Mutations in the Human Na-K-2Cl Cotransporter (NKCC2) Identified in Bartter Syndrome Type I Consistently Result in Nonfunctional Transporters
Patrick G.J.F. Starremans*,
Ferry F.J. Kersten*,
Nine V.A.M. Knoers,
Lambertus P.W.J. van den Heuvel and
René J.M. Bindels*
Departments of *Cell Physiology, Human Genetics, and Pediatrics, University Medical Centre Nijmegen, The Netherlands.
Correspondence to Dr. René J.M. Bindels, 160 Cell Physiology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3614211; Fax 31-24-3616413;
ABSTRACT. Bartter syndrome (BS) is a heterogeneous renal tubulardisorder affecting Na-K-Cl reabsorption in the thick ascendinglimb of Henle’s loop. BS type I patients typically presentwith profound hypokalemia and metabolic alkalosis. The maingoal of the present study was to elucidate the functional implicationsof six homozygous mutations (G193R, A267S, G319R, A508T, del526N,and Y998X) in the bumetanide-sensitive Na-K-2Cl cotransporter(hNKCC2) identified in patients diagnosed with BS type I. Tothis end, capped RNA (cRNA) of FLAG-tagged hNKCC2 and the correspondingmutants was injected in Xenopus laevis oocytes and transporteractivity was measured after 72 h by means of a bumetanide-sensitive22Na+ uptake assay at 30°C. Injection of 25 ng of hNKCC2cRNA resulted in bumetanide-sensitive 22Na+ uptake of 2.5 ±0.5 nmol/oocyte per 30 min. Injection of 25 ng of mutant cRNAyielded no significant bumetanide-sensitive 22Na+ uptake. Expressionof wild-type and mutant transporters was confirmed by immunoblotting,showing significantly less mutant protein compared with wild-typeat the same cRNA injection levels. However, when the wild-typecRNA injection level was reduced to obtain a protein expressionlevel equal to that of the mutants, the wild-type still exhibiteda significant bumetanide-sensitive 22Na+ uptake. Immunocytochemicalanalysis showed immunopositive staining of hNKCC2 at the plasmamembrane for wild-type and all studied mutants. In conclusion,mutations in hNKCC2 identified in type I BS patients, when expressedin Xenopus oocytes, result in a low expression of normally routedbut functionally impaired transporters. These results are inline with the hypothesis that the mutations in hNKCC2 are theunderlying cause of the clinical abnormalities seen in patientswith type I BS. E-mail: r.bindels@ncmls.kun.nl
Bartter syndrome (BS) is an autosomal recessive heterogeneousrenal tubular disorder in which one of the key transport proteinsinvolved in transcellular Na-K-Cl transport in the thick ascendinglimb of Henle’s loop (TAL) is impaired (1–4). Approximately23% of the body’s total NaCl reabsorption takes placethrough active transport pathways located in this nephron segment,emphasizing its physiologic importance. The primary mediatorfor Na-K-Cl uptake in the apical membrane of the TAL is thebumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), of whichcurrently six different isoforms have been identified (5–7).In rat, NKCC2a is present in both the medullary (mTAL) and cortical(cTAL) part of the thick ascending limb, whereas NKCC2f is mainlypresent in the inner part of the mTAL and NKCC2b in the outerpart of the cTAL and macula densa. Besides being present ina larger physical region of the TAL, NKCC2a also appeared tohave the largest Na+ transport capacity and would, therefore,be the predominant contributor to Na-K-Cl reabsorption in TAL(8). These cotransporters, in interplay with the basolaterallylocated chloride channel complex (CLC-Kb/Barttin) and the Na/K-ATPase,transport NaCl from the lumen back to the blood compartment.Another key component in this system is the apical inwardlyrectifying ATP-sensitive K+ channel, designated ROMK, whichensures adequate presence of luminal K+ critical for continuousNa-K-Cl uptake by NKCC2 and generates the lumen-positive electricalgradient driving paracellular Ca2+ and Mg2+ reabsorption (9,10).
So far, mutations in NKCC2, ROMK, ClC-Kb, and Barttin have beenlinked to the four types of BS that are currently distinguished.Types I and II BS correspond to mutations in NKCC2 (2) and ROMK(3,11), respectively. These two variants are life-threateningdisorders in which both the hypokalemic alkalosis as well asprofound systemic symptoms are present at birth (12–14).Some of these symptoms already arise in utero, where fetal polyuriacan cause polyhydramnios between 24 and 30 wk of gestation followedtypically by premature delivery. Affected neonates have severesalt wasting and hyposthenuria as well as hyperprostaglandinuriaand failure to thrive. An essential feature is marked hypercalciuria,which may lead to nephrocalcinosis and osteopenia (15,16). TypeIII BS is coupled to mutations in the basolateral CLC-Kb (4,17)and has a more heterogeneous phenotype. Usually, the clinicalfeatures formerly associated with the "Classical Bartter" typeare seen. However, mutations in the gene encoding ClC-Kb canalso cause an antenatal onset of BS or even display a more Gitelman-likephenotype with hypocalciuria and hypomagnesaemia (18). TypeIV BS is composed of a rare subset of patients with sensorineuraldeafness and has recently been linked to an essential chloridechannel -subunit, called Barttin, which is present not onlyin the kidney, but also in the inner ear (19,20). There arestill BS cases in which the four genes encoding the known iontransport proteins have been excluded as being the underlyingdisease genes, suggesting the presence of at least one othercausative gene.
The aim of the present study was to assess the functional consequencesof type I BS mutations selected from different regions in thehNKCC2 sequence. To this end, human wild-type NKCC2 and sixmutants identified in unrelated patients diagnosed with typeI BS were expressed in Xenopus laevis oocytes. hNKCC2 transporteractivity was determined, and mutant and wild-type expressionlevels were analyzed. Subsequently, their subcellular localizationwas visualized using immunocytochemical techniques.
Synthesis of hNKCC2 Constructs
hNKCC2a cDNA was obtained from a human kidney cDNA library (ClontechLaboratories Inc, Palo Alto, CA) by means of PCR and clonedinto a pGEM-Teasy vector (Promega Corp, Madison, WI). The codingsequence was subcloned into a custom oocyte expression vector,pTLN (21). A FLAG-epitope ("DYKDDDDK", IBI; Kodak, New Haven,CT) and Kozak sequence (22) were cloned into the construct replacingthe original ATG. Selected mutations in hNKCC2 were introducedby using the Quikchange Site-directed mutagenesis kit (Stratagene,La Jolla, CA), and all constructs were checked by double-strandedsequence analysis.
Preparation and Injection of Oocytes
Oocytes were obtained from Xenopus laevis and defolliculatedby incubation for 2 h in modified Barth’s solution (MBS:88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES-Tris [N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonicacid], pH 7.4, 0.8 mM MgSO4, 0.3 mM Ca(NO3)2, 0.4 mM CaCl2,and 25 µg/ml gentamycin) containing 2 mg/ml collagenaseA (Roche Molecular Biochemicals, Mannheim, Germany). Stage Vand VI oocytes were selected and stored at 18°C in MBS.Capped RNA (cRNA) transcripts were synthesized from Mlu I-linearizedhuman NKCC2 templates using SP6 RNA polymerase. Defolliculatedoocytes were injected with 50 nl of water containing 0 to 50ng cRNA and incubated 72 h at 18°C.
22Na+ Uptake Assay
The oocytes where transferred to a Cl--free medium (96 mM Na-gluconate,2 mM K-gluconate, 1.8 mM Ca-gluconate, 2.5 mM Na-pyruvate, 5mM HEPES-Tris, pH 7.4, 1 mM Mg(NO3)2, and 50 µg/ml gentamycin)16 to 20 h before the uptake experiment (23), and 10 to 15 Cl--depletedoocytes were then transferred to 500 µl of uptake medium(41 mM NMDG-HCl [N-Methyl-D-Glucamine], pH 7.4, 38 mM NaCl,10 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES-Tris, pH7.4, 100 µM amiloride, 100 µM hydrochlorothiazide,100 µM ouabain) containing 3 µCi 22Na+/ml, and incubatedat 30°C for 30 min, with or without 100 µM bumetanide(8). Oocytes were then washed five times with ice-cold uptakemedium without inhibitors, subsequently solubilized individuallyin 200 µl of 10% (wt/vol) SDS and counted in a BeckmannLS-600 liquid scintillation counter (Beckmann-Coulter, Fullerton,CA).
Isolation of Total Membranes
For isolation of total membranes (plasma and subcellular membranes),10 to 50 oocytes were homogenized in 1 ml of homogenizationbuffer (20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 mM NaH2PO4, 1mM ethylenediaminetetraacetate, 80 mM sucrose, 1 mM phenylmethylsulfonylfluoride,5 µg/ml leupeptin, 5 µg/ml pepstatin) and centrifugedtwo times at 100 x g at 4°C to remove yolk proteins. Next,membranes were isolated by centrifugation at 16,000 x g at 4°Cfor 30 min. Membranes were subsequently dissolved in Laemmli-buffer(2 µl/oocyte) and incubated 30 to 60 min at 37°C.
N-Glycosidase Treatments
Oocytes were injected with 3 ng of WT or 25 ng of mutant cRNA,and total membranes were isolated as described above and dissolvedin 0.3 µl/oocyte Laemmli-buffer. For the Peptide N-glycosidaseF (PNGase F) treatment (#P0704S; New England Biolabs Ltd., Hitchin,UK), 15 oocyte equivalents (10 µl) were supplemented with4 µl of G7-buffer, 4 µl of NP-40 (10% wt/vol), 1000U of PNGase F, and 30 µl of mQ to a total volume of 50µl. For the Endoglycosidase H (Endo H) treatment (#P0702S;New England Biolabs Ltd., Hitchin, UK), 15 oocyte equivalentswere supplemented with 4 µl of G5-buffer, 1000 U of EndoH, and 34 µl of mQ to a total volume of 50 µl. Asnegative controls, 15 oocyte equivalents were supplemented asdescribed for both PNGase F and Endo H, but no enzyme was addedtot the incubation mixture. All reactions were performed at37°C for 1 h and subsequently subjected to immunoblotting.
Immunoblotting
The dissolved membrane samples were subjected to electrophoresison 7% (wt/vol) SDS-polyacrylamide gels and blotted onto polyvinylidenedifluoride membranes (PVDF; Millipore Corp. Bedford, MA) bystandard procedures. Blots were incubated with mouse anti-FLAG-POantibodies (Sigma Chemical Co., St. Louis, MO) diluted 1:2000in Tris-buffered saline, pH 7.4, containing 0.2% (vol/vol) Tween-20and supplemented with 5% (wt/vol) nonfat dried milk. Finally,blots were washed and immunopositive bands were visualized usingan enhanced chemo-luminescence system (Pierce, Rockford, IL).Relative protein amounts were determined by analysis of immunopositivesignals of the films using a model GS-690 imaging densitometer(Bio-Rad, Richmond, VA) operated by Molecular Analyst software.The density of the wild-type protein was set at 100% after correctionfor background.
Immunocytochemistry
After removal of the follicle membranes, oocytes were fixatedin 1% (wt/vol) paraformaldehyde solution for 1 h (24,25), washedtwice in 80% (vol/vol) ethanol, and embedded in paraffin usinga Citadel Tissue Processor and Histocenter 2 (Shandon SouthernProducts Ltd, Cheshire, UK). Seven-micron sections were cut,deparaffinized, and incubated for 30 min in TN (100 mM Tris-HCl,pH 7.6, 150 mM NaCl) containing 0.2% (wt/vol) SDS and subsequentlyblocked in TNB (TN containing 0.5% [wt/vol] blocking reagentfrom Renaissance TSA-direct kit; NEN Lifescience Products Inc,Boston, MA) for 1 h at room temperature. Sections were subsequentlyincubated overnight at 4°C with 1:1000 diluted mouse M2anti-FLAG monoclonal (Sigma-Aldrich, St. Louis, MO) in TNB.After three washes in TNT (TN containing 0.05% [wt/vol] Tween-20)sections were stained for 1 h at room temperature with a 1:1000diluted Alexa 594 conjugated anti-mouse IgG (Molecular Probes,Eugene, OR) in TNB. Finally sections were washed three timesin TNT, dehydrated in subsequently 50% (vol/vol) and 100% (vol/vol)methanol and mounted in Vectashield (Vector Laboratories, Inc.Burlingame, CA). Digital images were made using a MRC-1000 confocallaser scanning microscope (Bio-Rad, Richmond, VA).
hNKCC2 Wild-Type and Mutant Constructs
In Figure 1, the predicted topology of hNKCC2 is shown (26).On the basis of the results of a recent NKCC2 study in mouseby Plata et al. (8), NKCC2a was selected as representative NKCC2isoform. We investigated six mutations located in various regionsof the hNKCC2 sequence, which were previously described, butnot functionally characterized (27). All patients presenteda similar clinical phenotype characteristic for antenatal BS(28). The studied mutations included missense, deletion, andnonsense mutations. Missense mutations G193R, A267S, and G319Rare situated in or near putative transmembrane regions (TM1–4),while A508T is located in an intracellular loop between TM8and 9. The del526N mutation is also located in this region;here the in-frame deletion of a nucleotide triplet causes theloss of an asparagine at position 526. Finally, the Y998X mutationintroduces a premature stop-codon at this position causing theformation of a truncated protein, missing the last 101 AA ofthe C-terminal tail.
Figure 1. Proposed topology of hNKCC2. The mutations investigated in the present study as well as potential glycosylation sites have been highlighted, and the location of the exon 4 cassettes and their relative sequence homology has been outlined as described by Gamba et al. (26) and updated in Swiss-Prot entry Q13621.
Bumetanide-Sensitive 22Na+ Uptake Studies
Because polyclonal antibodies directed against the C-terminalpart (AA921–1055) of human NKCC2 gave rise to nonspecificbackground on oocyte paraffin sections, a FLAG-epitope was incorporatedN-terminally to allow immunocytochemical detection. Both FLAG-taggedand untagged human NKCC2a were expressed in oocytes, and theirfunction was compared by means of 22Na+ uptake experiments.Upon injection of 25 ng of cRNA, a bumetanide-sensitive 22Na+uptake of 2.5 ± 0.5 nmol/oocyte per 30 min was observedfor both FLAG-tagged and untagged hNKCC2a as is demonstratedin Figure 2A. On the basis of these results, FLAG-tagged hNKCC2awas used in all further experiments. To optimize protein expressionfor 22Na+, uptake studies the cRNA injection levels were variedbetween 0.4 to 25 ng of hNKCC2a cRNA. At injection levels above1.5 ng of hNKCC2a cRNA, the 22Na+ uptake reached a plateau (Figure 2B).Furthermore, as shown in Figure 2C, at an injection levelof 25 ng 22Na+ uptake was linear up to 30 min of incubation.
Figure 2. Functional characterization of wild-type hNKCC2a by 22Na+ uptake studies. (A) Assessment of the influence of a FLAG-tag on the 22Na+ uptake of hNKCC2a. Oocytes were injected with 25 ng of either FLAG-tagged or untagged hNKCC2a cRNA. Displayed are the bumetanide-sensitive 22Na+ uptake rates of tagged and untagged hNKCC2a versus non-injected controls (Ni) both with (open bars) and without (filled bars) 10-5 M bumetanide. n= 15 oocytes per condition. (B) Analysis of the 22Na+ uptake of FLAG-tagged hNKCC2a as a function of the cRNA injection level. Shown is the bumetanide-sensitive 22Na uptake of oocytes injected with different cRNA amounts, measured after 30 min of incubation at 30°C. n= 15 oocytes per injected cRNA concentration. (C) Time-dependent 22Na+ uptake of FLAG-tagged hNKCC2a. Oocytes were injected with 25 ng FLAG-tagged hNKCC2a cRNA, and bumetanide-sensitive 22Na+ uptakes are displayed after 30, 60, 120, and 240 min of incubation at 30°C. n= 15 oocytes per time point.
Oocytes were then injected with 3 ng of either wild-type ormutant hNKCC2a cRNA, and the batches were divided for functionalanalyses and immunoblotting. None of the six tested mutantsexhibited a significant bumetanide-sensitive 22Na+ uptake whencompared with non-injected controls, and no immunopositive signalfor these mutants could be detected on immunoblot (data notshown). Injection levels were then raised to 25 ng of eitherwild-type or mutant hNKCC2a cRNA. Now, an immunopositive signalcould be detected for both wild-type and mutants, but the mutantsstill did not exhibit a significant bumetanide-sensitive 22Na+uptake when compared with non-injected controls (Figure 3).
Figure 3. Functional analysis of mutations in hNKCC2a. Oocytes were injected with 25 ng cRNA, and the bumetanide-sensitive 22Na+uptake was subsequently measured. 22Na+ uptake rates that are significantly different from the non-injected controls are marked with an asterisk. The data are the mean of two independent experiments with at least 15 oocytes per injected mutant.
Immunoblot Analysis of hNKCC2a Mutants
To determine the expression levels of the hNKCC2a mutants, totalmembranes isolated from oocytes expressing either wild-typeor mutant hNKCC2a were subjected to immunoblotting (Figure 4).The lane loaded with wild-type hNKCC2a showed a 125-kD and approximately170-kD band that were not present in the control lane. In thelanes loaded with mutants G193R, A267S, and G319R, the sametwo bands were detected. Interestingly, for mutants A508T anddel526N, only the lower 125-kD band was observed. Finally, thelane loaded with the Y998X mutant contained a specific bandat approximately 110 kD, corresponding to the size of the truncatedprotein, but also a band was observed at 125 kD. Densitometricanalysis showed that the protein abundance compared with wild-typewas 3.7% for G193R, 2.1% for A267S, 7.9% for G319R, 0.2% forA508T, 0.8% for del526N, and 0.2% for Y998X. Thus, all mutantsexhibited a significantly lower expression level than wild-typewhen an equivalent amount of oocyte membranes was loaded.
Figure 4. Immunoblot analysis of wild-type hNKCC2a and mutants. (A) Total membranes (plasma and intracellular membranes) were isolated from non-injected (Ni) controls and oocytes injected with 25 ng of FLAG-tagged hNKCC2a cRNA (WT) (1 and 5 oocyte equivalents) or 25 ng cRNA of mutants G193R, A267S, G319R, A508T, del526N, and Y998X (5 oocyte equivalents). They were subsequently loaded and separated on a 7% SDS-polyacrylamide gel and immunoblotted. (B) Total membranes (plasma and intracellular membranes) were isolated from oocytes injected with 3 ng of FLAG-tagged hNKCC2a cRNA (WT). On the left, 15 oocyte equivalents were treated with PNGase F (WT+F) while an equivalent amount was subjected to the same treatment in the absence of the enzyme as a negative control (WT-F). On the right, 15 oocyte equivalents were treated with Endo H (WT+H) while an equivalent amount was subjected to the same treatment in the absence of the enzyme as a negative control (WT-H). Samples were subsequently loaded and separated on a SDS-polyacrylamide gel and immunoblotted. (C) Total membranes (plasma and intracellular membranes) were isolated from oocytes injected with 25 ng of FLAG-tagged mutant cRNA (Y998X). On the left, 15 oocyte equivalents were treated with PNGase F (Y998X+F) while an equivalent amount was subjected to the same treatment in the absence of the enzyme as a negative control (Y998X-F). On the right, 15 oocyte equivalents were treated with Endo H (Y998X+H) while an equivalent amount was subjected to the same treatment in the absence of the enzyme as a negative control (Y998X-H). Samples were subsequently loaded and separated on a SDS-polyacrylamide gel and immunoblotted.
N-Glycosidase Treatment of hNKCC2a and Mutants
To gain more insight in the posttranslational modification ofhNKCC2a, N-glycosidase treatment of total membranes from oocytesexpressing hNKCC2a was performed. Upon incubation of the wild-typeprotein with PNGase F, which cleaves complex, hybrid, and high-mannoseglycosylations, the immunopositive bands at 125 kD and approximately170 kD were reduced to a single band at 120 kD representingthe core protein (Figure 4B, left). Upon treatment with EndoH, which cleaves high-mannose glycosylations, only the 125-kDband of wild-type was reduced, whereas the approximately 170-kDband was unaffected (Figure 4B, right). By a similar treatmentof the Y998X mutant, the 125-kD band shifted to 110 kD, whichcorresponds to the predicted unglycosylated size of the truncatedprotein (Figure 4C).
Immunocytochemical Analyses of hNKCC2a Mutants
To further determine whether absence of transport activity wasdue to misrouting or to functional impairment, paraffin sectionsof oocytes expressing the various mutants were stained withan anti-FLAG antibody and the subcellular localization of hNKCC2awas determined. In sections expressing wild-type hNKCC2a, injectedat 3 and 25 ng (Figure 5, A and B), immunopositive stainingwas observed at the plasma membrane, which was absent in non-injectedcontrols (Figure 5I). The sections expressing the mutants consistentlyshowed a significant immunopositive staining at the plasma membraneand in the cytoplasm (Figure 5, C through H).
Figure 5. Immunocytochemical analysis of wild-type hNKCC2a and mutants. Oocytes were injected with 25 ng wild-type hNKCC2a (A), 3 ng of wild-type hNKCC2a (B), 25 ng of G193R (C), 25 ng of A267S (D), 25 ng of G319R (E), 25 ng of A508T (F), 25 ng of del526N (G), and 50 ng of Y998X (H) or non-injected controls (I). Shown are typical observations of three independent experiments.
In the present study, the functional consequences of mutationsin hNKCC2 identified in type I BS were characterized. Wild-typehNKCC2a, heterologously expressed in Xenopus laevis oocytes,exhibited a significant bumetanide-sensitive 22Na+ uptake whencompared with non-injected controls, which is consistent withearlier studies on mouse and rabbit NKCC2 (7,29,30). hNKCC2ayielded two bands on immunoblot corresponding to a high-mannoseglycosylated form (125 kD) and a larger complex glycosylatedform of approximately 170 kD. Immunocytochemical analysis showedthat the immunopositive signal for hNKCC2 was predominantlypresent at the plasma membrane. All studied mutants, althoughdispersed over the hNKCC2 sequence, resulted in a low expressionof normally routed, but functionally impaired, transporters.
The studied mutants, including the truncated Y998X, were consistentlyprocessed into high-mannose glycosylated forms. Mutants A508T,del526N, and Y998X were not further processed, but G193R, A267S,and G319R appeared to be modified into complex glycosylatedforms. Interestingly, all mutants were routed predominantlyto the plasma membrane, irrespective of their posttranslationalmodification. Similar observations have been made for othermutated transport proteins, including the thiazide-sensitiveNaCl cotransporter, aquaporin-2, and several KCl cotransportermutants, where mutant transporters located at the plasma membranewere at least high-mannose glycosylated (35–37).
The expression level of the mutants was significantly lowerthan wild-type at similar cRNA injection levels. A decreasedprotein expression of mutants has also been observed for otherproteins such as thrombomodulin and protein S mutants (31,32).The exact mechanism responsible for this diminished proteinexpression remains to be elucidated, but we hypothesize thatthe hNKCC2 mutants are subject to early degradation by the cellularER quality control mechanism (33). This mechanism involves activationof the ubiquitin-proteasome proteolytic pathway by misfoldedproteins in the endoplasmic reticulum and rerouting from theGolgi complex and downstream vesicle systems to lysosomes (34).Future research is needed to identify the specific interactingcomponents responsible for this degradation.
Essentially, there are several basic levels at which mutationscan affect protein function. For instance, based on the analysisof cystic fibrosis transmembrane conductance regulator (CFTR)mutations observed in cystic fibrosis (CF) patients, five distinctcategories could be distinguished for which specific treatmentsare being developed (38). Class I (synthesis) mutations typicallyresult from premature stop codons or nonsense mutations. Theresulting mRNA is unstable and degraded. Class II (processing)mutations are normally synthesized, but retained in the endoplasmicreticulum because of protein folding defects and targeted forrapid degradation. This defect is often referred to as a "trafficking"defect because the protein is not transported to the plasmamembrane. Most mutations in this class are either missense ordeletion mutants. Class III (regulatory) mutations are defectivein the activation of transport, although they are present inthe proper location in the apical membrane. This class mainlyconsists of missense mutations in regulatory domains. ClassIV (function) mutations are characterized by a normal intracellularlocalization, but they have a reduced function. These classIV mutations are generally associated with milder phenotypes.Finally, class V mutations affect the structure of the geneand the efficiency of mRNA transcription and processing, markedlyreducing the levels of functional protein, and are mainly localizedin intronic or promoter regions (39). Applying the same classificationused for CFTR mutations on previous studies on BS type II (ROMK2)and Gitelman syndrome (NCC) would implicate that the three groupsof BS type II mutations belong to classes II, III, and IV, whereasthe Gitelman mutations group into classes II and IV (37,40,41).
All hNKCC2 mutations studied here can be categorized into asingle group, namely class III, because all proteins are ableto reach their appropriate location on the plasma membrane,but are not able to transport ions. These findings are ratherunexpected, considering that different types of mutations (missense,deletion, and nonsense) have been selected randomly over thehNKCC2 sequence. However, in recent studies on closely relatedKCl cotransporters, which are also part of the SLC12A solutecarrier family, a similar mechanism for KCC1 and KCC3 mutantswas shown (36,42). These mutants were able to reach the plasmamembrane but were nonfunctional. Therefore, on the basis ofthe similarities in the observations, we hypothesize that thestudied BS type I mutations apparently do not influence proteinfolding, routing, or processing in a significant way. Alternatively,these mutations may affect transporter affinity for one of itsions or negatively influence the proper regulation or activationof the transporter. The present study on hNKCC2 mutants as wellas the aforementioned studies on KCC1 and KCC3 mutants are basedon the Xenopus laevis oocyte expression system only, and theexistence of another mechanism in native TAL cells can, therefore,not be excluded.
To date, several approaches have been suggested for the clinicaltreatment of diverse genetic disorders like the use of amino-glycosidesfor class I mutants to skip aberrant stop-codons during translation,(chemical) chaperones to induce proper protein folding for classII mutants, and the use of butyrate-derivatives to increasethe plasma membrane presence of (partly) functional proteinsby overexpressing them for class IV and V. However, no functionalapproach is available yet for class III mutations, like thepresently studied hNKCC2 mutants. All therapies presently undergoingclinical trials are focused on routing a (partly) functionalprotein to its proper place in the plasma membrane in sufficientquantities and not in restoring function in an impaired, butcorrectly routed, protein.
In conclusion, human NKCC2a has been functionally expressedfor the first time, and six mutations identified in patientswith BS type I have been characterized. Expression levels forall studied mutants were significantly lower when compared withwild-type. Additionally, all mutants were correctly routed tothe plasma membrane, but remained nonfunctional. As such, theycan be grouped into a single category, equivalent to CFTR classIII mutants. This study functionally supports the hypothesisthat mutations in hNKCC2 are pathogenic and cause the phenotypeassociated with type I BS.
Acknowledgments
This study was supported by a grant from the Dutch Kidney Foundation(C97.1662).
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Received for publication September 27, 2002.
Accepted for publication February 15, 2003.
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Top
Abstract
Introduction
-subunit, called Barttin, which is present not only in the kidney, but also in the inner ear (19,20). There are still BS cases in which the four genes encoding the known ion transport proteins have been excluded as being the underlying disease genes, suggesting the presence of at least one other causative gene. 
