1 Department of Woman and Child Health, Karolinska Institute, Astrid Lindgren Children's Hospital, S-171 76 Stockholm, Sweden; and 2 Laboratory of Cytology and Genetics, Institute of Cytology and Genetics, and 3 Group of Functional Genomics, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia
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ABSTRACT |
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Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (Pf) was measured in individual LLC-PK1 cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH2 terminus with green fluorescent protein. Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased Pf of cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the Pf in transfected cells. The effect was abolished by the PKC inhibitor Ro 31-8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser180, a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser180 and that the effect is likely mediated by gating of the channel.
water channels; protein kinase C phosphorylation; LLC-PK1 cells; green fluorescent protein; water transport
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INTRODUCTION |
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CELLS THAT REQUIRE A HIGH water permeability, such as kidney epithelial cells, express specific water channels, aquaporins (AQPs; recently reviewed in Refs. 3 and 23). Because regulation of cell and total body water content is an essential homeostatic function, the question has been raised of whether the activity of AQPs is dynamically regulated by G protein-coupled receptors and intracellular messengers. Short-term regulation of the activity of AQPs by G protein-coupled receptors has until now mostly been studied for aquaporin-2 (AQP2) (18), the water channel that is expressed in kidney collecting duct and that is regulated by vasopressin (AVP) (6, 8, 9, 17, 27, 28, 40, 45, 48, 50).
Aquaporin-4 (AQP4) (22, 25) is expressed in collecting duct principal cells (13, 14, 55) and is important for concentration of urine (5). It has been shown that AQP4 can be phosphorylated by protein kinase C (PKC) in vitro, and, when expressed in Xenopus laevis oocytes, the water permeability of AQP4 is decreased by PKC activation (21). The physiological significance of these findings has not yet been revealed. Studies of the dynamic regulation of water permeability in well-differentiated mammalian cells have so far been associated with a number of methodological problems. Here we employ a method that allows simultaneous studies of water permeability of renal epithelial cells that do or do not express AQP4 tagged with green fluorescent protein (GFP). By using this method, we show that the water permeability of AQP4 is downregulated by PKC activation and by dopamine. To examine whether the effect of PKC was direct, or mediated via an intermediary protein, studies were also performed with AQP4, where the consensus site for PKC phosphorylation, Ser180, was mutated to alanine.
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METHODS |
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DNA constructs. Constructs encoding the short form of AQP4, AQP4-M23 (64), were used. cDNA encoding the whole AQP4-M23 was amplified by RT-PCR using total mouse brain RNA as a template. The cDNA fragments were subcloned in the frames of pEGFP-N1 and pEGFP-C2 plasmids (Clontech, Palo Alto, CA). The termination codon between AQP4-M23 and GFP in pEGFP-N1 was eliminated by using a U.S.E. Mutagenesis kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Inserts in the constructs expressing COOH- or NH2-terminal GFP-tagged AQP4 fusion protein were sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, UK). Analysis of AQP4 protein structure to predict PKC phosphorylation sites and to estimate their phosphorylation potential was performed using NetPhos 2.0 software (2). The point mutation of Ser180 to alanine (S180A) in NH2-terminal GFP-tagged AQP4 fusion protein was generated using a U.S.E. Mutagenesis kit (Amersham Pharmacia). The mutation and absence of other modifications were confirmed by sequence analysis of the whole insert.
Cell culture. For water permeability measurements, LLC-PK1 cells (European Collection of Cell Cultures, Center for Applied Microbiology & Research, Salisbury, Wiltshire, UK; subpassages 5-16) were grown on coverslips (Bioptechs, Butler, PA) in medium 199 (Sigma-Aldrich Sweden, Tyresö, Sweden) containing 50 U/ml penicillin and 50 µg/ml streptomycin supplemented with 10% FBS and 2 mM L-glutamine. On the second day of culture, the cells were transfected with AQP4 cDNA constructs (see above) using CLONfectin (Clontech) according to the manufacturer's protocol. Experiments were performed at the fourth day of culture, when the cells achieved 80-90% confluence.
In some experiments, LLC-PK1 cells were grown on permeable supports (0.2-µm Anopore membrane; 10-mm tissue culture inserts; Nunc, Roskilde, Denmark). Transfection of the cells was performed on the second day of culture as described above. Localization studies were performed on the fourth day of culture, when the cells reached confluency. In one protocol, rat astrocytes (CTX TNA2, European Collection of Cell Cultures, Center for Applied Microbiology & Research; subpassages 3-11) were used. The astrocytes were grown on coverslips in DMEM (Sigma-Aldrich Sweden) containing 0.5 U/ml penicillin and 50 µg/ml streptomycin supplemented with 10% FBS, 0.11 mg/ml sodium pyruvate, and 2 mM L-glutamine. According to our PCR studies, the astrocytes did not express AQP4. The cells were transfected on the second day of culture as described above. Experiments were performed on the fourth day of culture, when the cells achieved 80-90% confluency.Measurement of water permeability.
The water permeability was measured using a new approach, which
combines a method that we have previously developed (65) with the use of proteins carrying fluorescent tags. In the beginning of
every water permeability measurement, an image showing the distribution
of GFP-tagged proteins was recorded (Fig.
1A). Then, with the specimen
remaining on the stage of the microscope, the cells were loaded with
calcein (Fig. 1B) and the water permeability measurement was
performed (see details below). During the off-line analysis of the
series of the calcein images, the GFP and calcein images were
superimposed (Fig. 1C), which allowed us to identify the
transfected cells and to calculate the water permeability separately
for cells that did and did not express AQP4.
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Analysis of the subcellular distribution of GFP-tagged AQP4
proteins.
Coverslips with transfected cells were mounted in the same chamber as
was used for water permeability measurements (see above) in 300 mosM
PBS. A stack of images with a vertical displacement of 0.4 µm were
recorded. Then, the solution was changed to 300 mosM PBS containing
106 M PDBu, the cells were incubated for 3 min, and a new
stack of images were recorded. The ratio of GFP signal in the plasma
membrane to that in adjacent cytosol was measured using Scion Image
software (Scion, Frederick, MD). The subcellular localization of AQP4
was compared in the same cells before and after treatment with PDBu. Images from the same cell height were taken for the comparison. No
swelling or shrinkage of the cells was observed in response to PDBu.
Data presentation and analysis. Data are presented as means ± SE. Statistical analyses were made by using Student's t-test. A difference of P < 0.05 was considered statistically significant.
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RESULTS |
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Subcellular distribution and water permeability of AQP4 tagged with
GFP at the NH2 or COOH terminus.
The subcellular distribution of AQP4 tagged with GFP was studied using
confocal microscopy. NH2-terminal GFP-tagged AQP4 expressed in the LLC-PK1 cell line was targeted specifically to the
plasma membrane of the cells (Fig.
3A) with
very low signal present in cytoplasm. The XZ projection
image (Fig. 3B) shows that the protein is localized
exclusively in basal and lateral membranes of the cells. COOH-terminal
GFP-tagged AQP4 was also targeted to the basolateral membranes of
LLC-PK1 cells (Fig. 3, E and F) but
was to a fairly large extent present in the cytoplasm of the cells.
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Effect of PDBu on water permeability of LLC-PK1 cells
transfected with AQP4 tagged with GFP at the NH2 terminus.
Further experiments were performed using NH2-terminal
GFP-tagged AQP4, because its subcellular distribution resembled that in
kidney collecting duct cells. The water permeability of
LLC-PK1 cells transfected with NH2-terminal
GFP-tagged AQP4 was significantly decreased in response to the PKC
activator PDBu. No effect of PKC activation was observed in
untransfected cells from the same coverslips (Fig.
4A).
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Water permeability of GFP-tagged AQP4 mutated at Ser180 (GFP-AQP4 S180A). AQP4 amino acid sequence analysis using NetPhos 2.0 software (2) has shown that the water channel has a phosphorylation site for PKC at Ser180. High phosphorylation potential of this site raises a possibility that phosphorylation of Ser180 can mediate effects of PKC activation on the water permeability of AQP4. To test this hypothesis, the Ser180 residue was mutated to alanine. To confirm the mutation and absence of other modifications, the whole insert of GFP-AQP4 S180A construct was sequenced.
The subcellular distribution of GFP-AQP4 S180A protein was similar to that of wild-type NH2-terminal GFP-tagged AQP4 (Fig. 5, A and B). Most of the signal was present in the basolateral plasma membrane, with weak background fluorescence inside the cells. The water permeability of the cells expressing GFP-AQP4 S180A was similar to that of the cells that expressed wild-type GFP-tagged AQP4 (Fig. 5C), indicating that the mutation per se did not change the water channel properties of AQP4. The basal level of water permeability in this and following experiments was somewhat lower than in the previous series. This decrease is related to the change of the lot of CLONfectin used for transfection (see METHODS).
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Effect of dopamine on the water permeability of wild-type and
Ser180-mutated NH2-terminal GFP-tagged AQP4.
In cells that expressed GFP-AQP4, the water permeability was
significantly decreased by dopamine (Fig.
6A). In pilot studies, we have
found that in the concentration used (105 M), dopamine
specifically activates the dopamine receptors in LLC-PK1
cells and has no effect on other catecholamine receptors. There was no
effect of dopamine on the water permeability of untransfected cells.
Dopamine treatment had no effect on localization of GFP-AQP4. There
were no detectable changes in the M/C ratio after dopamine treatment in
cells grown on coverslips (data not shown).
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DISCUSSION |
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Evaluation of the method used for water permeability measurements. Employment of confocal microscopy for the water permeability measurements (65) allows the study of the dynamic regulation of the water channels in differentiated mammalian cells. Because water permeability can be measured in individual cells, transient transfection of the cells with GFP-tagged water channels can be used. Besides saving time, this allows comparison of the effects of experimental maneuvers in cells that express the water channel with those in neighboring untransfected cells.
Previous immunohistochemical studies have shown that in rats, AQP4 is localized on the basolateral membrane of principal cells in kidney collecting ducts (13, 14, 55), ependymal cells in the brain (13, 14, 47, 60), and airway epithelial cells in the lung (13, 31, 46). AQP4 distribution in astrocytes also has a highly polarized pattern (44, 47). In our experiments, AQP4 tagged with GFP at the NH2 terminus was targeted specifically to the plasma membrane of LLC-PK1 cells. COOH-terminal GFP-tagged AQP4 was also targeted to the plasma membranes but to a fairly large extent was present in the cytoplasm of the cells. This indicates that COOH-terminal GFP tagging can affect signals for plasma membrane targeting at the COOH terminus of the AQP4 molecule. The basolateral targeting of AQP4 is not a hindrance for the water permeability studies in the LLC-PK1 cell line. According to our observations, the subconfluent LLC-PK1 cells do not have tight contacts with each other and with the coverslip (Fig. 2). Wide intercellular spaces allow the perfusion solution to reach the basolateral surface of the cells. The transmembrane osmotic water transport is therefore significantly increased when the cells express AQP4 at the basolateral membrane. In different experiments, the increase in water permeability in transfected cells compared with untransfected ranged from 1.5- to 2-fold, which is comparable to the raise of water permeability in LLC-PK1 cells after transfection with AQP1 or AQP2 (11, 29, 62). This relatively modest increase may be attributable to the high intrinsic water permeability of LLC-PK1 cells. In astrocytes, where the basal water permeability is substantially lower than in LLC-PK1 cells, the expression of AQP4 increases water permeability more than fivefold. We have observed in ongoing studies that application of first messengers, which activate signaling pathways other than the PLC-PKC pathway, can result in an up to fourfold difference in water permeability of transfected and untransfected LLC-PK1 cells. Taken together, these data indicate that the influence of unstirred layers is minor in our experimental setup. It is the experience of our laboratory that LLC-PK1 cells transiently transfected with integral membrane proteins have a tendency to load with fluorophores (including calcein) to a somewhat lesser degree than neighboring untransfected cells. However, this does not compromise the water permeability measurements, because the change in calcein fluorescence after hyposmotic shock is calculated as relative to the initial fluorescence, and the obtained value does not by definition depend on the initial fluorescence intensity. Original signal-to-noise ratio, which is proportional to the initial fluorescence intensity, is lower in transfected than in untransfected cells (7.9 ± 0.4 and 10.7 ± 0.6, respectively; P < 0.001; n = 74 and 80 cells from 4 coverslips from independent experiments). However, in both groups of cells, the signal-to-noise ratio was high, and this difference did not lead to a higher variability of the data in transfected cells compared with untransfected cells. Loading of the cells with calcein is achieved by incubation with calcein-AM, which, being electrically neutral, freely diffuses into the cells. Once inside the cell, this nonfluorescent substrate is converted by intracellular esterases into a polar membrane-impermeant fluorescent product that is retained by cells. Because neither transfected nor untransfected cells lose the fluorescent signal during the control period before osmotic shock or after cell swelling, we can exclude that the fluorescent esterase product is leaking out of the transfected cells more intensively than out of the untransfected cells.Dynamic regulation of AQP4 in kidney epithelial cells. We show here that the activity of AQP4 can be dynamically regulated in a kidney epithelial cell line. Activation of PKC and dopamine decreased the water permeability of AQP4 but had no obvious effect on trafficking of the water channel. Mutation studies indicated that the downregulation of AQP4 water permeability is mediated by phosphorylation of the protein at Ser180.
Reversible phosphorylation is perhaps the most common way by which the function of proteins can be physiologically altered (7). The activity of many ion channels and pumps has been shown to be regulated this way (4, 53). Phosphorylation may alter the capacity of an integral membrane transporter either by an allosteric change that directly affects its permeability and/or by retrieval/recruitment of the protein from/to the plasma membrane. AQP2 is so far the only water channel in which functional regulation via phosphorylation reactions has been firmly established (6, 17, 26, 28, 36, 48). Phosphorylation of AQP2 influences trafficking but is not believed to have any gating effect (37). The results of the present study suggest that, in contrast to AQP2, AQP4 may be regulated via gating. AQP4 has been shown to be phosphorylated by PKC in vitro (21), but the phosphorylation site was not identified. We analyzed the sequence of AQP4 and found that there is only one PKC site, Ser180, with considerable phosphorylation potential. When this residue was mutated into alanine, the PKC-mediated decrease in the water permeability of AQP4 was abolished. This suggests that the effect of protein kinase C on AQP4 water permeability is not due to phosphorylation of an intermediary protein but rather to direct phosphorylation of AQP4, resulting in an allosteric change of the molecule that leads to decreased water permeability. AQP4 is expressed in principal cells of the collecting duct in the kidney (13, 14, 55). Deletion of AQP4 in mice leads to a defect in maximum urinary concentrating ability in response to water deprivation (5, 38). The water permeability of the basolateral membrane of collecting ducts is considerably higher than that of the apical plasma membrane (12). It has therefore been assumed that the apical membrane is the only rate-limiting barrier for water reabsorption in the collecting duct. However, studies of the AQP4 knockout mouse have indicated that the water channels in the basolateral membrane play a role in the regulation of the water reabsorption in the collecting duct (5). The observations that both in vivo and in vitro exposure of collecting ducts to AVP will cause a significant swelling of the collecting duct cells have also provided evidence for a barrier role of the basolateral membrane (12, 19, 32, 61). The present study shows that PKC activation can directly decrease permeability of AQP4 expressed in the basolateral membranes of kidney epithelial cells. This downregulation could represent a mechanism for repression of antidiuretic effects of AVP in collecting ducts by phorbol esters (1, 20), as well as by cholinergic and nucleotide receptor agonists (20, 33, 49). There is strong evidence that dopamine can also counteract the effect of AVP in collecting ducts (35, 42, 53). Dopamine actions are mediated via two types of receptors, D1 and D2, both of which have been shown to be able to signal via the phospholipase C and PKC pathway (reviewed in Ref. 41). Data from the present study indicate that dopamine will, by PKC-dependent downregulation of AQP4 water permeability, decrease the water permeability of the basolateral membrane of collecting duct epithelial cells. This should lead to a decrease in the efficacy of AVP-stimulated water reabsorption in this segment of nephron. In basolateral membranes of kidney collecting duct, AQP4 is coexpressed with another member of the aquaporin family, aquaporin-3 (AQP3) (10, 24). The relative contribution of these two water channels to the basolateral water permeability of the collecting duct remains to be determined. Recent studies indicate that the water permeability of AQP3 can be downregulated by acidic pH (66). Coexistence of these two water channels with presumably different dynamic regulation in the same membrane would give the principal cells an opportunity to regulate the water reabsorption in various conditions. Many cases of therapy-resistant water retention can be attributable to enhanced water permeability of the apical membrane of the collecting duct cells (34, 51). Further studies of factors that can downregulate AQP4 water permeability in the kidney might lead to new therapeutic strategies for the treatment of this condition. The results from this study may also have important implications for the physiological role of AQP4 in several other tissues. AQP4 is expressed in astrocytes and meningeal and ependymal cells in the brain (13, 14, 44, 47), where it has been proposed to play a role in the regulation of water permeability at the blood-brain barrier, in the regulation of brain interstitial fluid composition, and in osmosensory processes (25, 30, 39, 43, 44, 54, 57-59). It is also expressed in the epithelial cells of the trachea and terminal bronchi (13, 46), where it has been suggested to participate in lung-water clearance (31, 56, 63). In skeletal muscle, AQP4 is expressed in the sarcolemma of fast-twitch fibers and is supposed to be involved in the pathogenesis of muscular dystrophy (15, 16). The results of the present study may imply that activation of the PKC signaling pathway may have a negative effect on lung water clearance and osmotic equilibration in exercising skeletal muscle and may modulate brain water transport. ![]() |
ACKNOWLEDGEMENTS |
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We thank Eivor Zettergren Markus and Louise Gustafsson for expert assistance with cell culture.
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FOOTNOTES |
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This work was supported by Swedish Medical Research Council Grants 03644 and 13114, International Association for the Promotion of Cooperation with Scientists from the New Independent States of the Former Soviet Union Grant 97-11404, the Swedish Heart-Lung Foundation, the Märta and Gunnar V. Philipson Foundation, the Sällskapet Barnvård Foundation, and the Russian Foundation for Basic Research (project nos. 98-04-49369 and 01-04-49390).
Address for reprint requests and other correspondence: A. Aperia, Astrid Lindgren Children's Hospital, Q2:09, S-171 76 Stockholm, Sweden (E-mail: anita.aperia{at}ks.se).
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.
February 19, 2002;10.1152/ajprenal.00260.2001
Received 17 August 2001; accepted in final form 16 February 2002.
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