From the
Departments of Cell Physiology,
Biochemistry, ¶Pediatrics, and ||Human Genetics, University Medical Centre Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
Received for publication, March 26, 2003
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ABSTRACT |
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INTRODUCTION |
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Several studies indicated that members of the electroneutral cation-chloride cotransporter family could form oligomeric structures. Truncation studies performed in Xenopus laevis oocytes revealed that both N and C termini are required for KCl cotransport activity of the KCC1. Truncation of the first 117 amino acids of the N terminus of this cotransporter was shown to result in a dominant negative phenotype following co-injection with wild type KCC1 (12). Co-immunoprecipitation confirmed that truncated and wild type KCC1 formed multimers (12). In addition to KCC, NKCC1 was also suggested to exhibit a homodimeric structure. This conclusion was based on cross-linking experiments using plasma membranes isolated from rat parotid gland (13). Using the same approach, NCC was suggested to form high molecular weight complexes in rat renal membranes (14). However, the exact structural composition of these complexes was not determined.
Mutations in NCC have been implicated in the autosomal recessive renal tubular disorder Gitelman syndrome (GS, OMIM (Online Mendelian Inheritance in Man) 263800 [OMIM] ). The syndrome is characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria (15). In a previous study using X. laevis oocytes to assess the functional implications of mutations associated with this disorder, we identified two classes of GS mutants (16). Class I mutants exhibited no significant metolazone-sensitive 22Na+ uptake and a diffuse immunopositive staining just below the plasma membrane. Class II mutants showed a marked metolazone-sensitive 22Na+ uptake, which was, however, significantly lower than that obtained with the wild type transporter. Accordingly these mutants were detected both at and below the plasma membrane. Mutant NCCs were expressed in the absence of wild type NCC. Thus far, however, it is unknown whether mutant forms of NCC can interact with wild type NCC and, if so, whether such complexes are directed to the plasma membrane and what their transport activity will be. In this study, we analyzed representatives of class I (G741R) and class II (G980R) in more detail.
In about 40% of the GS patients only one affected allele was detected, which is not consistent with the recessive mode of inheritance of the disorder (3, 17, 18). There are several explanations for this finding (18). First, in the mutation detection strategy applied, PCR-single strand conformation polymorphism, it is known that mutations can be missed. Second, mutations may be present in non-coding regions such as promoter or enhancer segments, intron sequences, or 5' and 3' non-coding regions, which have not yet been screened. Third, detection analysis based on individual exons will not identify large heterozygous deletions. On the other hand, it cannot be excluded that these patients do express the normal protein, which is then, however, inactivated in one way or another by the mutant protein.
The aim of this study was to establish the structural composition of the functional NCC and to investigate whether mutant and wild type NCCs can form (in)active complexes.
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MATERIALS AND METHODS |
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Isolation and Injection of Xenopus OocytesOocytes were isolated from X. laevis and defolliculated by treatment with 2 mg/ml collagenase A for 2 h at room temperature. Stage V and VI oocytes were resuspended in modified Barth's solution containing 10 mM HEPES/Tris (pH 7.5), 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.8 mM MgSO4, 0.3 mM Ca(NO3)2, and 0.4 mM CaCl2 supplemented with 45 µg/ml gentamycin and stored at 18 °C until use. Oocytes were (co-)injected with 2.5, 5, or 10 ng of cRNA of wild type NCC (HA-wt or Flag-wt) and 10 ng of cRNA of mutant NCC (Flag-G741R or Flag-G980R) and analyzed 2 days after injection.
Isolation of Total MembranesFor isolation of total membranes (plasma and subcellular membranes), 50 oocytes were homogenized in 1 ml of homogenization buffer containing 20 mM Tris/HCl (pH 7.4), 5 mM MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 80 mM sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml leupeptin, and 5 µg/ml pepstatin A and centrifuged two times for 10 min at 3000 x g and 4 °C to remove yolk proteins. Subsequently membranes were isolated by a 30-min centrifugation at 14,000 x g and 4 °C.
Cross-linking of ProteinsMembranes of 15 oocytes were dissolved in 50 µl of solubilization buffer (SB) containing 20 mM Tris/HCl (pH 8.0), 0.5% (w/v) sodium deoxycholate, 5 mM EDTA, 10% (v/v) glycerol, 1 mM PMSF, 5 µg/ml leupeptin, and 5µg/ml pepstatin A for 1 h at 37 °C and centrifuged at 16,000 x g for 1 h at 4 °C to remove undissolved membranes. Membrane samples were treated with the cross-linking agent dimethyl-3,3'-dithiobispropionimidate (DTBP) (2 mM) for 30 min at room temperature following the manufacturer's protocol. Next samples were incubated in Laemmli buffer in the absence or presence (to break cross-links) of 10% (w/v) dithiothreitol and subjected to immunoblotting. DTBP covalently links proteins at distances comparable to its spacer arm length (11.9 Å), thereby yielding higher molecular weight complex.
ImmunoprecipitationMembranes of 15 oocytes co-expressing Flag-wt and HA-wt, Flag-G741R, or Flag-G980R were dissolved in 50 µl of SB for 1 h at 37 °C and centrifuged at 16,000 x g for 1 h at 4 °C to remove undissolved membranes as described previously (19). 20 µleqof protein A beads (Amersham Biosciences) were preincubated for 16 h at 4 °C with 2 µl of mouse anti-HA (Sigma) in IPP500 containing 10 mM Tris/HCl (pH 8.0), 500 mM NaCl, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) Tween 20, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 0.1% (w/v) bovine serum albumin. The solubilized membranes were diluted with 600 µl of sucrose buffer containing 20 mM Tris/HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, 0.1% (v/v) Triton X-100, 10% (w/v) sucrose, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A. Next antibody-bound protein A beads were washed three times with IPP100 containing 10 mM Tris/HCl (pH 8.0), 100 mM NaCl, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) Tween 20, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A and incubated with solubilized membranes for 416 h at 4 °C. After three washes with IPP100, the beads were dissolved in 25 µl of Laemmli buffer, incubated for 1 h at 37 °C, and subjected to immunoblotting.
Sucrose Gradient CentrifugationMembranes of oocytes expressing Flag-wt were dissolved in SB for 1 h at 37 °C and centrifuged at 100,000 x g for1hat4 °C to remove undissolved membranes. Solutions were prepared of 15, 20, 25, 30, and 35% (w/v) sucrose in 533 µl of gradient buffer containing 20 mM Tris/HCl (pH 8.0), 5 mM EDTA, 0.1% (v/v) Triton X-100, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A. Samples (300 µl) of total membrane proteins in SB were loaded on the gradient followed by centrifugation at 150,000 x g for 16 h at 8 °C. Then 200-µl fractions, designated AR, were carefully collected and analyzed by immunoblotting. Sedimentation markers were yeast alcohol dehydrogenase (150 kDa), -amylase (200 kDa), catalase (232 kDa), and apoferritin (443 kDa) (Sigma).
Expression of Concatameric cDNA ConstructsConcatameric constructs were obtained by linking the coding sequences of two NCC subunits in a head-to-tail fashion. To this end, two different NCC constructs were generated. The first construct was obtained by site-directed mutagenesis to remove the stop codon of the Flag-wt construct and introducing a unique EcoRV restriction site (Stratagene). The second construct was obtained by inserting a linker of 8 glutamines (20), acting as an intersubunit bridge, in front of the 5' ATG of pT7Ts-NCC. In brief, the N-terminal linker-NCC fragment containing a 5' BamHI and EcoRV restriction site was amplified with Pfu polymerase in a PCR using pT7Ts-NCC as a template followed by cloning into pT7Ts-NCC, pT7Ts-Flag-G741R or pT7Ts-Flag-G980R. Primers used were 5'-tggactagtcagatatctgctgctgctgctgctgctgctgctggcagtaaaaggtgagcacgtt-3' (forward primer) and 5'-ctcctggagcaggtcccg-3' (reverse primer). Concatameric constructs were obtained by digestion of the second construct with EcoRV and SpeI and insertion of the product into the first construct digested with the same enzymes. All cDNA constructs were verified by sequence analysis. Constructs were linearized with EcoRI to produce cRNA.
ImmunoblottingProtein samples were denatured by incubation for 30 min at 37 °C in Laemmli buffer containing 50 mM Tris/HCl (pH 6.8), 2% (w/v) SDS, 12% (v/v) glycerol, 0.01% (w/v) bromphenol blue, and 25 mM dithiothreitol, subjected to electrophoresis on a 6% (w/v) SDS-polyacrylamide gel, and immunoblotted onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) according to standard procedures. Blots were incubated with either mouse anti-FLAG or mouse anti-HA (both from Sigma) at dilutions of 1:8000 and 1:4000 in TBS-T containing 20 mM Tris/HCl (pH 7.6), 137 mM NaCl, 0.1% (v/v) Tween 20, and 5% (w/v) or 1% (w/v) nonfat dried milk, respectively. Subsequently blots were incubated with sheep horseradish peroxidase-conjugated anti-mouse IgG (Sigma) at a dilution of 1:2000. In the case of co-immunoprecipitation experiments, the immunoblots were probed with peroxidase-coupled anti-FLAG antibody (Sigma) at a dilution of 1:5000. NCC proteins were visualized using enhanced chemiluminescence (Pierce).
ImmunocytochemistryImmunocytochemistry was performed as described previously (16).
22Na+ Uptake AssayOocytes were transferred to a Cl-free medium containing 5 mM HEPES/Tris (pH 7.5), 96 mM sodium gluconate, 2 mM potassium gluconate, 1.8 mM calcium gluconate, 1 mM Mg(NO3)2, 2.5 mM sodium pyruvate, and 5 mg/dl gentamycin 24 h prior to the uptake assay (2). Fifteen to 20 Cl-depleted oocytes were transferred to 500 µl of uptake medium containing 5 mM HEPES/Tris (pH 7.5), 58 mM N-methyl-D-glucosamine HCl, 38 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.5 mM ouabain, 100 µM amiloride, 100 µM bumetanide, and 1 µCi/ml 22Na+ and incubated for 2 h at room temperature in the absence or presence of 100 µM hydrochlorothiazide. Ouabain was added to prevent Na+ exit via the Na+-K+-ATPase, bumetanide was added to inhibit the Na+-K+-2Cl cotransporter, and amiloride was added to block the Na+-H+ antiporter and Na+ channels. Hydrochlorothiazide was added to inhibit NCC. After 120 min, the uptake reaction was stopped by washing the oocytes five times in ice-cold uptake medium. Each oocyte was solubilized in 200 µl of 10% (w/v) SDS, and radioactivity was counted in a liquid scintillation counter.
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RESULTS |
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NCC Forms HomodimersBecause the above-mentioned experiments suggested that NCC forms oligomeric structures, we subsequently estimated the size of the complexes. To this end, membranes were isolated from oocytes expressing Flag-NCC, solubilized in 0.5% (w/v) sodium deoxycholate, and subjected to sucrose gradient centrifugation. Analysis of the immunoblot of 18 fractions (AR) collected from the gradient revealed that the peak intensity of NCC was present in fraction J (Fig. 3A). The sedimentation marker proteins (i.e. phosphorylase B, alcohol dehydrogenase, catalase, and apoferritin) that were loaded on a parallel sucrose gradient peaked in fractions F, H, I, and K-L, respectively (Fig. 3, arrows). A logarithmic plot of the molecular mass of the marker proteins as a function of the fraction of the gradient showed that NCC migrates as a complex with a molecular mass of about 310 kDa suggesting that NCC forms a dimeric complex. Sucrose gradient centrifugation in the presence of 0.1% (w/v) SDS revealed the monomeric NCC complex of 150 kDa (Fig. 3B).
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Concatameric NCC Exhibits Thiazide-sensitive 22Na+ UptakeTo investigate whether NCC is functional as a dimer we generated a concatameric NCC in which two NCC subunits are present in a head-to-tail fashion. Oocytes expressing concatameric NCC displayed a thiazide-sensitive 22Na+ uptake similar to that observed in oocytes expressing wild type NCC (Fig. 4A). Immunoblotting of total lysates of these oocytes showed a band of 240 kDa confirming the expression of the concatamer (Fig. 4B, lane 3).
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Wild Type NCC Co-immunoprecipitates with GS Mutant NCCMutations in NCC cause the recessive disease GS. Previously we demonstrated the existence of two different sorting mechanisms for mutant NCC expressed in oocytes (16). The role of dimerization in this differential sorting was investigated in the present study using the fully retarded G741R-NCC (class I) and the partially retarded G980R-NCC (class II). Western blot analysis of total membrane of oocytes co-expressing HA-tagged wild type NCC (HA-wt) and either FLAG-tagged wild type NCC (Flag-wt), G741R-NCC (Flag-G741R), or G980R-NCC (Flag-G980R) revealed a clear expression of the proteins (Fig. 5, A and B). The wt- and G980R-NCC were present in both the complex-(130150-kDa) and high mannose (100-kDa)-glycosylated form, whereas the G741R mutant was only present in the high mannose-glycosylated form. Fig. 5C shows that both Flag-G980R and Flag-G741R co-immunoprecipitated with HA-wt. Despite the abundance of the high mannose-glycosylated wt-NCC (Fig. 5, A and B, lane 2) only complex-glycosylated wt-NCC co-immunoprecipitated, indicating that dimeric wt-NCC is complex-glycosylated. Conversely Flag-G741R co-immunoprecipitated with HA-wt only in the high mannose-glycosylated form, and Flag-G980R co-immunoprecipitated with HA-wt in both the high mannose- and complex-glycosylated form.
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Functional Analysis of Concatameric Mutant-Wild Type NCCConcatameric constructs consisting of wild type and mutant NCC were generated to study the functionality and expression of wild type/mutant dimers. The wt/G741R concatamer did not exhibit a significant 22Na+ uptake (Fig. 6A). In contrast concatamer wt/G980R displayed a 22Na+ uptake that was similar to wt/wt concatamer. Immunoblotting of total lysates of these oocytes revealed a specific band with a molecular weight of 240 kDa (Fig. 6A, inset). The absence of Na+ transport in oocytes expressing wt/G741 concatamer suggests misrouting to the plasma membrane or disturbed activity of the transporter. To study these possibilities, sections of oocytes expressing the concatamer were stained with FLAG antibody to reveal the subcellular localization. The sections depicted in Fig. 6B show a plasma membrane staining of wild type NCC (middle panel) that was absent in oocytes expressing the wt/G741R concatamer (right panel). Oocytes expressing the wt/G741R concatamer displayed a noticeable intracellular staining, which indicates that the protein is misrouted and unable to reach the plasma membrane (right panel).
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Co-injection of Wild Type- and G741R-NCCBecause wild type-NCC and G741R-NCC co-immunoprecipitated and the wt/G741R concatamer lacked plasma membrane localization and thus transport activity, we next investigated whether G741R-NCC has the same effect on wild type function after co-expression of the monomeric constructs. Fig. 7A shows that 22Na+ uptake increased dose dependently with the amount of wt-NCC cRNA that was injected and that the half-maximal effect occurred at 2.5 ng of wt-NCC cRNA/oocyte. In addition, co-injection of 10 ng of G741R-NCC cRNA had no effect on the 22Na+ uptake values obtained with 2.5 and 5 ng of wt-NCC cRNA. Western blot analysis showed that mutant G741R-NCC had no effect on the overall expression of wt-NCC (data not shown). Subsequent immunocytochemistry of oocytes co-expressing wt- and G741R-NCC revealed that the presence of wt-NCC did not result in expression of G741R-NCC on the plasma membrane (Fig. 7B, right panel).
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DISCUSSION |
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The conclusion that the functional NCC is a homodimer is based upon a series of experiments starting with the finding that the cross-linker DTBP reversibly promotes the formation of a high molecular mass complex of 250 kDa followed by immunoprecipitation results, demonstrating the interaction between differentially tagged NCC proteins, and sucrose sedimentation centrifugation data showing a peak at
310 kDa and finally ending with the observation that a concatameric protein of two wt-NCCs mediates thiazide-sensitive Na+ transport. The idea that the functional NCC may consist of a multimeric complex was first suggested by cross-linking experiments in kidney membranes and urine samples of rat showing a specific band of
250 kDa (14). However, the exact structural composition of the functional transporter was not determined. In the present study, we used sucrose sedimentation centrifugation to establish the dimeric nature of the NCC multimer. We have previously used this technique to elucidate the structure of the epithelial calcium channels TRPV5 and -6 (22), the epithelial sodium channel ENaC (23), and the water channel aquaporin-2 (24). From its dimeric nature, NCC resembles the other members of the family of electroneutral cation-chloride cotransporters. In the case of NKCC1, quantitative analysis of the molecular size of oligomers formed by full-length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells provided evidence that the dominant structural unit of this transporter is a homodimer (13). Evidence that KCC1 forms multimers was provided by the observation that a dominant negative loss-of-function mutant co-immunoprecipitated with the full-length polypeptide (12). However, in the latter study the composition of the multimer was not further analyzed. The present finding that the functional NCC is a dimer is in agreement with the general idea that secondary transport proteins with 12 or 14 transmembrane-spanning domains including the tetracycline cation/proton antiporter TetA (25), the erythrocyte anion transport system Band 3 (26), and the renal Na+/H+ exchanger NHE1 (27) exist in the plasma membrane as oligomeric structures.
Our data show that wt-NCC is present in both the high mannose- and complex-glycosylated form. Importantly, however, only the complex-glycosylated form was co-immunoprecipitated. This indicates that the wt-NCC dimer consists of polyproteins that are complex-glycosylated. Using a purified plasma membrane preparation we have previously proven that only complex-glycosylated NCC is present on the plasma membrane (16). Taken together, these data convincingly demonstrate that complex-glycosylated dimeric wt-NCC is present on the plasma membrane. Recently it has been demonstrated that complex glycosylation, and therefore plasma membrane localization, is essential for NCC function (21). Here we demonstrated the functionality of dimeric NCC by expressing its concatameric form in oocytes. The same method was used to show that voltage-gated K+ channels (28), ENaC (20), and TPRV5 and -6 are functional as tetramers (22).
Mutations in NCC cause the recessive disease GS (3). Recently Ellison (29) proposed a straightforward classification of ion transporter defects including transporters that are normally synthesized and reach the cell surface but are inactive (type 1), transporters that are normally synthesized but do not traffic correctly to the plasma membrane (type 2), and transporters that are not properly transcribed or translated (type 3). Thus far, no type 1 mutants have been reported in GS. As far as type 2 mutants are concerned, we have recently demonstrated the existence of two classes consisting of fully (class I) and partially (class II) retarded mutant transporters, respectively (16). One of the hitherto unresolved questions is whether these mutant transporters do exhibit Na+ transport activity. In this context, the present finding that Na+ uptake values are not different between oocytes that express equal amounts of wt/wt or wt/G980R concatamer strongly suggests that the G980R mutant is fully active. This conclusion is compatible with individual subunits of the dimer being the minimum functional unit for Na+-Cl cotransport as has been demonstrated for NHE1 (27). Because the wt/G741R concatamer was not expressed on the plasma membrane its transport activity could not be determined. Our observations with mixed concatamers of wt- and mutant-NCC suggest that the fully retarded G741R-NCC mutant is dominant in its dimeric association with wt-NCC, whereas wt-NCC is dominant in its dimeric association with the partially retarded G980R-NCC mutant.
Comparison of Na+ uptake between oocytes expressing a certain amount of wt-NCC only and oocytes co-expressing the same amount of wt-NCC and an additional amount of G741R-NCC showed that Na+ uptake was not decreased in oocytes co-expressing wt- and mutant-NCC. From this finding it can be concluded that wt-NCC preferentially dimerizes with a wt-NCC. This then suggests that the G741R and the G980R mutations distort the tertiary structure that is required for sorting to the dimerization compartment or for physical interaction between NCC proteins. Similarly mutants of aquaporin-2 (AQP2) fail to tetramerize and therefore do not disturb oligomerization and subsequent membrane trafficking of wt-APQ2 (24). Intriguingly, however, some G741R-NCC was found to co-immunoprecipitate with wt-NCC in these oocytes demonstrating that wt/G741R dimers can be formed. NCCs comprising these dimers were high mannose-glycosylated. This substantiates our concatamer-based conclusion that wt/G741R dimers are retained in the endoplasmic reticulum. Indeed no staining of Flag-G741R was detected on the plasma membrane of these co-expressing oocytes. Therefore, we conclude that wt/G741R dimers, if formed, are degraded by the quality control system of the cell. In contrast to G741R-NCC, G980R-NCC co-immunoprecipitated with wt-NCC showed both high mannose and complex glycosylation indicating that only part of the wt/G980R dimers, if formed, are sorted to the plasma membrane. These findings are in agreement with previous results obtained with oocytes expressing either G741R or G980R alone (16).
One of the intriguing problems in GS research is that in 40% of the screened patients only one affected allele is identified (3, 17, 18). In principle, this finding can be explained in two ways (3, 17, 18). First, the transcript of the other, unidentified allele is not formed due to defects in promoter or intron regions or the transcript is formed but not properly translated (type 3). Second, the unaffected protein is normally formed but somehow inactivated by the mutant protein. It should be noted, however, that the latter explanation is in conflict with the recessive mode of inheritance known for GS. In light of the above discussion, the present finding that the wt/G741R concatamer did not reach the plasma membrane favors the second explanation. Indeed some G741R-NCC was found to co-immunoprecipitate in a high mannose-glycosylated form with wt-NCC. However, it is unlikely that this will happen predominantly in patients in whom hitherto only one affected allele is found. Therefore, we conclude that the second allele is also affected in the above-mentioned
40% of the GS patients.
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FOOTNOTES |
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** To whom correspondence should be addressed: 160 Cell Physiology, University Medical Centre Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-24-3614211; Fax: 31-24-3616413; E-mail r.bindels{at}ncmls.kun.nl.
1 The abbreviations used are: NCC, Na+Cl cotransporter; HA, hemagglutinin; KCC, K+Cl cotransporter; NKCC, Na+-K+-2Cl cotransporter; GS, Gitelman syndrome; PMSF, phenylmethylsulfonyl fluoride; DTBP, dimethyl-3,3'-dithiobispropionimidate; ENaC, epithelial sodium channel; AQP2, aquaporin-2.
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ACKNOWLEDGMENTS |
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REFERENCES |
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