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J. Biol. Chem., Vol. 277, Issue 43, 40610-40616, October 25, 2002

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Cloning and Functional Characterization of a Novel Aquaporin from Xenopus laevis Oocytes^*

Leila V. Virkki, Christina Franke^§, Petra Somieski^¶, and Walter F. Boron

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

Received for publication, June 20, 2002, and in revised form, August 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have cloned a novel aquaporin (AQP) from Xenopus laevis oocytes, which we have provisionally named AQPxlo. The predicted protein showed highest homology (39-50%) to aquaglyceroporins. Northern blot analysis showed strong hybridization to an ~1.4-kb transcript in X. laevis fat body and oocytes, whereas a weaker signal was obtained in kidney. We injected in vitro transcribed cRNA encoding AQPxlo into Xenopus oocytes for functional characterization. AQPxlo expression increased osmotic water permeability (P_f), as well as the uptake of glycerol and urea. However, AQPxlo excluded larger polyols and thiourea. An alkaline extracellular pH (pH_o) increased P_f and to a lesser extent urea uptake but not glycerol uptake. Remarkably, low HgCl₂ concentrations (0.3-10 µM) reduced P_f and urea uptake, whereas high concentrations (300-1000 µM) reversed the inhibition. We propose that AQPxlo is a new AQP paralogue unknown in mammals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Xenopus laevis oocyte is an important and powerful expression system for the study of foreign mRNAs and translated proteins from animals, plants, fungi, and prokaryotes. Xenopus oocytes are popular because of their large size (diameter 1.0-1.3 mm), robustness, the ease with which one can inject them with mRNA or other substances, and their suitability for a wide variety of experimental manipulations.

Because Xenopus oocytes are extensively used to study the properties of exogenous channels and transporters, an awareness and understanding of endogenous membrane protein expression is of paramount importance. The native oocyte expresses many ion channels (recently reviewed in Ref. 1) and transporters. Moreover, the overexpression of exogenous membrane proteins may change the expression of native oocyte proteins. If these proteins are not present in significant amounts in the control oocyte, it can be difficult to determine which properties can be attributed to a heterologously expressed protein and which are due to a change in the activity of endogenous protein.

Inasmuch as the water permeability of the native oocyte membrane is low, it was long thought that oocytes possess few endogenous water channels (i.e. aquaporins or AQPs).¹ Recently, Schreiber et al. (2) cloned an AQP3 orthologue (xAQP3) from the A6 X. laevis cell line (derived from kidney). They showed that, in oocytes expressing the cystic fibrosis transmembrane conductance regulator (CFTR), stimulation of CFTR activity with 3-isobutyl-1-methylxanthine (IBMX) increased the water and glycerol permeability of the oocyte membrane. They concluded that CFTR regulates the activity of endogenously expressed xAQP3, because IBMX failed to stimulate glycerol permeability in oocytes injected with antisense oligonucleotides complimentary to xAQP3. However, in the absence of exogenous CFTR expression, xAQP3 activity in the oocytes remained undetectable.

We report here the cloning of a novel member of the AQP family from X. laevis oocytes. In addition, we have studied the permeability properties of this novel clone by overexpressing it in its native host, the oocyte.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of AQPxlo

By searching the GenBank^TM data base for sequences with homology to known AQPs, we identified two novel EST sequences (GenBank^TM accession numbers AW200125 and AW199121) from X. laevis oocytes. To obtain the full-length coding region, we performed PCR on an X. laevis oocyte library (X. laevis oocyte 5'-STRETCH cDNA TriplEx library, Clontech Laboratories, Inc., Palo Alto, CA) using primers specific to the coding sequence and to the vector sequences flanking the 5' and 3' ends of the insert. The resulting PCR products were subcloned into a pCR 2.1 TOPO-vector (Invitrogen) and sequenced. We performed end-to-end PCR with primers designed to the 5' and 3' regions flanking the open reading frame to obtain a continuous cDNA fragment containing the entire coding region. The sense primer was 5'-ACTCGAGGCCGCGTCGACTGG and the antisense primer was 5'-TACTAGTGCCAATCAAGAAGCAGATTCCC, where the underlined sequence corresponds to engineered XhoI (sense) and SpeI (antisense) restriction sites. The resulting PCR product was cloned into pCR 2.1 TOPO-vector and sequenced. All sequencing was performed by the Keck Biotechnology Resource Laboratory (Boyer Center for Molecular Medicine, Yale University). The cDNA sequence was deposited into the GenBank^TM (accession number AY120934). We have provisionally named the clone AQPxlo (for Xenopus laevis oocyte).

Cloning of Rat AQP7

For the sake of comparing the functional properties of AQPxlo to those of a related mammalian AQP, we cloned rat AQP7. We performed PCR using sense primer 5'-GTGACCATGGGAATACATG and antisense primer 5'-GGATGCCTATCATGAGGG. As a template we used rat kidney cDNA, which was a kind gift or Dr. Inyeong Choi (Yale University). The PCR product was subcloned into a pCR 2.1 TOPO-vector and sequenced. The cDNA (GenBank^TM accession number AY120935) contained an 810-bp open reading frame encoding 269 amino acids. The deduced amino acid sequence was 97% identical to the published rat AQP7 sequence (GenBank^TM accession number NM_019157).

Northern Blot

Total RNA from oocytes, lung, skin, fat body, muscle, kidney, urinary bladder, stomach, intestine, and liver was extracted from adult female X. laevis using Trizol reagent (Invitrogen), according to manufacturer's directions. Total RNA (10 µg) was resolved by formaldehyde-agarose (1%) denaturing gels and blotted to positively charged nylon membrane (Hybond XL, Amersham Biosciences) by capillary elution. Blots were prehybridized by incubation in ExpressHyb hybridization solution (Clontech Laboratories, Inc) at 65 °C for 30 min and then hybridized with an -³²P-labeled probe (Random Primer labeling kit, Invitrogen) corresponding to the full-length AQPxlo sequence at 65 °C for 90 min. The blots were subjected to a high stringency wash (two 15-min washes at room temperature with 1× SSC, 0.1% SDS and two 30-min washes at 50 °C with 0.1× SSC, 0.1% SDS), after which they were mounted and autoradiographed.

Oocyte Expression

The AQPxlo fragment was excised from pCR 2.1 TOPO using XhoI and SpeI and subcloned into a KSM oocyte expression vector, in which the multiple cloning site is surrounded by the 5'- and 3'-untranslated regions for Xenopus -globin. This expression vector was a kind gift of Dr. William Joiner (Yale University). Rat AQP7 was excised from pCR 2.1 TOPO using XhoI and HindIII and ligated to KSM. Capped cRNA was transcribed in vitro using T3 Message Machine kit (Ambion, Austin, TX).

Stage V-VI oocytes were defolliculated with collagenase type Ia (Sigma) and stored in OR3 medium (Sigma) as described previously (3). On the day after isolation, oocytes were injected with 50 nl of cRNA encoding either AQPxlo (0.4 µg/µl), rat AQP3 (0.2 µg/µl), rat AQP7 (0.2 µg/µl), the rat urea transporter UT-A2 (0.2 µg/µl), or human AQP1 (0.05 µg/µl). Control oocytes were injected only with water. The cDNA encoding AQP1 (in the Xenopus expression vector pXG) was a gift from Dr. Peter Agre (The Johns Hopkins University, Baltimore, MD); AQP3 (in pSPORT) was a gift from Dr. Lawrence Palmer (Cornell University, New York, NY); and UT-A2 (in pBluescript) was a gift from Dr. Craig Smith (University of Manchester, UK).

Measurement of Oocyte Water Permeability

We used a volumetric assay to measure the osmotic water permeability (P_f) of oocytes injected with various cRNAs or water (control). The oocyte was placed in a perfusion chamber and illuminated from below. We acquired images of the oocyte silhouette every 2 s through a video camera attached to a stereomicroscope. The images were saved to a disk and analyzed using Optimas software version 5.2 (Media Cybernetics, Silver Spring, MD). The oocyte volume was calculated from the cross-sectional area of the oocyte, assuming the oocyte to be a perfect sphere. The volume was calibrated using, as an underwater standard, a brass ball bearing placed near the oocyte.

The oocyte was initially superfused with isotonic ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.5, osmolality 200 mOsm) at a solution flow of 4 ml min¹. To induce cell swelling, the perfusate was switched to a hypo-osmotic solution with half the osmolality of ND96, prepared by reducing the concentration of NaCl to 43 mM. Osmolalities were measured using a vapor pressure osmometer (Wescor, Inc. Logan, UT).

To study extracellular pH (pH_o) sensitivity of P_f in oocytes expressing AQPxlo, we preincubated the oocyte for 2 min in an iso-osmotic solution with the appropriate pH (5-11.5) before switching to the hypo-osmotic solution. For solutions with pH 5 or 6, we replaced HEPES with MES. For solutions with pH 7.5 or 8.5 we used HEPES. For solutions with pH 9.5, 10.5, or 11.5, we replaced HEPES with CHES. To avoid precipitation, we omitted MgCl₂ and CaCl₂ from the pH 11.5 solutions and increased the amount of NaCl to maintain osmolality.

To study the effect of HgCl₂ on P_f of oocytes expressing AQPxlo, we preincubated the oocytes for 5 min in ND96 solution containing the appropriate concentration of HgCl₂ (0.1-1000 µM) before switching to hypo-osmotic solution containing the same HgCl₂ concentration. We studied the reversibility of the HgCl₂ treatment by sequentially subjecting the same oocyte to cell swelling three times as follows: (i) without HgCl₂, (ii) with 1 µM HgCl₂, and (iii) without HgCl₂ but after a 5-min incubation period with -mercaptoethanol (5 mM).

The osmotic water permeability was calculated using Equation 1 (4, 5), where (d(V/V_o)/dt) is the initial rate of increase in relative oocyte volume following a hypo-osmotic challenge; V_o is the initial oocyte volume; S is the actual surface area; _osm is the osmotic gradient across the oocyte membrane, and V_w is the molar volume of water.

(Eq. 1)

S of the oocyte was obtained by multiplying the geometric surface area (calculated from the measured volume of each oocyte) by 8, as it has been shown that the actual surface area, accounting for the numerous infoldings and microvilli of the plasmalemma, is 8-9 times larger than the area of a sphere of equivalent size (5).

Measurement of Osmolyte Permeation in Oocyte

Radioisotope Uptake-- We measured unidirectional influx of urea and glycerol in oocytes using ¹⁴C-labeled urea ([¹⁴C]urea) and glycerol ([U-¹⁴C]glycerol; Moravek Biochemicals, Brea, CA). Four oocytes were placed in a 1.5-ml microcentrifuge tube in ND96 solution. Measurement of isotope uptake was started by replacing the ND96 solution with 700 µl of uptake solution, which consisted of ND96 containing 1 mM unlabeled analyte and 1 µCi/ml (37 kBq/ml) of the radioisotopically labeled analyte. Oocytes were incubated on a horizontal shaker for 2 min at room temperature. In preliminary experiments, we monitored the time course of ¹⁴C uptake to ensure that the uptake was linear during the first 2 min. Radioisotope uptake was stopped by washing oocytes three times in ice-cold ND96 containing 10 mM of the unlabeled analyte. Individual oocytes were transferred to scintillation vials and lysed in 400 µl of 10% SDS with continuous shaking. The ¹⁴C activity of individual oocytes was assessed by liquid scintillation counting (LKB-Wallac Rackbeta, Turku, Finland).

We calculated an apparent rate constant k using Equation 2,

(Eq. 2)

where t is the incubation time; A_OOCYTE is the radioactivity accumulated by an oocyte (assuming an oocyte volume of 1 µl) over the time of incubation (t), and A_U is the radioactivity of 1 µl of uptake solution.

When inhibitors were used, the oocytes were pretreated for 5 min in ND96 solution containing the inhibitor before adding the uptake solution (which also contained the inhibitor). Both HgCl₂ (1 and 300 µM) and phloretin (0.5 mM, final concentrations) were prepared fresh daily.

Reflection Coefficient -- The reflection coefficient  is an index of the effectiveness of a solute in generating an osmotic driving force across a semipermeable membrane. For  = 1, the membrane is impermeant to the solute, and for  = 0, the permeability of the solute is indistinguishable from that of the solvent. For a semipermeable solute, 0    1. To determine , we used a variation of the volumetric method (see above) that we used to measure P_f. For each oocyte, we obtained an initial P_f value by subjecting the oocyte to swelling by exposing it to a hypo-osmotic (100 mOsm) ND96 solution. After allowing the oocyte to recover in iso-osmotic ND96 solution for at least 5 min, we superfused the oocyte with a solution in which we iso-osmotically substituted 100 mM of the test solute for NaCl. When   1, the solute will enter the oocyte together with osmotically obliged water, leading to cell swelling. The oocyte was again allowed to recover before superfusing with another solute (see below). For AQPxlo-expressing oocytes we measured  for six different solutes as follows: the polyols glycerol (C-3), xylitol (C-5), ribitol (C-5), and mannitol (C-6), as well as urea and thiourea. Ribitol and xylitol were chosen because in ribitol, each of the -OH groups is on the same side of the carbon backbone, whereas in xylitol the -OH groups have a mixed stereospecific arrangement. In the bacterial glycerol facilitator GlpF, such stereoselectivity plays an important role in determining the permeability properties of the channel (6). As controls, we measured  for glycerol and urea in water-injected oocytes, as well as in oocytes expressing human AQP1 and rat AQP3.

We calculated  according to Equation 3,

(Eq. 3)

where (dV/dt) is the initial rate of increase in oocyte volume following an iso-osmotic challenge with a solute; S is the actual surface area; P_f is the osmotic water permeability (determined for the same oocyte as described above, using Equation 1); is the osmotic gradient for the analyte across the oocyte membrane, and V_w is the molar volume of water.

Measurement of Intracellular pH

An oocyte, placed in a perfusion chamber, was superfused at a rate of 4 ml min¹. pH-sensitive electrodes were fabricated and used as described previously (7, 8). Briefly, the oocyte was impaled with two microelectrodes, one for measuring the membrane potential (V_m) and the other one for measuring intracellular pH (pH_i). The tip of the pH electrode contained a pH-sensitive liquid membrane (Hydrogen Ionophore I, mixture B, Fluka Chemical Corp., Ronkonkoma, NY). Voltages were measured using an FD 223 Electrometer (World Precision Instruments, Inc., Sarasota, FL), and the voltage due to pH_i was obtained by an analogue subtraction of the signals from the V_m and pH electrodes. The pH calibration was performed as described previously (9).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence Data-- The cDNA encoding AQPxlo contains an open reading frame of 894 bp, which encodes 297 amino acids. Fig. 1A shows a sequence alignment with AQPxlo and human AQP3, AQP10, and the bacterial glycerol facilitator GlpF. Fig. 1B shows a dendrogram containing members of all vertebrate AQPs identified to date (AQPs 0-10). We also included xAQP3 and GlpF for comparison. An amino acid sequence-alignment analysis of these AQPs shows that AQPxlo is ~49% identical to mammalian and X. laevis AQP3; ~39-45% identical to mammalian AQPs 7, 9, and 10; and ~30% identical to the bacterial GlpF. Sequence identity to mammalian AQP1 is ~18%. Thus, it appears that AQPxlo is a member of the aquaglyceroporin subgroup of the AQP family.

View larger version (85K): [in this window] [in a new window]   Fig. 1.   Sequence analysis. A, alignment of amino acid sequences of AQPxlo, human AQP3, human AQP10, and the bacterial glycerol facilitator GlpF. Sequences were aligned using the Megalign module (Clustal method) of the Lasergene program suite (DNAstar). Membrane-spanning segments of GlpF, derived form the crystal structure (6), are underlined, and identical amino acids are highlighted. B, dendrogram of AQPs. We used Megalign (Clustal method) to align the amino acid sequences of AQPxlo, mouse AQP 0-5, 7, and 8, rat AQP 6 and 9, human AQP10, Xenopus AQP3, and GlpF. % divergence is indicated by the summed horizontal lengths of line segments between labels. C, hydropathy analysis of AQPxlo. Hydropathy analysis (Kyte-Doolittle algorithm, window size 9) is consistent with six membrane-spanning regions (numbered 1-6) and five connecting loops (A-E).

Fig. 1C shows a hydropathy plot for the deduced amino acid sequence of AQPxlo. The plot is similar to that of other members of the AQP family and is consistent with six transmembrane segments and five connecting loops. Loops B and E contain the aquaporin family signature motif, the amino acid sequence Asn-Pro-Ala. Loop B of AQPxlo contains a consensus protein kinase A/G phosphorylation motif (KKLS) at positions 94-97. Ser⁹⁷ is also part of a consensus protein kinase C phosphorylation motif (SWK) at residues 97-99. Loop C contains a consensus N-glycosylation site at position 134.

Northern Analysis-- Fig. 2 shows a Northern blot analysis of total RNA from multiple tissues from Xenopus. A strong ~1.4-kb signal is present in oocytes and fat body, and a somewhat weaker signal is seen in kidney. We detected no signal in lung, skin, skeletal muscle, urinary bladder, stomach, intestine, or liver.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis. X. laevis total RNA was probed with a 32P-labeled cDNA probe corresponding to the full coding region of AQPxlo. The RNA was run on two separate denaturing agarose gels, and the autoradiographs were apposed for display. The positions and sizes of molecular weight markers are indicated on the left. The molecular weight of the single band in fat body, oocyte, and kidney lanes is ~1.4 kb.

Water Permeability-- Fig. 3 shows the osmotic water permeabilities of control oocytes and of oocytes expressing human AQP1, rat AQP3, rat AQP7, and AQPxlo. For each clone, we injected the minimum amount of cRNA/oocyte required to produce a near-maximum P_f. In oocytes expressing the archetypal water channel AQP1, P_f was 30-fold higher than in water-injected oocytes, whereas in oocytes expressing the aquaglyceroporins AQP3 and AQP7, P_f was 15- and 4-fold higher, respectively. The P_f of AQPxlo-expressing oocytes increased 6-fold, compared with controls. Thus, AQPxlo appears to function as a water channel when overexpressed in the oocyte. Compared with oocytes expressing AQP1, the water permeability of oocytes expressing AQPxlo is low. However, because we have no information about the actual number of functional channels at the membrane, our data do not permit a direct comparison of the water permeabilities of the different AQPs.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Pf values of oocytes expressing different AQPs. Oocytes were injected with water or cRNA encoding AQP1 (2.5 ng/oocyte), AQP3 (10 ng/oocyte), AQP7 (10 ng/oocyte), or AQPxlo (20 ng/oocyte). Error bars, S.E., n = 5-7. Asterisk indicates that the difference is statistically significant (p < 0.05) compared with water-injected oocytes in a two-tailed t test.

Effect of pH_o Changes on Water Permeability-- Zeuthen and Klærke (10) have shown that the P_f and the glycerol permeability of AQP3 are virtually 0 at extracellular pH (pH_o) values below 6 and rise to maximum values at pH_o 6.5-7.0. Fig. 4 shows how changes in pH_o affect P_f in control oocytes and in oocytes expressing AQPxlo. As pH_o increases from 5.0, the P_f of AQPxlo-expressing oocytes increases markedly. Maximal P_f is observed at pH_o 9.5, which is 4-fold higher than the value at pH 7.5 and 8-fold higher than at 5.0. On the other hand, increasing pH_o from 9.5 to 10.5 or 11.5 caused small decreases in P_f. In water-injected oocytes, increasing pH_o from 5.0 to 11.5 did not have a statistically significant effect on P_f.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effect of pHo changes on Pf and pHi of AQPxlo-expressing oocytes. A, Pf. Oocytes were preincubated for 2 min at the appropriate pHo before switching to a hypotonic solution with the same pH. Closed circles, AQPxlo-expressing oocytes; closed diamonds, water-injected oocytes. n = 5 (H2O) and 8-14 (AQPxlo). B, pHi. Microelectrodes were used to measure pHi and Vm of oocytes exposed to solutions of different pHo values. Similar recordings were obtained in one other AQPxlo-expressing oocyte and two water-injected oocytes. A, asterisk indicates that the difference is statistically significant (p < 0.05) compared with the value obtained at pHo 7.5 in a two-tailed t test. Vertical bars indicate S.E. but are omitted when they are smaller than the symbol.

In order to determine whether shifts in pH_i may have played a role in the P_f changes shown in Fig. 4A, we measured pH_i and V_m of water-injected and AQPxlo-expressing oocytes exposed to ND96 at different pH_o values. Changing pH_o from 7.5 to 5.0, 9.5, or 10.5 resulted in changes in pH_i of less than 0.05 pH units (Fig. 4B). Lowering pH_o from 7.5 to 5.0 caused the oocyte to depolarize reversibly by ~15 mV, whereas raising pH_o from 7.5 to either 9.5 or 10.5 caused the oocyte to hyperpolarize reversibly by 10-15 mV. In contrast to raising pH_o to only 9.5 or 10.5, raising pH_o to 11.5 (Ca²⁺- and Mg²⁺-free) caused a large and rapid intracellular alkalinization and an irreversible depolarization. Our observation that oocyte pH_i is remarkably resistant to pH_o changes over the range from 5.0 to 10.5 implies that, over this range, the effects on the P_f of AQPxlo-expressing oocytes are mediated by extracellular protons.

We also examined P_f at pH_o values of 7.5 and 9.5 in oocytes expressing rAQP3 and rAQP7. As summarized in Fig. 5A, P_f is significantly increased at alkaline pH_o in AQPxlo-expressing oocytes but not in rAQP3- or rAQP7-expressing oocytes. In this batch of oocytes, the P_f of water-injected oocytes is significantly increased at alkaline pH_o, suggesting that they may have had a higher level of endogenous AQPxlo expression than those in Fig. 4.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of pHo changes on Pf, glycerol, and urea uptake in oocytes expressing different AQPs. A, Pf. B, glycerol uptake. C, urea uptake. Measurements were carried out on oocytes injected with water or expressing AQP3, AQP7, or AQPxlo. Dark gray bars, pHo 7.5; light gray bars, pHo 9.5. n = 7-12. Error bars, S.E. Asterisk indicates that the difference between control and AQP-expressing oocytes, at the relevant pHo, is statistically significant (p < 0.05). Dagger indicates that the difference between pHo 7.5 and 9.5, comparing oocytes expressing the same AQP, is statistically significant in a two-tailed t test.

Effect of pH_o Changes on Glycerol and Urea Uptake-- Because our sequence analysis placed AQPxlo among the aquaglyceroporins, we measured the uptake of glycerol and urea into water-injected oocytes, as well as oocytes expressing rAQP3, rAQP7, or AQPxlo. As shown in Fig. 5B, all of the AQPs tested substantially increased glycerol uptake (6-7-fold compared with water-injected oocytes). By contrast, Fig. 5C shows that only AQPxlo produced a substantial increase in urea uptake (11-fold). This compares to a 2.6-fold increase in urea uptake seen in rAQP3-expressing oocytes. In contrast to previous reports (11), we did not observe an increase in the rate of urea uptake in rAQP7-expressing oocytes.

Because raising pH_o from 7.5 to 9.5 substantially increased P_f in AQPxlo-expressing oocytes, we examined the effect of this pH_o on glycerol and urea uptake. As shown in Fig. 5B, increasing pH_o from 7.5 to 9.5 did not affect the rate of glycerol uptake in any of the oocytes expressing an AQP. Curiously, only the water-injected oocytes showed a significant increase in glycerol uptake at alkaline pH_o, perhaps reflecting the presence of an endogenous pH-sensitive glycerol transport pathway other than AQPxlo.

In oocytes expressing AQPxlo, the effect on urea uptake of raising pH_o seemed to be intermediate between that on P_f (2.7-fold; see Fig. 5A) and on glycerol uptake (no effect; see Fig. 5B). As shown in Fig. 5C, raising pH_o from 7.5 to 9.5 caused a significant increase in urea uptake (1.5-fold) in oocytes expressing AQPxlo. Raising pH_o failed to increase urea uptake in any of the other groups of oocytes tested.

Effect of HgCl₂ on Water Permeability-- Fig. 6 shows how HgCl₂ affects the P_f of water-injected oocytes and oocytes expressing hAQP1 or AQPxlo. For AQPxlo-expressing oocytes the interaction with HgCl₂ appears biphasic. At low concentrations of HgCl₂ (0.3-10 µM) the P_f is low, whereas at high HgCl₂ concentrations (300-1000 µM), P_f is similar to the value obtained in the absence of HgCl₂. In water-injected oocytes, 1 µM HgCl₂ produced a small but statistically significant inhibition of P_f. In contrast, the effects of HgCl₂ on the water permeability of AQP1-expressing oocytes are monophasic. Correcting for the background P_f in water-injected oocytes, 0.3 µM HgCl₂ reduced the AQPxlo-dependent P_f by 78%, and 300 µM HgCl₂ reduced the AQP1-dependent P_f by 82%.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of HgCl2 on Pf. Oocytes injected with water (closed diamonds) or expressing either AQP1 (open squares) or AQPxlo (closed circles) were treated with the indicated concentrations of HgCl2. Data are expressed as % of control (i.e. absence of HgCl2, indicated by a dotted line). n = 6-9 (H2O), 4-6 (AQP1), and 5-12 (AQPxlo). Error bars, S.E. Asterisk indicates that the difference between (un-normalized) values in HgCl2 and controls is statistically significant (p < 0.05) in a two-tailed t test.

To investigate whether HgCl₂ has time-dependent effects on P_f, we added 1 or 300 µM HgCl₂ and then measured P_f after 2, 5, and 20 min. The time of preincubation did not affect P_f at either concentration of HgCl₂ (not shown).

Because alkaline pH_o increases the P_f of AQPxlo-expressing oocytes (Fig. 4), we investigated whether HgCl₂ at low concentration (1 µM) still blocks P_f when pH_o is increased from 7.5 to 9.5. Because HgCl₂ may act as a Cl/OH ionophore (12), we decided not to examine P_f at different pH_o values in the presence of HgCl₂. Instead, we first pretreated AQPxlo-expressing oocytes with 1 µM HgCl₂ for 5 min in iso-osmotic ND96 at pH_o 7.5. We then exposed the oocytes to either (i) a hypo-osmotic solution at pH_o 7.5 with HgCl₂, (ii) a 2-min wash with an iso-osmotic HgCl₂-free solution at pH_o 7.5, followed by the comparable hypo-osmotic solution, or (iii) a 2-min wash with an iso-osmotic HgCl₂-free solution at pH_o 9.5, followed by the comparable hypo-osmotic solution. Fig. 7A shows that, at pH_o 7.5, HgCl₂ reduced P_f by ~70% whether it was present or absent from the hypotonic swelling solution. In contrast, when P_f was measured at pH_o 9.5, HgCl₂ pretreatment reduced P_f by only 43%.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   HgCl2 inhibition of Pf, effect of pHo changes, and reversibility. A, effect of pHo. AQPxlo-expressing oocytes were either untreated or pretreated with 1 µM HgCl2 at pHo 7.5 for 5 min. Subsequently, Pf was measured (i) in untreated oocytes at pHo 7.5 or 9.5 (black bars); (ii) in pretreated oocytes in the continued presence of HgCl2 at pHo 7.5 (light gray bar); or (iii) in pretreated oocytes in the absence of HgCl2 either at pHo 7.5 or 9.5 (dark gray bars). B, reversibility of HgCl2 inhibition. Pf was measured in the same oocyte before HgCl2 treatment, after treatment with HgCl2 (1 µM), and after treatment with -mercaptoethanol (5 mM). Error bars, S.E. Asterisk indicates that the difference between HgCl2-treated and -untreated oocytes is statistically significant (p < 0.05) in a two-tailed t test.

Fig. 7A shows that, in AQPxlo-expressing oocytes, inhibition of P_f by HgCl₂ is not reversed by washing. We therefore determined whether inhibition by HgCl₂ could be reversed using a reducing agent by superfusing an oocyte for 5 min with 5 mM -mercaptoethanol (after first measuring P_f before and after treatment with 1 µM HgCl₂). Fig. 7B shows that -mercaptoethanol treatment completely reversed the inhibition of AQPxlo by HgCl₂.

Effect of Inhibitors on Glycerol and Urea Uptake-- We investigated whether HgCl₂ has a similar biphasic inhibition profile on glycerol and urea uptake as it has on P_f (Fig. 6). Fig. 8, A and B, shows that a low (1 µM) concentration of HgCl₂ reduces both glycerol and urea uptakes of AQPxlo-expressing oocytes. In contrast, a high (300 µM) concentration of HgCl₂ inhibits glycerol uptake but has no effect on urea uptake. Thus, HgCl₂ acts with a different concentration profile on P_f and urea uptake than on glycerol uptake. In water-injected oocytes or rAQP3-expressing oocytes, 300 µM HgCl₂ had no effect on glycerol uptake. In contrast, in water-injected oocytes, urea uptake was reduced by the low (1 µM) concentration of HgCl₂ but was increased by the high (300 µM) HgCl₂ concentration.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Effect of HgCl2 and phloretin on glycerol and urea uptake. A, effect of HgCl2 on glycerol uptake in water-injected oocytes and oocytes expressing AQP3 or AQPxlo. B, effect of HgCl2 on urea uptake in water-injected oocytes and in oocytes expressing AQPxlo. Black bars indicate control condition; light gray bar, 1 µM HgCl2; and dark gray bars, 300 µM HgCl2. C, effect of phloretin on urea uptake in water-injected oocytes and oocytes expressing UT-A2 or AQPxlo. Black bars indicate control condition; gray bars indicate 500 µM phloretin. A and B, error bars, S.E. n = 6-16; C, n = 4-8. Asterisk indicates that the difference between that value and water-injected control is statistically significant (p < 0.05) in a two-tailed t test. Dagger indicates that the difference between that value and no-drug control (black bar) is statistically significant (p < 0.05) in a two-tailed t test.

Of the 11 mammalian AQPs cloned to date, only AQP9 (13) and AQP10 (14) show substantial glycerol and urea permeabilities, raising the possibility that AQPxlo is the amphibian orthologue of one of these. Because phloretin blocks the urea permeabilities of both AQP9 and AQP10, we investigated the effect of phloretin (500 µM) on the urea uptake of AQPxlo. Fig. 8C shows that phloretin strongly inhibits urea uptake in both water-injected oocytes and oocytes expressing the rat urea transporter rUT-A2 but has no effect on AQPxlo-expressing oocytes.

Measurement of Reflection Coefficient -- Fig. 9A shows the reflection coefficient of AQPxlo-expressing oocytes for glycerol, xylitol, ribitol, mannitol, urea, and thiourea. Of these, glycerol is the only solute with a  (0.57 ± 0.09) substantially smaller than 1, indicating that glycerol permeates AQPxlo-expressing oocytes. This result is in good agreement with the isotopic flux data in Fig. 5B. The  obtained for urea in AQPxlo-expressing oocytes (0.95 ± 0.02) is only slightly less than 1, but the difference is statistically significant (p < 0.0089). Neither xylitol, ribitol, mannitol, nor thiourea had  values significantly lower than 1, indicating that they are not able to permeate AQPxlo-expressing oocytes.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Reflection coefficients  for osmolytes. A,  measured for straight chain polyols (glycerol, xylitol, ribitol, and mannitol), urea and thiourea in AQPxlo-expressing oocytes. B,  measured for glycerol and urea in water-injected oocytes (black bars) and oocytes expressing AQP1 (light gray bars) or AQP3 (dark gray bars). A, error bars, S.E. n = 6; B, n = 4-6. Asterisk indicates that the value of  is significantly (p < 0.05) smaller than 1.

Fig. 9B shows  measurements for water-injected oocytes as well as oocytes expressing hAQP1 and rAQP3. rAQP3-expressing oocytes had a  of 0.37 ± 0.01, in agreement with the isotopic flux data in Fig. 5B. Interestingly, water-injected oocytes had a  (0.68 ± 0.18) for glycerol that was similar to that of AQPxlo-expressing oocytes, consistent with the presence of endogenous AQPxlo.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have cloned from Xenopus oocytes a novel AQP that is predominantly expressed in fat body, oocytes, and kidney. AQPxlo is permeable to water, glycerol, and urea but excludes the larger polyols xylitol, ribitol, and mannitol, as well as thiourea. Judging from its strong expression in fat body, AQPxlo may play a role in the triglyceride metabolism in this tissue by promoting glycerol transport. The fat body, an organ associated with the anterior testis or ovary, serves as a storage organ for fat and undergoes marked changes in size in many anurans, depending on the season and the nutritional status of the animal.

Sensitivity to Changes in pH_o-- The P_f of AQPxlo falls sharply as one lowers pH_o below 9.5 and more gradually as one raises pH_o above 9.5 (Fig. 4A). Regarding other AQPs, both the P_f and glycerol permeability of AQP3 fall sharply at acidic pH_o values, with a pK of 6.4 for P_f and 6.1 for glycerol (10). According to one report, the P_f of bovine major intrinsic protein (AQP0) is maximal at pH_o 6.5 and falls at both more acidic and more alkaline pH_o values (15).

The biphasic pH_o dependence of P_f in AQPxlo-expressing oocytes is consistent with the idea that AQPxlo has more than one titratable site that influences the permeability pathway for water, and that the sum of effects is positive in its influence from pH 5.0 to 9.5 and negative above pH 9.5. Because oocyte pH_i is remarkably resistant to pH_o changes between pH_o 5.0 and 10.5 (Fig. 4B), it is likely that these sites are located where they are accessible to extracellular protons.

In stark contrast to P_f, which increased 4-fold as we raised pH_o from 7.5 to 9.5, glycerol uptake in AQPxlo-expressing oocytes was unaffected (Fig. 5B). The effect of pH_o on urea uptake in AQPxlo-expressing oocytes falls between that of P_f and glycerol uptake in that increasing pH_o from 7.5 to 9.5 produced a small but significant increase in the rate of urea uptake (Fig. 5C). Our observation that the movement of the small H₂O is very pH_o-sensitive, whereas the uptake of the much larger glycerol is not pH_o-sensitive at all, suggests that pH_o sensitivity does not depend in any simple way on molecular size. Rather, the complex pattern of pH_o sensitivities may reflect interactions between the transported species and specific titratable residues lining the channel.

Sensitivity to HgCl₂-- The effect of HgCl₂ on the P_f (Fig. 6) and urea uptake (Fig. 8B) of AQPxlo-expressing oocytes is novel. A low (1 µM) HgCl₂ concentration reduces both P_f and urea uptake, whereas a higher (300 µM) concentration reverses the inhibition. In contrast, HgCl₂ inhibited glycerol uptake equally well at both low (1 µM) and high (300 µM) HgCl₂ concentrations (Fig. 8A).

The simplest way to interpret the data is to assume that the action of HgCl₂ involves at least two reactive groups. One (a high affinity site) reacts at low concentrations of Hg²⁺, resulting in a marked decrease in the permeability of AQPxlo to H₂O, glycerol, and urea. The other (a low affinity site) reacts only when the HgCl₂ concentration is elevated, and its effect is to reverse only the inhibition of P_f and urea uptake. Presumably the reaction of the first Hg²⁺ partially obstructs the pore of AQPxlo, reducing the transport of all substances. The second Hg²⁺ might then cause a conformational change that relieves the obstruction just enough to let H₂O and urea (but not glycerol) pass. We do not know the nature of the Hg²⁺-reaction sites, but they are likely to be thiol groups of cysteine residues, as is the case for several other AQPs. AQPxlo has six cysteines, at positions 24, 40, 74, 105, 174, and 210. Preston et al. (16) showed that of the four cysteines present in AQP1, Cys¹⁸⁹ mediates the blocking effect of mercurials. AQPxlo has a tyrosine (Tyr²¹²) at the position equivalent to Cys¹⁸⁹ in AQP1 but has a cysteine nearby (Cys²¹⁰).

Interestingly, Yasui et al. (17) showed that HgCl₂ causes an ~10-fold increase in the P_f of oocytes expressing human AQP6, and also induces an ion conductance. The authors showed that the double mutant Cys¹⁵⁵-Cys¹⁹⁰ is unresponsive to stimulation by HgCl₂. AQPxlo has a cysteine (Cys¹⁷⁴) at the position equivalent to Cys¹⁵⁵ in AQP6 (Cys¹⁵² in AQP1), but has the aforementioned Tyr²¹² at the position equivalent to Cys¹⁹⁰ in AQP6 (Cys¹⁸⁹ in AQP1). Thus, of the six cysteine residues in AQPxlo, Cys¹⁷⁴ and Cys²¹⁰ are good candidates for HgCl₂ reaction sites.

Does AQPxlo Contribute to Transport in Native Oocytes?-- Because the water permeability of native Xenopus oocytes is low, it is perhaps surprising that they should contain significant amounts of mRNA encoding an endogenous water channel, as indicated by our Northern blot analysis (Fig. 2). On the other hand, the AQPxlo protein may not be expressed at the cell membrane or at all. Indeed, oocytes contain large amounts of stored mRNA that are not translated into protein until the egg starts developing. Our data indicate that native AQPxlo expression in the oocyte is, at most, low. For example, the P_f of water-injected oocytes showed little (Fig. 5A) or no (Fig. 4) change in response to changes in pH_o. Also, glycerol uptake in water-injected oocytes was not blocked by HgCl₂. On the other hand, HgCl₂ at 1 µM reduces both P_f and urea uptake in water-injected oocytes, consistent with native AQPxlo expression. It is possible that if oocytes express the AQPxlo protein at the cell membrane, then the level of expression might vary among batches of oocytes.

However, native AQPxlo might contribute significantly to transport in oocytes heterologously expressing other membrane proteins. Several groups (18-20) have reported examples in which the heterologous expression of a membrane protein in Xenopus oocytes has led to the up-regulation of native Xenopus membrane proteins. For example, the heterologous overexpression of any of several membrane proteins leads to the appearance of a native nonspecific cation channel in Xenopus oocytes (18). In addition, expression of rBAT or 4F2hc in Xenopus oocytes induces amino acid transport, although the proteins themselves do not possess intrinsic transport activity (19). Instead, they associate with native Xenopus membrane proteins to form a heteromeric amino acid transporter (20).

The up-regulation of endogenous AQPxlo could account, at least in part, for novel transport activity observed in oocytes heterologously expressing two other membrane proteins. First, expressing major intrinsic protein (AQP0) in oocytes leads to an increase in glycerol permeability (21-23), even though major intrinsic protein in its native environment in the lens does not contribute to significant glycerol permeability (24). Second, Schreiber et al. (25) have shown that IBMX triggers an increase in water and glycerol permeability in oocytes expressing CFTR. In principle, the up-regulation of AQPxlo could account for this observation. On the other hand, Schreiber et al. (2) eliminated the IBMX-stimulated water and glycerol permeabilities by injecting antisense oligonucleotides directed against a Xenopus AQP3 and concluded that CFTR up-regulates endogenous xAQP3. We were unable to obtain from oocytes a PCR product corresponding to xAQP3²; however, it is possible that the Xenopus used by Schreiber et al. (2) contained a significant amount of mRNA encoding xAQP3, whereas those used by us did not.

Is AQPxlo a New Aquaporin?-- Our sequence and functional data indicate that AQPxlo is an aquaglyceroporin. Four paralogous aquaglyceroporins are known in mammals, AQPs 3, 7, 9, and 10. The dendrogram in Fig. 1 suggests that AQPxlo is most closely related to AQP3 and AQP10, sharing ~49% amino acid sequence identity with mammalian and Xenopus AQP3 and ~45% identity with AQP10. This level of sequence similarity is less than seen between mammalian AQP2 and AQP5 (~60%), which are recognized as distinct AQP paralogues. Thus, a comparison of deduced amino acid sequences suggests that AQPxlo represents a new paralogous AQP. However, searching the human and mouse genome data bases has thus far failed to reveal a mammalian orthologue of AQPxlo.

The permeability properties of AQPxlo are unique. Unlike AQP3 and AQP7, AQPxlo is permeable to urea. Unlike AQP9 (26), AQPxlo does not serve as a pathway for mannitol or thiourea and is not sensitive to inhibition by phloretin. Unlike the recently cloned AQP10 (14), which takes up glycerol 7-fold faster than urea, AQPxlo takes up glycerol and urea equally fast (as measured by ¹⁴C uptake). Also unlike AQP10, whose urea uptake is blocked by phloretin, AQPxlo-mediated urea uptake is not inhibited by phloretin. Thus, the conclusion that AQPxlo is a new AQP paralogue is supported by our observation that the permeability properties of AQPxlo are unlike those of any of the known mammalian aquaglyceroporins.

	FOOTNOTES

^* This work was supported in part by the National Institutes of Health and by the Office for Naval Research.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.

Supported by the American Heart Association. To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, P. O. Box 208026, 333 Cedar St., New Haven, CT 06520-8026. Tel.: 203-785-6609; Fax: 203-785-4951; E-mail: leila.virkki@yale.edu.

^§ Present address: Medizinische Hochschule Hannover, Vegetative Physiologie OE 4220, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.

^¶ Present address: Dept. of Physiology, University of Munich, 80336 Munich, Germany.

Published, JBC Papers in Press, August 20, 2002, DOI 10.1074/jbc.M206157200

² L. V. Virkki and G. J. Cooper, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: AQP, aquaporin; CFTR, cystic fibrosis transmembrane conductance regulator; IBMX, isobutyl-1-methylxanthine; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; TOPO, topoisomerase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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