Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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It is generally accepted that gases such as
CO2 cross cell membranes by
dissolving in the membrane lipid. No role for channels or pores in gas
transport has ever been demonstrated. Here we ask whether expression of
the water channel aquaporin-1 (AQP1) enhances the
CO2 permeability of
Xenopus oocytes. We expressed AQP1 in
Xenopus oocytes by injecting AQP1
cRNA, and we assessed CO2
permeability by using microelectrodes to monitor the changes in
intracellular pH (pHi) produced
by adding 1.5% CO2/10 mM
to (or removing it from) the
extracellular solution. Oocytes normally have an undetectably low level
of carbonic anhydrase (CA), which eliminates the
CO2 hydration reaction as a
rate-limiting step. We found that expressing AQP1 (vs. injecting
water) had no measurable effect on the rate of
CO2-induced
pHi changes in such low-CA
oocytes: adding CO2 caused
pHi to fall at a mean initial rate
of 11.3 × 10
4 pH
units/s in control oocytes and 13.3 × 10
4 pH units/s in oocytes
expressing AQP1. When we injected oocytes with water, and a few days
later with CA, the CO2-induced
pHi changes in these water/CA
oocytes were more than fourfold faster than in water-injected oocytes
(acidification rate, 53 × 10
4 pH units/s).
Ethoxzolamide (ETX; 10 µM), a membrane-permeant CA inhibitor, greatly
slowed the pHi changes (16.5 × 10
4 pH
units/s). When we injected oocytes with AQP1 cRNA and then CA, the
CO2-induced
pHi changes in these AQP1/CA
oocytes were ~40% faster than in the water/CA oocytes (75 × 10
4 pH units/s), and ETX
reduced the rates substantially (14.7 × 10
4 pH units/s). Thus, in
the presence of CA, AQP1 expression significantly increases the
CO2 permeability of oocyte
membranes. Possible explanations include
1) AQP1 expression alters the lipid
composition of the cell membrane, 2)
AQP1 expression causes overexpression of a native gas channel,
and/or 3) AQP1 acts as a
channel through which CO2 can
permeate. Even if AQP1 should mediate a
CO2 flux, it would remain to be
determined whether this CO2
movement is quantitatively important.
intracellular pH
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INTRODUCTION |
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A VARIETY OF BIOLOGICALLY IMPORTANT, low-molecular-weight molecules including water, NH3, CO2, and urea were once thought to cross cell membranes by diffusing through the lipid bilayer. However, two lines of evidence have challenged the generality of this principle. First, some cell membranes have a very low permeability to one or more of these substances. For example, apical membranes of renal medullary collecting duct cells have a very low water permeability in the absence of arginine vasopressin. Xenopus oocytes also have a low water permeability. Apical membranes of the renal thick ascending limb (12, 18), gastric glands (28, 38), and colonic crypts (35) all have very low NH3 permeabilities. Finally, apical membranes of gastric glands have a very low permeability to CO2 (38). Thus some membranes have a very low permeability to molecules previously thought to penetrate virtually all membranes. The second line of evidence is that water and urea transport in many cell types is facilitated by specific membrane proteins. For example, the cloning and expression of aquaporin-1 (AQP1) water channels (25, 26) and the UT2 urea transporter (40) demonstrated that routes (other than diffusion through the lipid bilayer) may contribute substantially to the transport of small molecules.
At the present time, seven members of the AQP family, as well as the closely related major intrinsic protein (of the lens fibers of eye), have been identified in vertebrates (9, 11, 16, 17, 22, 25, 29; for reviews, see Refs. 5, 19, 20). The cloning and expression of the cDNAs encoding these proteins have led to more information about their functions and distributions, as well as the mechanisms regulating their activities. Less is known, however, about the permeability of these channels to small molecules other than water, to which the permeability is high (26). Several studies indicate that at least some AQPs have a finite, though very low, permeability to other small molecules. Although AQP1 appears to be impermeable to urea and H+ (3, 41, 42), glycerol, ethylene glycol, and 1,3-propanediol have been reported to permeate AQP1 reconstituted into proteoliposomes (1). A report that expressing AQP1 increases the cation conductance of Xenopus oocytes (39) has not been confirmed (2, 7, 33, 36). AQP3, a water channel present at the basolateral membrane of the collecting duct, is permeable to glycerol and urea (8). However, the permeability of the AQPs to water is far higher than to these other solutes. For example, for AQP3, the water-to-glycerol permeability ratio is ~103 and the water-to-urea ratio is ~104.
The above data on the permeability of AQPs to several small organic molecules raised the possibility that the AQPs may also be permeable to CO2, which has a minimum molecular diameter that is even smaller than that of water. To test this possibility, we expressed AQP1 in Xenopus oocytes and assessed CO2 permeability by using pH-sensitive microelectrodes to measure the initial rate at which the CO2 entry caused a decrease in intracellular pH (pHi). Our results indicate that the overexpression of AQP1 in oocytes significantly increases the apparent permeability to CO2 and are consistent with the possibility that AQP1 may act as a gas channel.
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METHODS |
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Solutions. The
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-buffered ND96 medium contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH 7.5). The
CO2/ solutions contained 10 mM
(NaHCO3 replacing NaCl) and were
equilibrated with 1.5% CO2-98.5%
O2.
Isolation of oocytes. Adult female Xenopus laevis, obtained from Nasco (Fort Atkinson, WI), were kept in Plexiglas tanks containing carbon-filtered water and were fed frog chow twice a week. Oocytes in stage V-VI were harvested according to established procedures (13). Briefly, the frog was anesthetized by immersing in water containing 0.2% tricaine (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO) for 5 min, followed by hypothermia induced by placing the frog over ice. An ~1-cm incision was made in the abdominal wall, and a lobe of the ovary was excised. The wound was closed with a few stitches of 5-0 cat gut in the muscular wall, followed by 3-4 stitches in the skin with 6-0 silk. Frogs, which recovered from the operation in ~45 min, were not used again for 2 mo. The excised piece of ovary containing oocytes was rinsed several times with Ca2+-free ND96 solution until the solution was clear. The tissue was then agitated in ~15 ml of sterile filtered Ca2+-free ND96 solution containing collagenase type I (Sigma) for 30-40 min. Free oocytes were rinsed several times with sterile OR3 medium (GIBCO BRL Leibovitz media containing glutamate, 500 units each of penicillin and streptomycin, with the pH adjusted to 7.5 and osmolarity adjusted to 200-205 mosM), sorted, and then stored at 18°C.
Preparation of cRNA. cDNA encoding
AQP1 was a gift from Dr. Peter Agre. It had been cloned into the
expression plasmid pSP64T, a Bluescript-derived vector containing the
3' and 5' untranslated regions of the X. laevis -globin gene (26). The cDNA was cut to
linearize and was transcribed in vitro with T7 RNA polymerase, using a
Stratagene mCapping kit (Stratagene, La Jolla, CA), and the cRNA was
purified by phenol-chloroform extraction (21). The RNA concentration
was determined by ultraviolet absorbance, and its quality was assessed
by gel electrophoresis (32).
Injection of oocytes. Oocytes isolated the previous day were visualized with a dissecting microscope and were injected with 50 nl of cRNA for AQP1 (0.02 µg/µl, for a total of 1 ng of RNA). Control oocytes were injected with 50 nl of sterile water. In many experiments, 2-4 days after injecting either water or AQP1 cRNA, we injected oocytes with carbonic anhydrase [CA; 50 ng/oocyte of bovine erythroid CA (C-3934, Sigma) contained in 50 nl of water]. Sterile pipettes used for injecting water, cRNA, or CA had tip diameters of 20-30 µm, were backfilled with paraffin oil, and were connected to a Drummond 10-µl positive-displacement pipette. Oocytes were used 3-5 days after injection with cRNA and 1 day after injection of CA. To verify AQP1 expression, we placed oocytes in deionized water and measured the time for the oocytes to burst. Water-injected oocytes or uninjected ("native") oocytes showed few signs of swelling even after 20 min of incubation in deionized water. Oocytes expressing AQP1 burst in <35 s.
Electrophysiological measurements. We
measured pHi and membrane
potential (Vm)
using microelectrodes. The single-barreled pH microelectrodes contained
a liquid membrane H+ cocktail
(cocktail B, 95293; Fluka Chemical, Ronkonkoma, NY) and were prepared
as described previously (31, 34). Briefly, aluminosilicate glass
capillaries (1.5 mm OD, 0.96 mm ID; Frederick Haer, Brunswick, MD) were
pulled on a Brown-Flaming puller (model P80/PC; Sutter Instruments, San
Raphael, CA), dried in an oven at 200°C for >2 h, and vapor
silanized with bis(dimethylamino)-dimethyl silane in a closed vessel.
Exchanger was introduced into the electrode tip. pH electrodes
were backfilled with a buffer solution (4), connected to a
high-impedance electrometer (FD-223; World Precision Instruments,
Sarasota, FL), and the slopes were determined (53 to
59
mV/pH unit) in commercial buffers of pH 6 and 8 (Fisher Scientific,
Pittsburgh, PA). In each experiment, we performed a single-point
calibration in the pH 7.5 ND96 solution.
Vm electrodes,
filled with 3 M KCl and connected to the above electrometer, had
resistances of 1-10 M and tip potentials of <4 mV. Oocytes
were simultaneously impaled with pH and
Vm electrodes. The extracellular reference electrode was a free-flowing calomel half
cell filled with saturated 3 M KCl. The data were sampled approximately
once per second by an analog-to-digital converter interfaced to an IBM
personal computer.
For experiments, oocytes, visualized with a dissecting microscope, were held on a nylon mesh in a special perfusion chamber, through which solutions at 22°C flowed continuously at ~4 ml/min. Solutions were switched using a pneumatically activated five-way valve with minimal dead space (37). Chamber volume was ~300 µl. Electrode tips were placed into the most superficial portion of the oocyte.
Statistics. The initial rate of change
of pHi
(dpHi/dt)
was determined from the slope of a linear regression line fitted to a
portion of the pHi vs. time data
obtained immediately after adding
CO2/.
Statistical significance was judged from Student's
t-tests. Differences between means
were considered not significant (NS) if the
P value was >0.05.
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RESULTS |
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CO2-induced
pHi changes in the absence of injected CA.
We assessed CO2 permeability by
monitoring the decrease in pHi
evoked by switching the extracellular solution from one buffered to pH
7.5 with HEPES (CO2 free) to
another buffered to the same pH with 1.5%
CO2/10 mM
. As the
CO2 diffuses into the cell, it
undergoes the following series of reactions
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CO2-induced
pHi changes in the presence of injected CA.
The low rates of CO2-induced
acidification in the above experiments suggested to us that CA activity
in Xenopus oocytes might be so low
that the CO2 hydration reaction
becomes rate limiting for the
CO2-induced fall in
pHi. Indeed, injecting the CA or
expressing the cRNA encoding CA II increased the rate of
CO2-induced acidification by more
than fourfold (24). To investigate the possibility that low CA activity
may have masked an effect of AQP1 expression on the
CO2-induced acidification rate, we
injected oocytes with water or AQP1 cRNA, and then, 1 day before the
experiment, we injected each oocyte again with CA. Figure
2A shows
the results of an experiment on a water/CA oocyte in which we exposed
an oocyte to
CO2/ three times: twice under control conditions and once in the presence of
the membrane-permeant CA inhibitor ethoxzolamide (10 µM). In the
absence of inhibitor, the acidifications induced by applying CO2 and the alkalinizations
elicited by removing the CO2 were substantially faster than in water/non-CA oocytes (see Fig.
1A). The first two
CO2 exposures in Fig.
2A caused
pHi to fall at initial rates of 54 and 44 × 10
4 pH
units/s. Ethoxzolamide caused the rates of
pHi change to decrease substantially. In Fig. 2A,
pHi fell at the rate of only 18 × 10
4 pH units/s with
the final CO2 application. This
last observation confirms earlier work, performed on oocytes not
preinjected with water, showing that ethoxzolamide largely reverses the
effects of CA injection on
CO2-induced
pHi transients (24). The inset in
Fig. 2A compares the
CO2-induced acidifications for the
final two CO2 pulses.
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DISCUSSION |
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The present study demonstrates that expressing AQP-1 in oocytes enhances CO2 transport across the cell membrane, provided that the oocyte, which lacks significant native CA activity (24), has been injected with CA. Based on the rates of CO2-induced acidification and alkalinization summarized in Fig. 2C, we conclude that expressing AQP1 increases the effective CO2 permeability of the oocyte membrane by ~40%.
Role of CA. The AQP1-dependent
acceleration of CO2 transport, as
evidenced by increases in the rates of
pHi changes produced by these
CO2 fluxes, required the presence
of CA in our experiments. If we omitted the injection of CA (Fig.
1B) or used ethoxzolamide to block
the injected CA (Fig. 2B),
expression of AQP1 had no significant effect on the measured rates of
pHi change. It is likely that AQP1
expression increases the effective permeability of the membrane to
CO2, regardless of the status of
CA. However, in the absence of CA, the
CO2-induced acidification will be
slowed by two factors (24). First, the
H+ that we measure with our pH
microelectrodes will be generated more slowly from the
CO2 that enters the cell. Second,
the slow conversion of CO2 and
water to and
H+ will secondarily lead to an
accumulation of CO2 in the
unstirred layer near the intracellular surface of the cell membrane.
Thus, even though AQP1 increases the
CO2 permeability, the out-to-in CO2 gradient driving
CO2 influx will collapse more
rapidly in the absence of CA.
Advantages of the oocyte preparation. We used Xenopus oocytes in the present study because they provide distinct advantages over other preparations. First, the oocyte is a powerful system for expressing heterologous RNA (for reviews, see Refs. 15 and 30). Injecting cRNA for AQP1 has proven to be very effective for expressing this water channel (27). Second, oocytes tolerate sequential injections of cRNA and other substances (e.g., CA) and still have excellent membrane integrity, as judged by initial membrane-potential values in our experiments. Third, because it is easy to make long-lasting microelectrode impalements, it is possible to monitor pHi with microelectrodes, which are inherently more stable and accurate than dyes. Fourth, because the oocyte is a very large cell, the CO2-induced pHi changes are orders of magnitude slower than in most mammalian (i.e., small) cells, slow enough to be measured accurately under the conditions of the present experiments. In proteoliposomes containing reconstituted AQP1 (14), for example, which have been used to investigate the selectivity of water channels to other solutes, CO2-induced pH changes would be extremely fast.
Permeability properties of the oocyte
membrane. A fifth advantage of the
Xenopus oocyte preparation is that its
cell membrane appears to have a low baseline permeability to small
molecules, including CO2. From the
initial rate of CO2-induced
pHi decrease, as well as the
oocyte's buffering power and the volume-to-surface area ratio, we
estimate that the CO2 permeability
of a control (i.e., water-injected) oocyte containing CA is ~6 × 103 cm/s.
Because expressing AQP1 in the presence of CA increases the
CO2 permeability by ~40%, we
estimate that AQP1 increases the
CO2 permeability by ~2 × 10
3 cm/s under the
conditions of our experiments.
The lipids in the cell membrane of native Xenopus oocytes have a very low permeability to water, a property that is essential for their survival in pond water. However, like the apical membrane of gastric gland cells (37, 38) and the apical membrane of colonic crypt cells (Ref. 35 and S. K. Singh and W. F. Boron, unpublished observations), membranes that face markedly inhospitable environments, the oocyte membrane also has an exceedingly low permeability to H+ (10) and NH3 (S. K. Singh, A. Cimini, and W. F. Boron, unpublished observations). In fact, that the native oocyte membrane has even a modest permeability to CO2 is a major exception to the rule that membranes facing inhospitable environments (deionized or nearly deionized water in the case of a mature oocyte) seem to have a negligible permeability to the small molecules that have been tested: H+, water, CO2, and NH3. Thus it is intriguing to speculate that the oocyte has a small-molecule-impermeable membrane that is rendered permeable to CO2 and O2 by a native gas channel.
Explanations for the enhanced CO2 permeability provided by AQP1. Extremely high levels of expression of a foreign integral membrane protein (e.g., AQP1) could lead to an increase in apparent CO2 permeability of a Xenopus oocyte by at least three mechanisms. First, overexpression of AQP1 could lead to a change in the lipid composition of the cell membrane (e.g., shorter or more unsaturated fatty acid chains), which could in turn increase the CO2 permeability through the lipid portion of the membrane. Second, overexpression of AQP1 could lead to the increased expression of a native gas channel in the oocyte. Finally, AQP1 might itself mediate the movement of CO2 across the cell membrane. It is not unreasonable to postulate a movement of CO2 through the same pore that admits water; the minimum molecular diameter of the linear CO2 molecule is actually less than that of the angled water molecule.
Additional work is required to distinguish among the above three competing hypotheses. If water channels actually permit the passage of CO2, then inhibiting these channels should slow the CO2-induced pHi changes. Preliminary work suggests that p-chloromercuribenzenesulfonic acid (a mercurial inhibitor of AQP1) blocks the AQP1-dependent component of CO2 permeability in AQP1-expressing oocytes (23). If CO2 does indeed permeate the oocyte membrane in part through AQP1, then a question that would still remain is whether the CO2 permeability is a mere biophysical curiosity or whether it has biological significance. However, even if the CO2 permeability of AQP1 were not quantitatively important, it would provide an additional tool to probe the pore of the AQP1 and related membrane channels. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Gordon J. Cooper for helpful scientific discussions, Emilia M. Hogan for excellent technical assistance, and Duncan Wong for extensive help with computing.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Program Project Grant PO1-DK-17433. N. L. Nakhoul was supported by NIDDK Training Grant T32-DK-07259 and American Heart Association (Louisiana Affiliate) Grant-in-Aid LA-96-GS-18. M. F. Romero was supported by the National Kidney Foundation and by NIDDK National Research Service Award DK-09342.
Portions of this work have been published in preliminary form (23).
Present address of M. F. Romero: Dept. of Physiology and Biophysics, Case Western Reserve Univ., Cleveland, OH 44106.
Address for reprint requests: N. L. Nakhoul, Dept. of Medicine, Section of Nephrology, SL45, Tulane Medical School, 1430 Tulane Ave., New Orleans, LA 70112.
Received 24 September 1997; accepted in final form 21 November 1997.
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