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Importance of aquaporin-2 expression levels in genotype -phenotype studies in nephrogenic diabetes insipidus

E. J. Kamsteeg and P. M. T. Deen

Department of Cell Physiology, University of Nijmegen, 6500HB Nijmegen, The Netherlands


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus. Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect (DNE) on wild-type (WT) AQP2 water permeability (Pf) but only at low expression levels. Here, we determined the cell biological basis for this requirement. Injection of different amounts of WT-AQP2 cRNAs revealed that a correlation between AQP2 protein levels and Pf is only obtained with low expression levels. In coexpression studies of WT- and mutant AQP2 proteins, higher expression levels of AQP2-R187C also exerted a DNE on the Pf of WT-AQP2. Immunoblot and immunoprecipitation analysis revealed that this DNE was caused by competitive inhibition of WT-AQP2 expression and escape of AQP2-R187C from the endoplasmic reticulum, resulting in oligomerization with WT-AQP2. Because many disease-related mutants of multimeric renal membrane transporters and channels are likely to be identified, our data provide important information for studying the effects of such mutants on the activity of WT transporters and channels in oocytes.

recessive nephrogenic diabetes insipidus; overexpression; water channel; endoplasmic reticulum; oocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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TO STUDY FUNCTION AND ROUTING of wild-type (WT) and mutant proteins, they are often heterologously expressed in cells derived from organisms as diverse as bacteria, yeast, plants, insects, toads, and mammals. Of these, one of the best-exploited cell systems is the Xenopus laevis oocyte (9). This success is due to the fact that these cells have a diameter of 1 mm, which facilitates injection of cRNAs, they are easily isolated from the toad and can be cultured in simple medium for nearly 2 wk. In addition, these cells are equipped with a translational machinery equivalent to 50,000 somatic cells (2). This has been essential in the functional cloning of many membrane transporters and channels, because these efforts often started with very small amounts of a particular transcript (11, 13). Nowadays, oocytes are mostly used to study the topology, routing, and function of WT and mutant channels and transporters (12, 16, 19, 23, 26, 30).

We have extensively used oocytes to study WT and mutant aquaporin-2 (AQP2) water channels. Aquaporins are of prime importance in water balance, which is a primary function of the kidney. On dehydration, the antidiuretic hormone vasopressin is released from the pituitary and binds to its V2 receptor, which is located in the basolateral membrane of renal collecting duct cells. This binding initiates a signaling cascade, resulting in the redistribution of AQP2 from intracellular vesicles to the apical membrane, rendering these cells water permeable. Consequently, urine is concentrated (4, 20). In nephrogenic diabetes insipidus (NDI), the kidney is unable to concentrate urine in response to vasopressin. In most families, congenital NDI is inherited as an X-chromosomal trait and is caused by mutations in the V2 receptor (22, 27). In some families, NDI segregates as an autosomal recessive or dominant disease, and in both forms AQP2 mutations have been identified (5, 17). Expression of maximal protein levels in oocytes revealed that all AQP2 missense mutants in recessive NDI were retained in the endoplasmic reticulum (ER) and that the small amount that reached the plasma membrane conferred a reduced or no water permeability (Pf) to the oocytes (3, 18, 28). Expression of a mutant encoded in dominant NDI (AQP2-E258K) revealed that this mutant was functional and retained in the Golgi complex (17). The ability of AQP2-E258K, but not a mutant in recessive NDI (AQP2-R187C), to form heterotetramers with WT-AQP2 and to inhibit further routing of WT-AQP2 to the plasma membrane explained the observed dominant-negative phenotype (12). This specific "dominant-negative effect" (i.e., occurring with AQP2-E258K, but not with AQP2-R187C), however, was only observed when low amounts of AQP2 proteins were expressed. In this study, we investigated why low expression levels are necessary to reveal a specific dominant-negative effect of AQP2-E258K. The results have implications for future studies into genetic defects of transporters.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
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Constructs and in vitro transcription. Constructs of the oocyte expression vector pTsT7 containing cDNAs encoding WT-AQP2, AQP2-R187C, AQP2-E258K, FLAG-tagged WT-AQP2-F (WT-AQP2-F), or AQP2-R187C tagged with a vesicular stomatitis virus glycoprotein (VSV-G) peptide (AQP2-R187C-V) were as described (5, 12, 17). The pT7TS-constructs were linearized with Sal4I, and g-capped cRNA transcripts were synthesized in vitro as described (3). The cRNAs were purified and dissolved in diethylpyrocarbonate-treated water. The integrity of the cRNAs was checked by agarose gel electrophoresis, and their concentrations were determined with a spectrophotometer.

Isolation, injection, and Pf measurements of X. laevis oocytes. Oocytes were isolated from X. laevis and defolliculated by digestion at room temperature for 2 h with 2 mg/ml collagenase A (Boehringer Mannheim, Mannheim, Germany) in modified Barth's solution [MBS; (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 10 HEPES (pH = 7.5), 0.82 MgSO4, 0.33 Ca(NO3)2, and 0.41 CaCl2 as well as 25 µg/ml gentamicin]. Stage V and VI oocytes were selected and stored at 18°C in MBS. Oocytes were (co-)injected and 2 days after injection were analyzed in a standard swelling assay (5).

Isolation of total membranes. For the isolation of total membranes, oocytes were homogenized in 20 µl HbA [(in mM) 20 Tris (pH = 7.4), 5 MgCl2, 5 NaH2PO4, 1 EDTA, 80 Sucrose, and 1 phenylmethylsulfonyl fluoride (PMSF) as well as 5 µg/ml leupeptin and pepstatin] per oocyte and centrifuged for 2 × 5 min at 200 g at 4°C. Next, membranes were isolated by 20-min centrifugation at 4°C at 14,000 g and resuspended in 15 µl of Laemmli buffer.

Isolation of plasma membranes. Oocytes were stripped from their vitelline membrane and rotated in 1% colloidal silica, ludox Cl (Sigma-Aldrich, St. Louis, MO) in MES- buffered saline for silica (MBSS; 20 mM MES, 80 mM NaCl, pH = 6.0) for 30 min at room temperature, washed three times in MBSS, rotated in 0.1% polyacrylic acid (Sigma-Aldrich) in MBSS for 30 min at room temperature and washed three times in MBS. Subsequently, 25 oocytes were homogenized in 1,200 µl HbA at 4°C and centrifuged for 30 s at 13.5 g at 4°C, after which 1 ml of the top of the sample was removed and 1 ml of HbA was added. This centrifugation and exchange of HbA were repeated four times, but centrifugation changed from twice at 13.5 g via once at 24 g to once at 38 g. After the last centrifugation step, HbA was removed and plasma membranes were spun down for 20 min at maximum speed at 4°C and resuspended in 15 µl of Laemmli buffer.

Immunoblotting. Protein samples were denatured by incubation for 30 min at 37°C in Laemmli buffer, subjected to electrophoresis on a 12% SDS-polyacrylamide gel, and immunoblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) by standard procedures. Membranes were blocked for 1 h in 5% nonfat-dried milk in TBS-T (TBS with 0.1% Tween 20). Blots were incubated with 1:3,000-diluted affinity-purified rabbit AQP2:257-271 antibodies, raised against the 15 COOH-terminal amino acids of rat AQP2 (3), 1:1,000-diluted chicken alpha -FLAG (Aves Lab, Tigard, OR), or a 1:10,000 mouse alpha -VSV-G in TBS-T supplemented with 1% nonfat-dried milk. Subsequently, blots were incubated with 1:5,000-diluted goat alpha -rabbit IgG (Sigma), 1:3,000-diluted goat alpha -chicken IgY (Aves Lab), or a 1:2,000-diluted sheep alpha -mouse IgG (Sigma), which were all coupled to horseradish peroxydase. The latter two were used in TBS-T supplemented with 1% nonfat-dried milk. Finally, proteins were visualized by using enhanced chemiluminescence (Pierce, Rockford, IL).

Immunocytochemistry. Oocytes were stripped from their vitelline membranes, fixed in 1% wt/vol paraformaldehyde-lysine-periodate (15), dehydrated, and embedded in paraffin. Five-micrometer sections were cut, stretched in 37°C water, dried on gelatin-coated object glass (Menzel Gläser) for at least 1 h at 37°C, deparaffinated with xylol, and rehydrated with a 100, 96, 90, 80, 70, and 50% ethanol series and, finally, water. Oocyte sections were treated with 0.1% Triton X-100, quenched with 50 mM NH4Cl in TBS, blocked for 30 min in 10% goat serum in TBS, and incubated with 1:300-diluted affinity-purified rabbit 7 AQP2:257-271 antibodies for 16 h in TBS with 10% goat serum. Subsequently, the sections were washed three times in TBS and incubated in 1:300-diluted goat alpha -rabbit IgG, coupled to alexa-594 (Molecular Probes) in TBS with 10% goat serum for 1 h. After three TBS washes and dehydration with ethanol, the sections were mounted in vectashield (Vector Laboratories, Burlingame, CA).

Immunoprecipitation of AQP2 proteins with FLAG antibodies. Membranes of 60 oocytes (co-)expressing AQP2 mutants were dissolved in 200 µl solubilization buffer [4% Na-deoxycholate, 20 mM Tris (pH = 8.0), 5 mM EDTA, 10% glycerol, 1 mM PMSF, 5 µg/ml leupeptin and pepstatin A] for 1 h at 37°C and centrifuged at 100,000 g for 1 h at 4°C to remove undissolved membranes as described (12). Fifteen-microliter equivalents of protein G-agarose beads (Pharmacia, Uppsala, Sweden) were preincubated for 12 h at 4°C with 1 µl monoclonal FLAG antibody (m2; Sigma) in IPP500 [500 mM NaCl, 10 mM Tris (pH = 8.0), 0.1% NP-40, 0.1% Tween 20, 1 mM PMSF, and 5 µg/ml of leupeptin and pepstatin A] and 0.1% BSA. The solubilized membranes were diluted with 600 µl 10% sucrose, and NaCl was added to a final concentration of 100 mM. Next, the washed antibody-bound protein G beads were incubated for 4 h at 4°C with the solubilized membranes, washed three times with IPP100 [100 mM NaCl, 10 mM Tris (pH = 8.0), 0.1% NP-40, 0.1% Tween 20, 1 mM PMSF, and 5 µg/ml of leupeptin and pepstatin] and resuspended in 30 µl Laemmli buffer. After denaturation, the coupled proteins were subjected to immunoblotting.

Sedimentation by sucrose gradient centrifugation. Oocyte total membranes were dissolved in solubilization buffer for 1 h at 37°C and freed from undissolved membranes as described above. Sedimentation by gradient centrifugation was done as described (12). Briefly, 5-17.5% sucrose gradients were prepared of 533 µl of 5, 7.5, 10, 12.5, 15, and 17.5% sucrose each in 20 mM Tris (pH = 8.0), 5 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, and 5 µg/ml of leupeptin and pepstatin. Three hundred-microliter samples of solubilized membrane proteins were loaded and subjected to 150,000 g centrifugation for 16 h at 8°C. Two hundred-microliter fractions were taken off carefully, designated A through S, and analyzed by immunoblotting with or without previous immunoprecipitation. As sedimentation markers, a mixture of ovalbumin (43 kDa), bovine serum albumin (67 kDa), phosphorylase B (97 kDa), yeast alcohol dehydrogenase (150 kDa), and catalase (232 kDa) was used. All markers were from Sigma.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Only at low expression levels, AQP2-E258K confers a specific dominant-negative effect on the function of WT-AQP2. To be able to detect a change in WT-AQP2-conferred Pf by interaction with AQP2-E258K, expression levels of WT-AQP2 should be in a linear relation with the Pf. Therefore, a concentration series of WT-AQP2 cRNAs was injected into oocytes. Two days after injection, Pf appeared to increase linearly with expression levels for injections up to 3 ng of cRNA, at which the Pf reached a plateau phase (Fig. 1). Immunoblot analyses of these proper oocytes revealed that, unlike Pf, the total membrane expression still increased with injections above 3 ng (Fig. 1, inset). Therefore, to determine under which condition AQP2-E258K, but not AQP2-R187C, exerted a dominant-negative effect on WT-AQP2, oocytes were injected with 0.3 ng cRNA of FLAG-tagged WT-AQP2 (WT-AQP2-F) together with 0.3, 1, 3, or 10 ng cRNA of VSV-G-tagged AQP2-R187C or AQP2-E258K (AQP2-R187C-V or AQP2-E258K-V, respectively). As reported before (12), the Pf of oocytes coinjected with 0.3 ng cRNAs of WT-AQP2-F and AQP2-E258K-V was reduced compared with oocytes injected with 0.3 ng WT-AQP2-F cRNA alone or together with 0.3 ng AQP2-R187C-V cRNA (Fig. 2A). However, the Pf of oocytes injected with 0.3 ng of WT-AQP2-F cRNA and 1, 3, or 10 ng cRNA of AQP2-R187C-V was decreased compared with the Pf of oocytes injected with 0.3 ng of WT-AQP2-F cRNA alone, thus also showing a dominant-negative effect. In contrast, the Pf of oocytes injected with 0.3 ng of WT-AQP2-F cRNA and 1, 3, or 10 ng of AQP2-E258K-V cRNA was elevated compared with the Pf of oocytes injected with 0.3 ng of both WT-AQP2-F and AQP2-E258K-V and even exceeded the value of oocytes expressing 0.3 ng of WT-AQP2-F alone (Fig. 2A). Coinjections of 10 ng of WT-AQP2-F cRNA with either 10 ng of AQP2-R187C-V cRNA or 10 ng of AQP2-E258K-V cRNA revealed a dominant-negative effect of both mutants on WT-AQP2 (Fig. 2B). These results clearly show that a specific dominant-negative effect of AQP2-E258K is only apparent when low amounts (0.3 ng) of cRNAs encoding WT- and mutant AQP2 are coinjected.


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Fig. 1.   Expression-function relation for wild-type aquaporin-2 (WT-AQP2). Oocytes were injected with 0, 0.1, 0.3, 1, 3, or 10 ng WT-AQP2 cRNA. Two days later, the osmotic water permeability (Pf) of these oocytes was determined in a standard swelling assay. Values are means ± SE of at least 12 oocytes. Inset: of these oocytes, total membranes were isolated and 0.5 oocyte equivalents were immunoblotted for AQP2 (longer exposure times also showed a signal for oocytes injected with 0.3 ng cRNA).



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Fig. 2.   Pf of oocytes (co)-expressing WT- and mutant AQP2 proteins. Oocytes were not injected (control) or injected with the indicated amounts of cRNAs encoding FLAG-tagged wild-type AQP2 (WT-F), VSV-G-tagged AQP2-R187C (RC-V), or VSV-G-tagged AQP2-E258K (EK-V) or were coinjected with cRNAs of WT-AQP2 and either 1 of the mutants. Oocytes were (co) injected with low (A) amounts or high (B) amounts of WT-AQP2 cRNA. Two days after injection, the Pf values were determined in a standard swelling assay. Values are means ± SE of at least 12 oocytes.

The causes of a dominant-negative effect obtained with higher expression levels of AQP2-R187C. On the basis of the appearance of a 32-kDa high-mannose AQP2 band in immunoblots and a dispersed ER-like immunocytochemical staining pattern of oocyte sections, it was concluded that misfolding and subsequent retention of AQP2-R187C in the ER is likely to be the cause of recessive NDI (3). Later, sucrose gradient centrifugation and mutant-specific immunoprecipitation analyses on oocytes coexpressing low levels of WT-AQP2/AQP2-E258K or WT-AQP2/AQP2-R187C revealed that AQP2-E258K, but not AQP2-R187C, was able to heterotetramerize with WT-AQP2, thereby providing an explanation for dominant or recessive NDI, respectively (12). To investigate why a specific dominant effect was only obtained with low injection levels (Fig. 2), total membranes and plasma membranes of the oocytes used above were isolated and subjected to immunoblotting. Additionally, total membranes were subjected to coimmunoprecipitation assays with FLAG antibodies (which recognize WT-AQP2-F) and the precipitate was immunoblotted to determine whether AQP2-R187C-V and/or AQP2-E258K-V formed heterooligomers with WT-AQP2-F. Immunoblot analysis of the total membranes (which shows relative total expression levels) revealed that, with injections above 0.3 ng of AQP2-R187C-V or AQP2-E258K-V cRNA, the amounts of these proteins gradually increased, whereas the expression of WT-AQP2-F, derived from 0.3 ng of cRNA, gradually decreased (Fig. 3, total membranes). In parallel, the amount of WT-AQP2-F in the plasma membrane decreased gradually with increasing amounts of expressed AQP2-R187C-V with injections above 0.3 ng. In contrast, WT-AQP2-F expression in the plasma membrane in coinjections with AQP2-E258K-V cRNAs was already low when 0.3 ng of AQP2-E258K-V cRNA was coinjected and sustained at a low level for higher expression levels of AQP2-E258K-V. Analysis of the plasma membrane expression of the AQP2 mutants revealed that the expression of AQP2-E258K-V was already visible at 0.3-ng injection and steadily increased with higher expression levels, whereas AQP2-R187C-V was only detectable with 3- and 10-ng injections (Fig. 3, plasma membranes).


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Fig. 3.   Immunoblot analysis of AQP2 proteins in oocytes coexpressing different amounts of tagged WT- and mutant-AQP2 proteins. Oocytes were injected with indicated cRNA amounts of WT-F, RC-V, or EK-V alone or in combination. Of these oocytes, total and plasma membranes (indicated) were isolated and subjected to immunoblotting for AQP2. For immunoprecipitation assays, total membranes were solubilized in deoxycholate and subjected to immunoprecipitation with FLAG-antibodies (IP alpha -FLAG) and subsequently immunoblotted. To detect VSV-G-tagged AQP2 mutants or WT-AQP2-F, VSV-G, or FLAG antibodies (alpha -VSV or alpha -FLAG) were used, respectively. For (co-) injections with 0.3 or 10 ng cRNA WT-AQP2-F, equivalents of 4 or 1 oocytes of total and plasma membranes were immunoblotted, respectively. For the immunoprecipitations, equivalents of 60 oocytes were used.

At these expression levels, coimmunoprecipitation analysis on solubilized oocyte membranes, using FLAG antibodies, revealed no coprecipitation of AQP2-R187C-V with WT-AQP2-F, whereas in all coinjections, AQP2-E258K-V coimmunoprecipitated with WT-AQP2-F. With 10-ng (co-)injections of WT-AQP2-F and mutant cRNAs, AQP2-E258K-V again coprecipitated with WT-AQP2-F, but now also AQP2-R187C-V oligomerization with WT-AQP2-F could be visualized (Fig. 3, IP alpha -FLAG). However, the signal of AQP2-R187C-V was weaker than that of AQP2-E258K-V in the immunoprecipitates, whereas their expression in total membranes was similar. The absence of AQP2-R187C-V in immunoprecipitates from oocytes only expressing AQP2-R187C-V shows the specificity of the FLAG immunoprecipitations (Fig. 3, IP alpha -FLAG).

To alternatively determine the subcellular localization of AQP2-R187C at different expression levels, immunocytochemistry was performed on oocytes injected with 0.3, 1, 3, or 10 ng AQP2-R187C cRNA or 1 ng of WT-AQP2 cRNA. As reported before, WT-AQP2 was only expressed at the plasma membrane (Fig. 4A). With 0.3- and 1-ng injections, AQP2-R187C staining was dispersed throughout the cell only, which is a typical distribution for ER-retarded proteins (Fig. 4B for 1 ng). In oocytes injected with 3 or 10 ng of AQP2-R187C cRNA, however, a clear plasma membrane staining was obtained, besides the dispersed intracellular staining (Fig. 4C for 10 ng). Noninjected oocytes revealed no staining (Fig. 4D).


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Fig. 4.   Immunocytochemistry of oocytes expressing different amounts of AQP2-R187C or WT-AQP2. Oocytes were injected with 1 ng WT-AQP2 cRNA (A), 1 ng AQP2-R187C cRNA (B), 10 ng AQP2-R187C cRNA (C), or not injected (D). Two days after injection, the oocytes were fixed and embedded in paraffin. Five-micrometer sections were cut and expressed AQP2 was visualized by using rabbit AQP2 antibodies followed by ALEXA594-conjugated anti-rabbit IgGs. Arrows, plasma membrane.

Oligomerization of AQP2-R187C. Because at high expression levels, AQP2-R187C was expressed at the plasma membrane and was able to oligomerize with WT-AQP2, the oligomerization state of AQP2-R187C was determined. For this, solubilized membranes of oocytes injected with 0.3, 1, 3, or 10 ng AQP2-R187C cRNA were subjected to sucrose gradient centrifugation. Subsequent immunoblotting of the fractions revealed that with all injections, the majority of AQP2-R187C was found in fractions G and H, which correspond to a 30- to 60-kDa size (Fig. 5). As reported for injections of low amounts (12), the presence of peak levels of AQP2-R187C in these fractions indicates that it is expressed as a monomer. Even with high expression levels of AQP2-R187C, the peak fractions were not shifted to fraction K, in which WT-AQP2 and AQP2-E258K homotetramers were found (12). Of special interest is the change in the level of high-mannose glycosylation. With low amounts, nearly all AQP2-R187C was expressed as an ER-glycosylated form (32-kDa band in Fig. 5), whereas with high amounts, unglycosylated AQP2 (29 kDa) became more pronounced. In fact, densitometric analysis revealed that the mean 32/29-kDa-band ratios were 23, 1.8, 0.9, and 0.6 with 0.3-, 1-, 3-, or 10-ng AQP2-R187C cRNA injections, respectively.


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Fig. 5.   Immunoblot analyses of the oligomerization state of AQP2-R187C expressed at different levels in oocytes. Total membranes of oocytes injected with 0.3, 1, 3, or 10 ng of AQP2-R187C cRNA (indicated) were solubilized in 4% deoxycholate and subjected to sucrose gradient centrifugation. Fractions (C-O) were collected and immunoblotted for AQP2. AQP2-RC and h-AQP2-RC, unglycosylated and high-mannose glycosylated AQP2-R187C bands, respectively; 1-5, fractions with peak intensities of the marker proteins ovalbumin (43 kDa), BSA (67 kDa), phosphorylase B (97 kDa), yeast alcohol dehydrogenase (150 kDa), and catalase (232 kDa), respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

X. laevis oocytes are easy to handle, inject, and analyze and are therefore widely used to study the regulation and routing of channels and transporter and mutants thereof. Although targeting of an epithelial protein to a basolateral or apical membrane cannot be studied in oocytes, because these cells are not polarized, effects of disease-causing mutations are often manifested similarly in oocytes and mammalian cells on expression. For example, many mutants of the cystic fibrosis transmembrane conductance regulator, which are found in cystic fibrosis, and of the low-density lipoprotein receptor in familial hypercholesterolemia were found to be misfolded and retained in the ER in oocytes and in mammalian cells (10, 14). Similar results were obtained for AQP2 mutants in recessive NDI (3, 18, 25). Also, AQP2-E258K, which is retained in the Golgi complex in oocytes (17), appears to be retained in the Golgi complex in mammalian cells (unpublished observations).

Although these data were obtained at unrestrained expression levels, it was necessary to control the expression levels tightly at low levels to observe a specific dominant effect of AQP2-E258K on WT-AQP2 function. In this study, we determined the underlying mechanism, using tags flanking the NH4-terminus of the AQP2 proteins. These tags have been shown not to disturb the routing or function of AQP2 in oocytes (12, 29). For convenience, the AQP2 proteins used in this study will further be annotated without the indication of the tag.

Increasing expression levels of the AQP2 mutants reduce WT-AQP2 expression. At a fixed injection of 0.3 ng cRNA of WT-AQP2, increasing amounts of coinjected mutant AQP2 above 0.3 ng cRNA caused a reduced expression and consequently reduced WT-AQP2 levels in the plasma membranes (Fig. 3). Because AQP2-E258K is not retained intracellularly as severely as AQP2-R187C (Fig. 3, total and plasma membranes) and is, in contrast to AQP2-R187C, a functional water channel (Fig. 2), the resulting Pf of oocytes increased with elevating amounts of expressed AQP2-E258K, but not AQP2-R187C (Fig. 2A). These data indicated that with injections of increasing amounts of mutant cRNA, the translational machinery becomes saturated, which is likely to be the major reason for the observed dominant-negative effect of AQP2-R187C on WT-AQP2.

At high expression levels, AQP2-R187C oligomerizes with WT-AQP2. At low (0.3 ng) injection levels, at which the expression level of WT-AQP2 is not reduced by coexpression of the mutants, the plasma membrane expression of WT-AQP2 is severely reduced with AQP2-E258K, but not with AQP2-R187C. As reported earlier (12), this specific dominant-negative effect is caused by the ability of AQP2-E258K, but not AQP2-R187C, to heterotetramerize with WT-AQP2 at these expression levels (Ref. 12, Fig. 3). At higher injection levels of AQP2-E258K (with reducing WT-AQP2 expression), however, some WT-AQP2 was still found in the plasma membrane, whereas a similar reduction in WT-AQP2 expression in AQP2-R187C resulted in the total absence of WT-AQP2 in the plasma membrane. This indicates that at high AQP2-R187C expression levels, this mutant in recessive NDI is also able to interfere the routing of WT-AQP2 to the plasma membrane. The possibility that, with 3- and 10-ng injections, AQP2-R187C displaces WT-AQP2 from the plasma membrane is unlikely, because the plasma membrane expression of AQP2-E258K is considerably higher than that of AQP2-R187C when 10-ng mutant cRNA is coinjected, whereas WT-AQP2 is still present in the plasma membrane (Fig. 3). The most likely explanation is that at higher expression levels, AQP2-R187C is also able to oligomerize with WT-AQP2. This could not be visualized in immunoprecipitations in 0.3-ng WT-AQP2 cRNA coinjections but was clearly observed for the 10-ng coinjections (Fig. 3).

These data are of special interest, because the ability of AQP2-E258K, but not AQP2-R187C, to oligomerize with WT-AQP2 at low expression levels provided a cell biological explanation for the occurrence of autosomal recessive and dominant NDI (12). Because AQP2-E258K is localized in the Golgi complex whereas AQP2-R187C is retained in the ER, it was hypothesized that either AQP2 mutants in recessive NDI are excluded from oligomerization with WT-AQP2, because AQP2 oligomerization occurs in the Golgi complex, or that AQP2 oligomerization occurs in the ER but compartmentalization of WT-AQP2 and ER-retained mutants prevents their oligomerization. Although the present finding does not address this dilemma and the oligomerization of WT-AQP2 with AQP2-E258K occurs more easily than with AQP2-R187C (compare immunoprecipitation signals in relation to expression levels in total membranes), it proves that a recessive mutant in NDI is physically able to oligomerize with WT-AQP2. It is unclear, however, whether the observed interaction between AQP2-R187C and WT-AQP2 differs from interactions between WT-AQP2 monomers.

At high expression levels, AQP2-R187C escapes the ER and is expressed at the plasma membrane. Immunocytochemical analysis revealed that with injections of 0.3 and 1 ng AQP2-R187C cRNA, this mutant was predominantly retained in the ER (Fig. 4B). This organelle exerts a quality control on newly synthesized proteins in that it retains misfolded proteins after which they are targeted for degradation (21). With injection of 10 ng cRNA, however, AQP2-R187C was, besides in the ER, also clearly expressed in the plasma membrane (Figs. 3 and 4C), indicating that at this expression level AQP2-R187C escapes the ER. This escape phenomenon has also been observed for other mutant channels expressed in oocytes and sometimes led to the finding that the retained mutant protein was functional when expressed at the plasma membrane (6, 7, 18). Escape from the ER might also explain the change in glycosylation of AQP2-R187C with higher injections (Fig. 5). Newly synthesized WT-AQP2 (29 kDa) can be N-glycosylated with high-mannose groups in the ER (32 kDa) and trimmed and complex glycosylated in the median Golgi complex, after which it continues its routing to the plasma membrane as a complex-glycosylated protein of 40-45 kDa. For unexplained reasons, however, more than one-half of WT-AQP2 as found in rat and human kidneys, urine, transfected cell lines, and injected oocytes is unglycosylated (1). As seen in Fig. 5, with low (0.3 ng) injections, the majority of AQP2-R187C migrates as an ER-glycosylated form, whereas with higher injections AQP2-R187C expression clearly shifts to the unglycosylated 29-kDa form. Possibly, this shift with injection of higher amounts is caused by the escape of ER-glycosylated AQP2-R187C from the ER and consequent trimming to a core-glycosylated AQP2 protein of 29 kDa in the Golgi complex. Alternatively, with higher injections, much AQP2-R87C might have already been exported from the ER before full glycosylation can occur, whereas with injections of 0.3 ng cRNA, the full retention of the mutant in the ER provides the glycosylation machinery ample time to glycosylate all AQP2-R187C moieties. In the first situation, less ER-glycosylated mutant AQP2 protein is expected in plasma membrane fractions compared with total membranes, whereas no difference in the ratio of 29- to 32-kDa AQP2 bands would be expected in the latter situation. The fact that expression of high amounts of AQP2 mutants in recessive NDI results in a similar 29/32-kDa signal ratio for plasma membranes and total membranes (3, 18) underscores the latter hypothesis.

In conclusion, our results clearly show that for a specific dominant-negative effect of AQP2-E258K, low expression levels of WT- and mutant AQP2 proteins are needed. This appeared to be caused by the fact that, with increased expression of the ER-retained mutant AQP2-R187C, WT-AQP2 expression was reduced and AQP2-R187C could not be fully retained in the ER and eventually oligomerized with WT-AQP2. Low expression levels in oocytes also appeared to be essential for a specific enhancement of band 3-mediated anion transport on coexpression with glycophorin A (8) and for the HUT-3 urea transporter to confer physiologically relevant transport characteristics (24) and indicate that data derived from oocytes injected with high amounts of cRNAs should be considered with caution. Because many disease-related mutations in multimeric membrane transporters and channels are likely to be identified, our data may constitute important information to study the effects of these mutants on the activity of these transporters and channels in oocytes.


    ACKNOWLEDGEMENTS

We thank Dr. C. H. van Os for critical reading of this manuscript and constructive comments.


    FOOTNOTES

This study was supported by grants from the Dutch Kidney Foundation (C95.5001). P. M. T. Deen is an investigator of the Royal Netherlands Academy of Arts and Sciences.

Address for reprint requests and other correspondence: P. M. T. Deen, Univ. of Nijmegen, 162, Dept. of Cell Physiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands (E-mail: peterd{at}sci.kun.nl).

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.

Received 18 January 2000; accepted in final form 2 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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