RAPID COMMUNICATION
Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes

Nazih L. Nakhoul, Bruce A. Davis, Michael F. Romero, and Walter F. Boron

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB> 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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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/HCO<SUP>−</SUP><SUB>3</SUB> solutions contained 10 mM HCO<SUP>−</SUP><SUB>3</SUB> (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 beta -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 MOmega 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/HCO<SUP>−</SUP><SUB>3</SUB>. Statistical significance was judged from Student's t-tests. Differences between means were considered not significant (NS) if the P value was >0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB>. As the CO2 diffuses into the cell, it undergoes the following series of reactions
CO<SUB>2</SUB> + H<SUB>2</SUB>O → H<SUB>2</SUB>CO<SUB>3</SUB> → H<SUP>+</SUP> + HCO<SUP>−</SUP><SUB>3</SUB>
The first reaction in this sequence (CO2 hydration) is very slow. The enzyme CA, which is present in many biological systems, substantially speeds the overall conversion of CO2 to HCO<SUP>−</SUP><SUB>3</SUB>. If the hydration of CO2 is not rate limiting, we would expect that greater influxes of CO2 would produce higher rates of H+ production, which we would measure as faster pHi decreases.

Figure 1 illustrates the effects of CO2/HCO<SUP>−</SUP><SUB>3</SUB> on two oocytes, one previously injected with cRNA encoding AQP1 and the other injected with water. Neither had been injected with CA. Applying CO2/HCO<SUP>−</SUP><SUB>3</SUB> causes a slow and sustained decrease in pHi in both the control oocyte (Fig. 1A) and the one injected with AQP1 cRNA (Fig. 1B). The CO2/HCO<SUP>−</SUP><SUB>3</SUB> also causes modest, slowly developing depolarizations (not shown). These effects are completely reversed on removal of the CO2/HCO<SUP>−</SUP><SUB>3</SUB>. Figure 1C, which summarizes a larger group of experiments, shows that the mean initial rate of CO2-induced acidification was 11.3 × 10-4 pH units/s for water-injected controls and was 13.3 × 10-4 pH units/s with expression of AQP1 (NS). The mean control rate of alkalinization elicited by CO2/HCO<SUP>−</SUP><SUB>3</SUB> removal, 7.4 × 10-4 pH units/s, was substantially less than the corresponding rate of acidification in control oocytes. However, it too did not change significantly with AQP1 expression (mean value, 6.2 × 10-4 pH units/s; NS). Finally, the magnitude of the CO2/HCO<SUP>−</SUP><SUB>3</SUB>-induced pHi decrease was unaffected by AQP1 expression (see legend to Fig. 1).


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Fig. 1.   Effect of aquaporin-1 (AQP1) expression on CO2-induced acidification in oocytes without added carbonic anhydrase. A: water-injected oocyte. During indicated interval, the oocyte was exposed to an extracellular solution buffered with 1.5% CO2 /10 mM HCO<SUP>−</SUP><SUB>3</SUB> rather than HEPES; extracellular pH was 7.5 throughout. B: AQP1-injected oocyte. Steady-state change in intracellular pH (Delta pHi) caused by applying CO2/HCO<SUP>−</SUP><SUB>3</SUB> was 0.233 ± 0.009 (n = 22) for water-injected oocytes and 0.241 ± 0.016 (n = 15) for oocytes injected with AQP1 cRNA (not significant). C: data summary. For each pair of bars, solid bar represents mean rate of pHi decrease elicited by applying CO2/HCO<SUP>−</SUP><SUB>3</SUB>, whereas open bar represents mean rate of pHi increase caused by removing CO2/HCO<SUP>−</SUP><SUB>3</SUB>. Numbers in parentheses are numbers of observations (means ± SE).

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/HCO<SUP>−</SUP><SUB>3</SUB> 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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Fig. 2.   Effect of AQP1 expression on CO2-induced acidification in oocytes subsequently injected with carbonic anhydrase (CA). A: oocytes injected with water and then CA. During the 3 indicated intervals, the oocyte was exposed to an extracellular solution buffered with 1.5% CO2/10 mM HCO<SUP>−</SUP><SUB>3</SUB> (extracellular pH = 7.5). Inset: CO2-induced acidifications for 2nd and 3rd CO2/HCO<SUP>−</SUP><SUB>3</SUB> pulses; accompanying numbers are rates of pHi decrease in pH units/s × 10-4. Mean pHi decrease caused by CO2/HCO<SUP>−</SUP><SUB>3</SUB> in 36 water/CA oocytes, including those not treated with ethoxzolamide (ETX), was 0.284 ± 0.008. B: oocytes injected with AQP1 cRNA and then CA. Protocol was same as in A. Mean pHi decrease caused by CO2/HCO<SUP>−</SUP><SUB>3</SUB> in 25 AQP1/CA oocytes, including those not treated with ETX, was 0.315 + 0.009. C: data summary. Effect of AQP1 expression on initial rates of acidification caused by CO2 application (solid bars) and CO2 withdrawal (open bars). First pair of bars reproduces data summarized in Fig. 1C for oocytes injected with water and AQP1 cRNA, but not CA. Second pair of bars summarizes data for oocytes injected with water and then CA and for those injected with AQP1 cRNA and then CA (means ± SE). D: data summary. Effect of ETX on initial rates of acidification caused by CO2 application in paired experiments. Solid bars, acidification rates in absence of inhibitor; cross-hatched bars, in presence of 10 µM ETX. First pair of bars summarizes data for oocytes injected with water and then CA; 2nd pair of bars, for oocytes injected with AQP1 cRNA and then CA (means ± SE).

Figure 2B shows an experiment similar to the one in Fig. 2A, but on an AQP1/CA oocyte. Here, the uninhibited CO2-induced pHi changes were even faster than in the presence of CA alone. The first two CO2 exposures in Fig. 2B caused pHi to fall at initial rates of 74 and 60 × 10-4 pH units/s. Ethoxzolamide reduced the rate to 17 × 10-4 pH units/s, virtually the same as in the water/CA oocyte after ethoxzolamide in Fig. 2A. From other work, we know that ethoxzolamide has no effect on CO2-induced pHi changes in oocytes that have not been injected either with CA or with cRNA encoding CA (24).

Figure 2C summarizes the mean data for oocytes injected with either water or AQP1 cRNA, followed by CA. For comparison, the first two double bars reproduce the data from Fig. 1C. The mean acidification rate with CO2/HCO<SUP>−</SUP><SUB>3</SUB> application for water/CA oocytes (53 × 10-4 pH units/s) was significantly lower than for AQP1/CA oocytes (75 × 10-4 pH units/s, P approx  0.005). Similarly, the mean alkalinization rate with CO2/HCO<SUP>−</SUP><SUB>3</SUB> withdrawal for water/CA oocytes (26 × 10-4 pH units/s) was significantly lower than for AQP1/CA oocytes (36 × 10-4 pH units/s, P < 0.05). Thus expression of AQP1 significantly increased the rates of pHi changes caused by CO2 application and withdrawal.

Figure 2D summarizes paired data for the CO2-induced acidifications in ethoxzolamide experiments. The drug reduced the mean acidification rate from 44 to 16.5 × 10-4 pH units/s (P = 0.005) in water/CA oocytes (Fig. 2A) and from 60 to 14.7 × 10-4 pH units/s (P < 0.01) in AQP1/CA (Fig. 2B). Thus AQP1 significantly increased CO2 permeability in CA-injected oocytes, and the effect was reversed by ethoxzolamide.

    DISCUSSION
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Methods
Results
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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 HCO<SUP>−</SUP><SUB>3</SUB> 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 × 10-3 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

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.

    FOOTNOTES

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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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 274(2):C543-C548
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