenopus oocytes markedly increases the H2O permeability of these cells (20) confirmed that H2O does pass through channels. The cloning of AQP2 from the renal collecting duct (7), as well as its association with the membrane vesicles inserted into the cell apical membrane under the direction of vasopressin (6), showed how H2O transport could be regulated. The only other example of a membrane protein that can mediate substantial transport of a small, neutral molecule is the UT family of urea transporters (28).
As far as gas transport is concerned, the first suggestion that gases
might not cross all membranes simply by dissolving in the membrane
lipid was the observation (13) that
NH3/NH+4, when applied to the lumen of the renal thick ascending limb, acidifies the cells (due to NH+4 uptake) rather than
alkalinizes them (due to NH3
uptake). Later work demonstrated that the apical membranes of
gastric-gland cells have an immeasurably low permeability to both
NH3 and
NH+4 as well as to both
CO2 and HCO3 (27) and that apical membranes of
colonic-crypt cells similarly have no measurable permeability to
NH3 or
NH+4 (24) or to
CO2 or
HCO
3 (25).
The above studies demonstrate that cell membranes exist that are impermeable to H2O or gases and that either expressing AQPs or inserting preexisting AQPs into the membrane can increase the permeability to H2O. However, still unresolved is the question of whether channels might increase the gas permeability of a membrane in the same way that the AQPs increase H2O permeability. A recent study on Xenopus oocytes injected with carbonic anhydrase (CA) demonstrated that expressing AQP1 increases by ~40% the rate at which exposing the cell to CO2 causes intracellular pH (pHi) to fall (15). Although it is possible that AQP1 serves as a conduit for CO2, this previous study could not rule out the possibility that overexpressing AQP1 increases the apparent permeability to CO2 by 1) stimulating injected CA, 2) enhancing the CO2 solubility of the membrane's lipid, or 3) increasing the expression of a native "gas channel."
The aim of the present study was to determine whether CO2 can actually pass through AQP1. We eliminated CA as a variable by performing the experiments in the absence of injected CA; oocytes have no detectable, native CA activity (16). We found that the CO2 permeability of oocytes increases with the degree of AQP1 expression and that the AQP1-dependent CO2 permeability is blocked by the mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS). Mercurials are known to reduce the H2O permeability of AQP1 (20) and do so by interacting with the cysteine at position 189 (21). We found that the C189S mutant of AQP1 induces the same CO2 permeability as the wild-type AQP1 but that the C189S-dependent increase in CO2 permeability is insensitive to PCMBS.
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METHODS |
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Solutions. The control amphibian
Ringer solution ND-96 contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH adjusted to
7.50 using NaOH or HCl). In
HCO3-buffered solutions, 10 mM NaCl
was replaced with NaHCO3, and the
solution was equilibrated with a gas mixture of 1.5%
CO2-98.5%
O2 to gave a solution pH of 7.50. In the 0 Ca2+ ND-96 Ringer
solution used in oocyte isolation,
CaCl2 was omitted and replaced by
NaCl. The OR3 culture medium contained 6.85 g/l of Leibovitz L-15 cell
culture medium (GIBCO-BRL, Gaithersburg, MD), 10,000 U/ml penicillin G
sodium, 10,000 mg/ml streptomycin sulfate (GIBCO-BRL), and 5 mM HEPES
(pH adjusted to 7.50 with NaOH or HCl). The osmolarity of all solutions
was adjusted to between 195 and 200 mosmol/l with NaCl or
H2O. PCMBS (Sigma, St. Louis, MO)
was powdered into solutions to give a final concentration of 1 mM on
the day of experiments.
cRNA preparation. The cDNA encoding
AQP1 and C189S was a generous gift of Dr. P. Agre. In both cases, the
cDNA had been inserted in the plasmid pXbG-ev1, flanked by the 3'
and 5' untranslated regions of the
Xenopus -globin gene
(20). The plasmid was linearized by cutting with XBA1, and cRNA was
made using a T3 polymerase kit (Ambion, Austin, TX). The cRNA encoding
ROMK1 was a gift from Drs. Carmel McNicholas and Gerhard Giebisch.
Oocyte preparation. Stage V-VI oocytes were isolated from Xenopus laevis. Toads were anesthetized by immersion in 0.2% tricaine (buffered to pH 7.50 with 5 mM HEPES). After the anesthetized animals were placed on ice, a 1-cm incision was made in the abdominal wall, and a lobe of an ovary was removed. The abdominal muscle was sutured using 5-0 cat gut, and the skin was sutured using 6-0 silk. The removed ovarian tissue was cut into sections of ~5 × 5 × 5 mm and agitated in 0 Ca2+ ND-96 for 1 h. The oocytes were incubated two times for 20 min in 0 Ca2+ ND-96 containing 2 mg/ml type I collagenase (Sigma), separated by a 15-min wash in 0 Ca2+ ND-96. At the end of the second collagenase treatment, the oocytes were washed in 0 Ca2+ ND-96 for 30 min. This was followed by a further 30-min wash in the standard Ca2+-containing ND-96. The oocytes were then transferred to OR3 media and sorted by size and stage. The sorted oocytes were kept in OR3 at 18°C. On the day after isolation, oocytes were injected with 50 nl of sterile H2O (GIBCO-BRL) or 50 nl of a cRNA solution containing either 1 ng of AQP1 cRNA, 1 ng of C189S cRNA, or 12.5 ng of ROMK1 cRNA. Oocytes were used in physiological experiments 3-10 days after injection. Just before each experiment, the vitelline membrane of a selected oocyte was removed by manual dissection. All experiments were performed at 22°C.
Quantification of AQP1 expression. The expression level of AQP1 or its C189S mutant was determined by measuring the time taken for the oocyte to "lyse" when we switched the bathing solution to deionized H2O. Oocytes with intact vitelline membranes swell and explode dramatically (20). Devitellinized oocytes also swell but, instead of exploding, gently emit a plume of debris ("lysis"). Because cell pressure probably rises less during cell swelling in devitellinized oocytes, it is likely that the relationship between lysis time and H2O permeability is more linear than in oocytes with an intact vitelline membrane. We did not note a substantial change in lysis time during the 3-10 days after injection of H2O or cRNA.
Electrophysiological measurements. Oocytes were transferred to a perfusion chamber that had a long, thin channel and a volume of ~200 µl. Solutions constantly flowed down the length of the channel, delivered by syringe pumps (Harvard Apparatus, South Natick, MA) at a rate of 4 ml/min. The switching of solutions was controlled by pneumatically operated valves (Clippard Instrument Laboratory, Cincinnati, OH). Two thin strands of nylon were stretched across the top of the channel and formed an "X" when viewed from the top of the chamber. The solution flowing down the channel pushed the oocyte against the notch formed by the intersection of the nylon strands and thus held the oocyte in place.
pHi and membrane potential
(Vm) were
measured using microelectrodes, as described in detail previously (22).
Briefly, electrodes were pulled from borosilicate capillary glass
(Warner Instruments, West Haven, CT) using a horizontal puller (Sutter
Instrument, Novato, CA). The
Vm electrodes
were filled with 3 M KCl and had resistances of 2-5 M. The pH
microelectrodes were silanized at 200°C for 5 min, using
bis-di-(methylamino)dimethylsilane (Fluka Chemical, Ronkonkoma,
NY). The tips of the electrodes were filled with hydrogen ionophore I
cocktail B (Fluka) and then backfilled with a buffer containing 0.04 M
KH2PO4,
0.023 M NaOH, and 0.150 M NaCl (pH 7.0). The electrodes were calibrated
at pH 6 and 8 and had slopes of 55-60 mV/pH unit. An additional,
single-point adjustment was made in the perfusion chamber by
calibrating the electrode against the pH 7.50 ND-96 solution, just
before impalement.
Both the Vm and pH microelectrodes were connected to high-impedance electrometers. The voltage due to pH was obtained from the subtraction of the pH and Vm signals (see Ref. 23). Vm was the difference between the Vm electrode and an external calomel reference electrode. The signals were digitized and recorded by an 80486-based personal computer.
Statistics. The initial rate of intracellular acidification was calculated by linear regression. Significance is assumed at the 5% level. Analysis was performed using paired t-tests or ANOVA, as appropriate, in SigmaStat for Windows. If ANOVA indicated a difference, comparison between groups was performed using the Student-Newman-Keuls method.
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RESULTS |
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Determination of oocyte viability. We assessed the viability of the oocytes from the measurement of Vm, rejecting oocytes if Vm was more positive than 30 mV. We found that neither removing the vitelline membrane nor expressing AQP1 had a statistically significant effect on Vm. The mean Vm of H2O-injected oocytes was 52.2 ± 3.3 mV (n = 14) with the vitelline membrane intact and 48.8 ± 2.1 (n = 36) with the vitelline membrane removed. AQP1-expressing oocytes had a Vm of 49.0 ± 4.9 mV (n = 10) with the vitelline membrane intact and 53.2 ± 2.2 mV (n = 34) with the vitelline membrane removed.
Effect of AQP1 expression on rate of CO2-induced acidification. In oocytes in which vitelline membranes had been removed, the rate of CO2-induced acidification increased in an expression-dependent manner (Fig. 1, A-C). Figure 1A shows experiments from three individual oocytes with different expression levels, as judged by lysis time in deionized H2O. As the level of expression increases, the rate of acidification also increases. Data from 34 experiments showed a good correlation between lysis time and rate of acidification (Fig. 1B). We grouped these data into three bins on the basis of cell lysis time: AQP1/Hi for lysis times <60 s, AQP1/Mid for lysis times of 61-120 s, and AQP1/Low for lysis times of 121-180 s. Compared with the rate of acidification in H2O-injected controls (Fig. 1C), the rate was significantly higher in the AQP1/Hi group and AQP1/Mid group. The rate of acidification for the AQP1/Low group was not different from the H2O-injected controls. As observed in a previous study (15), expression of AQP1 had no effect on the rate of CO2-induced acidification in oocytes with intact vitelline membranes.
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Effect of expressing ROMK1 on rate of CO2-induced acidification. In oocytes expressing the K+ channel ROMK1 (12), the rate of CO2-induced acidification was not significantly different from that in controls. Expression of ROMK1 caused a shift in the resting Vm from 48.8 ± 2.1 mV (n = 36) to 96.8 ± 3.0 mV (n = 6, P < 0.0001), which is close to the predicted equilibrium potential for K+ under the conditions used.
Effect of PCMBS on the rate of CO2-induced acidification. If CO2 passes through AQP1, then one might expect the CO2-induced acidification to be inhibited by mercurial compounds, which are known to block the H2O permeability of AQP1 (20). We examined this possibility by twice exposing an oocyte to 1.5% CO2, first in the absence and then in the presence of PCMBS, an organic mercury derivative. In H2O-injected oocytes (Fig. 2A), a 15-min incubation in 1 mM PCMBS produced a small but statistically significant decrease in the rate of the CO2-induced acidification1 compared with the matched control (P < 0.0001, paired t-test). In oocytes expressing AQP1 (Fig. 2B), a 15-min incubation in PCMBS also significantly reduced the rate of the CO2-induced acidification (P = 0.0009). However, the magnitude of the PCMBS effect was 3.5-fold greater in AQP1-expressing oocytes than in H2O-injected controls. As expected, the treatment with PCMBS also reduced the H2O permeability of AQP1-expressing oocytes.2
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Effect of expressing C189S on the rate of
CO2-induced acidification.
The C189S mutant of AQP1 has a normal
H2O permeability but is immune to
inhibition by mercurial derivatives (21). The triangles in Fig.
3A
represent CO2-induced
acidification rates in oocytes with different levels of C189S
expression. These data fall on the same line (taken from Fig.
1B) as that derived for oocytes expressing wild-type AQP1. Moreover, comparing oocytes that lysed in
the middle time range (60- 120 s), we find no significant difference in the CO2-induced acidification
rates between oocytes expressing wild-type AQP1 (21.7 ± 1.1 × 104
pH · s
1,
n = 15) and those expressing the C189S
mutant (23.6 ± 0.4 × 10
4
pH · s
1,
n = 7). Thus oocytes expressing the
C189S mutant display the same CO2
permeability properties as those expressing the wild-type AQP1 channel.
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Effect of PCMBS on the rate of CO2-induced acidification in C189S-expressing oocytes. Exposing an oocyte expressing C189S to 1 mM PCMBS for 15 min produced a small reduction in the rate of CO2-induced acidification compared with the preceding CO2 exposure in the same cell (Fig. 3B). PCMBS also did not affect lysis time.2 In six such paired experiments, PCMBS produced a decrease in the CO2-induced acidification rate (Fig. 3, C and D) that was statistically significant and virtually identical to that produced by PCMBS in H2O-injected control oocytes (Fig. 3, C and D). Indeed, there was no difference between the effects of PCMBS in the control and C189S groups as judged by one-way ANOVA. However, the effect of PCMBS on the rate of the CO2-induced acidification was significantly greater in AQP1 oocytes (Fig. 3, C and D) than in either the control or C189S groups (degrees of freedom = 2,17; F = 20.4; P < 0.005 in both cases).
Determination of CO2 permeability.
Of the CO2 molecules entering the
cell, the fraction that forms
H2CO3
and then dissociates into H+ and
HCO3 is
K/(H + K), where H is intracellular H+ concentration and
K is the apparent dissociation
constant (i.e., 7.24 × 10
7) for the
equilibrium CO2 + H2O
HCO
3 + H+ (8). Thus the
CO2 influx
(JCO2)
is the product of (H + K)/K
and the rate of intracellular H+
formation (JH;
see Ref. 2). JH
is the product of the initial rate of intracellular acidification on
exposure to CO2
(dpHi/dt), the intrinsic intracellular buffering power, and the volume-to-surface ratio estimated by Preston et al. (20) to be 0.02 cm. From the steady-state CO2-induced
pHi decrease evoked by applying
1.5% CO2 (23), we obtained a mean
intrinsic buffering power of 19.9 ± 1.0 mM
(n = 25) in the present experiments.
There is no evidence that resting oocytes express any significant
acid-base transporters, suggesting that our estimates of intrinsic
buffering power are reliable. The
CO2 permeability is
JCO2
divided by the initial transmembrane
CO2 concentration gradient. We
assumed that the extracellular solution was equilibrated with 1.5%
CO2, that the intracellular
solution was equilibrated with 0.03%
CO2, and that the
CO2 solubility coefficient was
0.0364 mM/mmHg (9).
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DISCUSSION |
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The oocyte is a convenient system for studying membrane
CO2 permeability. Our approach was
to measure the rate of intracellular acidification produced by exposing
an oocyte to a solution, at constant pH, containing 1.5%
CO2. As the
CO2 enters the cell, it slowly
combines with H2O to form
H2CO3.
The
H2CO3
then rapidly dissociates to yield HCO3
and the proton that we detect. CA catalyzes
CO2 + OH
HCO
3, which has the effect of
bypassing the slow step in the above reaction sequence. Because the
oocyte is devoid of both native CA and
HCO
3 transporters (22), the initial
rate at which pHi falls in the
presence of a
CO2/HCO
3
solution is proportional to rate at which
CO2 enters the cell.
In agreement with a previous study by Nakhoul et al. (15), we observed
that expression of AQP1, by itself, had no effect on the rate of
CO2-induced acidification. Those
authors were able to unmask an AQP1-dependent increase in
CO2 permeability by injecting CA
protein into oocytes (15). However, this result left open the
possibility that AQP1 acted by stimulating the injected CA. In the
present study, we did not inject the oocytes with CA, and thus the
oocytes had negligible CA activity. We found, instead, that we could
unmask the effect of AQP1 by stripping away the vitelline membrane.
Thus our data rule out the possibility that, in the experiments of
Nakhoul et al. (15), AQP1 acted by stimulating injected CA. Our data
also suggest that there are two rate-limiting steps in the
intracellular acidification produced by exposing an oocyte to
CO2 as follows:
1) the flux of
CO2 across the vitelline membrane
and 2) the conversion of
intracellular
H2CO3
to HCO3 and
H+.
One of our goals was to determine whether the effect of AQP1 was specifically related to AQP1 or merely a consequence of inserting large numbers of any channel into the cell membrane. We found that expressing the K+ channel ROMK1 produced no alteration in CO2 permeability, indicating that there is at least some specificity for the class of channel expressed in the cell membrane.
To address further the issue of whether CO2 passes through AQP1, we examined the effect of the mercury derivative PCMBS, which does not permeate cell membranes (26), on the CO2-induced acidification. PCMBS produced a small reduction in the rate of CO2-induced acidification in control oocytes, an effect that might be explained in the following two ways: 1) PCMBS interacts with the membrane lipids, decreasing the permeability of this pathway to CO2, or 2) the Xenopus oocyte contains a native gas channel that is partially sensitive to mercurials. PCMBS also decreased the CO2-induced acidification rate in AQP1-expressing oocytes, but to a much greater extent (3.5-fold) than in control oocytes.
The above results are consistent with the hypothesis that CO2 can pass through AQP1 but do not rule out the possibility that AQP1 is somehow increasing CO2 permeability by either altering the composition of the membrane lipids or inducing a native CO2 pathway that is sensitive to PCMBS. To rule out these two options, we expressed the mercury-insensitive mutant of AQP1, C189S. This mutant increased the H2O and CO2 permeability to the same extent as the wild-type channel (Fig. 3A). However, neither the C189S-dependent H2O permeability nor the C189S-dependent CO2 permeability was sensitive to PCMBS (Fig. 3, B-D). Thus, unless the C189S mutation not only knocks out the ability of mercury to inhibit the H2O permeability of AQP1 but also knocks out AQP1's ability to alter membrane lipid composition and/or recruit native gas channels, we can conclude that CO2 does indeed pass through AQP1. These data, complemented by those in the earlier paper by Nakhoul (15), provide the first evidence for a gas crossing a membrane by a route other than the membrane lipid.
The conclusion that CO2 can pass
through the AQP1 channel raises the following two questions.
1) Compared with
H2O, do significant amounts of
CO2 pass through the channel?
2) Are movements of
CO2 through AQP1 physiologically
significant? To answer the first question, we have estimated the
AQP1-dependent CO2 permeability of
the oocyte and found it to be 18.4 × 104
cm · s
1
(see Determination of
CO2
permeability). The AQP1-dependent osmotic H2O permeability of oocytes is 129 × 10
4
cm · s
1
(20). Thus the
CO2-to-H2O
permeability ratio is ~0.14. This figure is orders of magnitude
larger than for any other solute suggested to pass through an AQP-type
channel. For example, in AQP1 the
H2O-to-solute permeability ratio is ~0.0008 for glycerol and ~0.0001 for urea (1). Thus AQP1 is far more permeable to CO2 than to any other molecule
aside from H2O.
The second question must still be answered. We would expect AQP1-dependent CO2 permeability to be most important in cells with a low intrinsic CO2 permeability, those with high levels of AQP1 expression, and/or cells that mediate very high CO2 fluxes. Indeed, AQP1 is present at high levels in tissues or cells with high gas-transport rates: pulmonary capillary endothelium (17), renal proximal tubule (4), choroid plexus, placenta (10), and erythrocytes (19). It is intriguing to speculate that AQP1 homologues with no known function, such as major intrinsic protein, which is the major membrane protein in the lens of the eye (3), or Nodulin-26, which is similarly abundant in the membranes of legume root nodules (5), may be gas channels.
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ACKNOWLEDGEMENTS |
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We thank Dr. Paul De Weer for suggesting to us that a channel might be permeable to CO2. We also are extremely grateful to Dr. P. Agre for the gift of the cDNA encoding AQP1 and the C189S mutant. We also thank Drs. B. A. Davis and M. F. Romero for help in generating the cRNA for AQP1 and C189S, Drs. G. Giebisch and C. M. McNicholas for providing the cRNA for ROMK1, and E. M. Hogan for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Neurological and Communicative Disorders and Stroke Grant NS-18400 (to W. F. Boron) and by the Human Frontiers Scientific Program Organization (to G. J. Cooper).
Portions of this work have been published in preliminary form (3a).
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. §1734 solely to indicate this fact.
1 In all three groups of oocytes treated with PCMBS (i.e., H2O-injected oocytes, oocytes expressing AQP1, and oocytes expressing the C189S mutant), a 15-min exposure to PCMBS had no significant effect on the pHi prevailing just before application of CO2. However a 15-min exposure to PCMBS produced a significant depolarization in all three groups. The magnitude of this depolarization was the same for each group: 12.9 ± 4.4 mV (n = 6) for H2O-injected oocytes, 17.2 ± 1.7 mV (n = 6) for AQP1-expressing oocytes, and 15.1 ± 3.5 mV (n = 6) for C189S-expressing oocytes (degrees of freedom = 2,17, F = 0.45, P = 0.63).
2 From the rates of CO2-induced acidification in the absence of PCMBS, as well as the regression line describing the relationship between lysis time and acidification rate (Fig. 1B), we were able to predict the lysis time in deionized H2O, if the oocyte had not subsequently been treated with PCMBS. In AQP1-expressing oocytes, this computed control lysis time was more than threefold greater than the actual lysis time after treatment with PCMBS. However, in oocytes expressing the C189S mutant, the computed control and actual PCMBS lysis times were identical.
Address for reprint requests: G. J. Cooper, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.
Received 8 June 1998; accepted in final form 31 August 1998.
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