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N-Glycosylation at Two Sites Critically Alters Thiazide Binding and Activity of the Rat Thiazide-sensitive Na⁺:Cl^- Cotransporter

Robert S. Hoover, Esteban Poch^¶, Adriana Monroy^*, Norma Vázquez^*, Toshiyuki Nishio, Gerardo Gamba^* and Steven C. Hebert

*Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico; Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, Connecticut; Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan; and ^¶ Servei de Nefrologia, Hospital Clinic, Universitat de Barcelona, Barcelona, Spain.

Correspondence to Dr. Steven C. Hebert, Professor and Chairman, Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street P.O. Box 208026, New Haven, CT 06520-8026. Phone: 203-785-6696; Fax: 203-785-7678;

	Abstract

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ABSTRACT. The rat thiazide-sensitive Na-Cl cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N-linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated cotransporter. Functional expression of cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive ²²Na⁺ uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)–tagged cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl^- affinity. Thus, we have demonstrated that rNCC is N-glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site. E-mail: steven.hebert@yale.edu

	Introduction

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Sodium chloride cotransport sensitive to thiazides is the dominant mechanism mediating Na⁺ and Cl^- reabsorption in the early distal convoluted tubule of the mammalian kidney, as has been demonstrated by in vivo microperfusion studies (1). The thiazide-sensitive Na-Cl cotransporter (NCC) has been cloned by our group from the winter flounder urinary bladder (2) and from the rat kidney (3). More recently the cotransporter was cloned from human (4) and mouse (5) kidney. NCC belongs to the electroneutral cotransporter family of proteins that also includes the cloned bumetanide-sensitive Na-K-2Cl cotransporters (3,6,7) and the more recently cloned K⁺-Cl^- cotransporters (8–11). The members of this family share a great degree of similarity in amino acid sequence and proposed topology. Functional expression of NCC in Xenopus laevis oocytes yielded ²²Na⁺ uptake that was Cl^--dependent and thiazide-sensitive (2,3,12), and a polyclonal antibody raised against the N-terminus of the rNCC protein immunolocalized the cotransporter to the apical membrane of the distal convoluted tubule (13). In addition, several mutations in the coding sequence of the human NCC gene have been genetically linked to the Gitelman syndrome variant of familial hypokalemic metabolic alkalosis, demonstrating the relevance of this cotransporter to human pathophysiolgy (4). Moreover, this cotransporter is the site of action of one of the most commonly used classes of anti-hypertensive agents, the thiazide diuretics.

On the basis of hydropathy analysis, the basic topology of the Na-Cl and Na-K-2Cl cotransporters consists of a central hydrophobic core region containing 12 putative transmembrane-spanning segments between long hydrophilic, putatively intracellular amino and carboxyl termini. Some experimental evidence exists that this is the correct topology for the Na-K-2Cl cotransporter (14). The predicted molecular weight of the core NCC protein is 110 kD (3). However, the higher molecular weight band observed on Western blot analysis (between 135 and 150 kD) using anti NCC antibody (13) suggested that the protein is glycosylated. N-glycosylation is a co-translational event in which an oligosaccharide chain is transferred to the nascent polypeptide at asparagine residues within a specific consensus sequence [Asn-Xaa-Ser/Thr, Xaa Pro; (15)]. Recently, the reported nucleotide sequence of rNCC was found to contain errors, one of which resulted in the loss of a consensus sequence for N-glycosylation (N424 in the corrected sequence). The corrected nucleotide and amino acid sequences were submitted to Genbank (accession number U10097.2). The corrected amino acid sequence of rNCC includes six consensus sequences for N-glycosylation, only two of which are present within a putative extracellular loop (N404 and N424). Both of these sites are predicted to be in a large extracellular loop between trans-membrane (TM) segments seven and eight. Several studies indicate that N-glycosylation in proteins may play a role in modulating their biologic activity and half-life, in directing protein folding, and in regulating cell-surface expression (16). However, there is very little information in the literature regarding glycosylation preventing or decreasing inhibitor binding to transport proteins. In fact, the snake alpha neurotoxin binding to the nicotinic acetylcholine receptor provides an extremely rare example of glycosylation effects on inhibitor binding (17) in a membrane-bound protein of any kind.

In vitro translation experiments by Gamba et al. (3) provided the first indication that rNCC may be glycosylated. They noted an increase in the apparent molecular weight of rNCC in the presence of canine microsomal membranes, which express oligosaccharyl transferase activity. This effect was reversed by enzymatic deglycosylation with endoglycosidase H (Endo H). Although results obtained in cell-free, in vitro translation systems may not be directly applicable to the in vivo situation (15,18), these data provided an impetus for further experimentation. Additionally, it has recently been shown that the mouse homolog of NCC is glycosylated in an oocyte expression system (5). However, glycosylation in native tissue, the sites of glycosylation, and the functional impact of glycosylation have yet to be demonstrated.

The present study was conducted to elucidate whether or not the rNCC cotransporter is glycosylated in vivo, the sites at which it is glycosylated, and the influence of this process on the expression and function of the protein. Site-directed mutagenesis was performed on the putatively extracellular N-glycosylation sites of rNCC to analyze the glycosylation pattern of the wild-type and the mutants, as well as to elucidate the consequences of the disruption of these sites on its transport activity, surface expression, and thiazide sensitivity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Sulfo-N-succinimidyl-6-biotinamido hexanoate was from Pierce (Rockford, IL). Tunicamycin, phenylmethylsulfonyl fluoride, leupeptin, amiloride, bumetanide, ouabain, gentamicin, and immobilized streptavidin were from Sigma. Horseradish peroxidase–linked anti-rabbit IgG and ECL reagent were from Amersham Life Science (Arlington Heights, IL). Endoglycosidase H, collagenase type B, and T7 RNA polymerase were from Boehringer Mannheim (Germany), and recombinant N-glycanase (Peptide N-glycosidase F) was from Genzyme (Cambridge, MA). Enhanced green fluorescence protein (EGFP) was from CLONTECH (Palo Alto, CA). FM 4-64 was from Molecular Probes (Eugene, OR).

Site-Directed Mutagenesis and Construction of Tagged Cotransporters
Site-directed mutagenesis of rNCC Asn 404 glycosylation site was performed in rNCC/pSPORT1 as a template according to the method of Kunkel (19). The oligonucleotide 5'-ATGCCTCAGGGGACGTGCAAGACACC-3' was used to mutate the asparagine at position 404 to glutamine (N404Q) in a region flanked by unique ClaI-NheI sites in the rNCC clone. A silent Bsu36I site was created upstream to the point mutation with the above primer to confirm the subcloning of the PCR product. The oligonucleotide 5'-CTGCGGTTATGGCTGGCAATTCACGG-3' was used to mutate the asparagine at position 424 to glutamine (N424Q). The latter primer was used on the wild-type rNCC/pSPORT1 clone and on the N404QrNCC/pSPORT to create the single mutant N424Q and double mutant N424, 404Q respectively. The mutants were verified by nucleotide sequence analysis.

To assess the surface expression of rNCC and the glycosylation mutants N404Q, N424Q and N404Q/N424Q, the ORF of EGFP (Clontech) was ligated, in frame, to the 5' end of the open reading frame of the cotransporters in the vector pSport. EGFP (GFPmut1) is a mutated form of the Aequoria victoriae protein, GFP that exhibits single excitation and emission peaks at 490 and 509 nm, respectively, fluorescing 35 times more intensely than wild-type GFP when excited at 488 nm (20). The constructs were verified by nucleotide sequence analysis utilizing an Applied Biosystems 377 slab gel and 3700 and 3100 capillary instruments. Then cRNA was synthesized for injection as described below.

Xenopus laevis Oocyte Preparation and Injection
The methods for the preparation, handling, and injection of oocytes have been described previously (2). Briefly, oocyte clusters were incubated during 1 h with vigorous shaking in a Ca²⁺-free medium (82.5 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl₂, and 5.0 mM HEPES/Tris, pH 7.4) containing 2 mg/ml of collagenase type B. Then, stage V-VI oocytes were transferred to the regular frog Ringer ND96 (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, and 5.0 mM HEPES/Tris, pH 7.4), manually defolliculated with fine forceps and incubated overnight at 18°C. The next day, survival oocytes (usually > 95%) were injected with 50 nl of water as control or with water containing 0.5 µg/µl of cRNA that was in vitro transcribed using the T7 RNA promoter in the presence of CAP-analog from either wild-type or mutant rNCC clones previously linearized with NotI (Boehringer Mannheim). Given the decreased expression level of the double glycosylation mutant, 50 ng of this cRNA as well as the standard 25 ng was injected for measurement of the IC50. For the tunicamycin co-injection studies X. laevis oocytes were coinjected with rNCC cRNA and 50 µg/ml (final, 2.5 ng/oocyte) of tunicamycin (dissolved in DMSO). Control (Co) oocytes were coinjected with rNCC cRNA and DMSO alone.

Sodium Transport Measurements
Ten to twenty oocytes for each group of mutant or wild-type rNCC (WTrNCC) cRNA-injected oocytes were incubated in ND96 supplemented with Na⁺-pyruvate (2.5 mM) and gentamycin (5 mg/ml) for the first 3 d and then in Cl-free ND96 (96 mM Na⁺ isethionate, 2.0 mM K⁺-gluconate, 1.8 mM Ca²⁺ gluconate, 1.0 mM Mg-gluconate, and 5.0 mM HEPES/Tris, pH 7.4) for the fourth day. Chloride-free incubation was done to reduce cell Cl^- activity, thus increasing the driving force for tracer uptake. ²²Na⁺ uptake was assessed in individual oocytes 4 d after injection using the following protocol: a 30-min incubation in a Cl^--free ND96 medium (96.0 mM Na⁺-gluconate, 2.0 mM K⁺-gluconate, 6.0 mM Ca²⁺-gluconate, 1.0 mM Mg-gluconate, and 5.0 mM HEPES/Tris, pH 7.4) containing ouabain (1 mM), amiloride (0.1 mM), and bumetanide (0.1 mM), followed by a 60-min uptake period in a K⁺-free, NaCl medium (80.0 mM NaCl, 16.0 mM Na-gluconate, 1.8 mM CaCl₂, 1.0 mM MgCl₂, and 5.0 mM HEPES/Tris, pH 7.4), containing ouabain, amiloride, bumetanide, and 2.5 µCi of ²²Na⁺ per ml (NEN). Ouabain was added to prevent ²²Na⁺ exit via Na⁺-K⁺-ATPase and amiloride to prevent ²²Na⁺ uptake via Na⁺ channels or Na⁺/H⁺ antiport. Removal of extracellular K⁺ and addition of bumetanide to the uptake medium prevented ²²Na⁺ uptake via the Na⁺-K⁺-2Cl^- cotransporter endogenously expressed in oocytes (3). This same protocol was followed for ²²Na⁺ uptakes of EGFP-tagged wild-type and mutant cotransporters. To determine the Cl^--dependent fraction of ²²Na⁺ uptake, paired groups of oocytes were incubated in uptake media with Cl^- (as above) or without Cl^- (96.0 mM Na⁺-gluconate, 6.0 mM Ca²⁺-gluconate, 1.0 mM Mg-gluconate, and 5.0 mM HEPES/Tris, pH 7.4). For kinetic analysis, uptakes were performed with a fixed concentration of Na⁺ or Cl^- at 40 mM, with changing concentrations of counterion from 0 to 20 mM for Na⁺ and 0 to 40 mM for Cl^-. NMDG was used as Na⁺ substitute and gluconate as a Cl^- substitute to maintain osmolality and ionic strength. Thiazide sensitivity was assessed by measuring Na⁺ uptake in paired groups of oocytes with or without metolazone (0.1 mM) in the incubation and uptake medium. The IC50 of the cotransporters to metolazone was determined by exposing groups of rNCC cRNA-injected oocytes to the diuretic at concentrations varying from 10^-9 to 10^-4 M. The diuretic was present in both the incubation and uptake periods. All experiments were performed at 32°C.

Membrane Preparation
Superficial cortex was dissected from kidneys of 250- to 300-g male Sprague-Dawley (Charles River Labs, Wilmington, MA) and homogenized in 9 volumes of ice-cold homogenization buffer (0.32 M sucrose, 5 mM Tris-HCl, pH 7.5, and 2 mM EDTA) with a Teflon homogenizer and centrifuged at 3000 x g for 10 min. The supernatant was removed and centrifuged at 100,000 x g at 4°C for 30 min. The pellet was resuspended in a buffer containing 5 mM Tris-HCl, pH 7.5, and 2 mM EDTA. Protein concentration was determined with the Protein Assay kit (Bio-Rad, Hercules, CA) using bovine -globulin as a standard.

Oocyte homogenates were obtained 3 to 4 d after injection with water or cRNA. Groups of 10 to 20 oocytes were washed once in ND96 solution, once in oocyte homogenization buffer (80 mM sucrose, 1 EDTA, 20 mM Tris/HCl, pH 7.4, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], and 1 µg/ml leupeptin) and homogenized in 20 µl/oocyte of homogenization buffer. Homogenization was performed by passaging the oocytes through a 25-gauge needle at least 20 times. Homogenates were centrifuged twice at 200 x g for 5 min at 4°C to remove yolk and cellular debris. To obtain the membrane fraction, the supernatant was centrifuged at 14,000 x g for 20 min, and the pellet was resuspended in 4 µl of 1X SDS Laemmeli buffer per oocyte. Sixteen microliters of these samples were loaded onto the gel as described below.

Western Blotting
Samples for immunoblotting were heated at 37°C in sample buffer containing 2% SDS, 120 mM Tris-HCl, pH 6.8, and 2% -mercaptoethanol resolved on 6 to 7% SDS-polyacrylamide gels according to Laemmli (21) and electrophoretically transferred in 25 mM Tris-HCl, 192 mM glycine, and 20% methanol to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). An amino-terminal polyclonal antibody generated against a rNCC-MBP fusion protein was used for immunodetection (13). The immune serum was affinity-purified in a CNBr-activated Sepharose 4B coupled with fusion protein (Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions. This antibody has been demonstrated to recognize specifically NCC protein by preabsorption experiments with specific rNCC antigen and by the fact that pre-immune serum does not recognize the immunoreactive protein (13). Membranes were blocked for 1 h in 5% milk powder/TBS-T (10 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0.1% Tween 20) and exposed to either affinity-purified anti-NCC antibody diluted 1/50 to 1/100 in 5% milk powder/TBS-T or anti-NCC immune serum (1:500 dilution) overnight at 4°C. After washing in TBS-T, membranes were exposed to horseradish peroxidase-linked anti-rabbit IgG secondary antibody for 1 h at room temperature. Antigen-antibody complexes were detected by enhanced chemiluminescence with the ECL Western blot analysis system (Amersham). Prestained molecular mass markers were purchased from Bio-Rad. Kodak X-Omat AR or Biomax MR film was used to detect chemiluminescence. Molecular masses were determined with the use of Lab Works Image Acquisition and Analysis software (UVP Inc., Upland, CA).

Enzymatic Deglycosylation
For Peptide N-glycosidase F digestion, 80 to 100 µg (20 µl) of protein from kidney membranes were denatured in 0.5% SDS, 50 mM -mercaptoethanol, 0.55 M Tris-HCl, pH 8.6, and 1 mM EDTA for 1 to 2 h at 4°C and diluted with Nonidet P-40 to a final concentration of 1.5% in the presence of 1 µg/ml leupeptin and 0.2 mM PMSF. The samples were split into two, and either 0.25 U of N-glycanase or water (control) were added and incubated for 12 to 15 h at 37°C. Laemmli sample buffer was added to the mixture and electrophoresed as described above.

For endoglycosidase H digestion, 80 to 100 µg of protein (20 µl) were denatured in 0.5% SDS, 50 mM -mercaptoethanol, 100 mM sodium acetate, pH 5, and 1 mM EDTA for 1 h at 4°C and then diluted with Triton X-100 (final concentration of 1%) in the presence of 1 µg/ml leupeptin and 0.2 mM PMSF. The samples were split into two and either 10 mU of endoglycosidase H or water (control) were added and incubated for 12 to 15 h at 37°C. Samples were handled as indicated above.

Confocal Laser-scanning Microscopy, Membrane Colocalization, and Fluorescence Image Analysis
Laser-scanning confocal microscopy was used to determine the surface expression of rNCC-EGFP, N404Q-EGFP, N424Q- EGFP, and N404Q/N424Q-EGFP in oocytes. Oocytes were procured and prepared for injection as described above. Oocytes were then injected with 40 ng of cRNA from each EGFP-tagged cotransporter. Then, 3 to 4 d after injection, whole unfixed oocytes bathed in ND-96 solution were visualized through an Acrostigmat 10x objective lens (NA = 0.25; Carl Zeiss, Inc.) using an LSM410 microscope (excitation with 488 nm line of an Omnichrome series 43 multiline Ar ion laser; Carl Zeiss, Inc.). Fluorescence emissions were passed through a 515- to 565-nm bandpass filter. For membrane colocalization, oocytes were bathed in ND-96 at 4 to 6°C with 4 um FM 4-64, a fluorescence membrane dye and visualized in the confocal microscope with excitation as above at 488 nm. For determination of fluorescence secondary to FM 4-64 membrane labeling, emissions were passed through a 590- to 640-nm bandpass filter. Background autofluorescence of water-injected oocytes was minimized by adjusting brightness and contrast settings at a constant pinhole size. These settings were then used to assess fluorescence of NCC and mutant injected oocytes. Plasma membrane fluorescence was quantified at equatorial focal sections of oocytes using SigmaScan Pro (Jandel Scientific, San Rafael, CA) image measurement software. A total of at least twelve oocytes from two different frogs were analyzed for each group. Pairs of fluorescence images from the same oocyte with the different emission spectra were analyzed for colocalization and surface plotting utilizing Image-Pro Plus (Media Cybernetics, Silver Spring, MD) software.

Statistical Analyses
All data are expressed as means ± SEM (SEM). Data comparisons among groups were performed by t test, ANOVA one way with Bonferroni correction, or by ANOVA on Ranks (only for comparison of EGFP-tagged cotransporters versus control). Data were analyzed and plotted with the SigmaStat and Sigmaplot programs (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously developed a polyclonal antibody raised against a 110–amino acid segment of the amino terminus of rNCC (13). The antibody has subsequently been affinity purified. Immunodetection of rNCC protein from rat superficial renal cortex membranes with this antibody (Figure 1) reveals a major broad band at 135 to 150 kD and a minor band at 115 kD. After enzymatic deglycosylation with Protein-N-glycosidase F (N-glycanase, 0.25 U), the molecular weight of the rNCC protein is brought down from 135 to 150 kD to 113 kD (Figure 1), the latter representing the unglycosylated, core protein. Control protein exposed to 37°C for 15 h without N-glycanase showed clear presence of glycosylated protein.

View larger version (47K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Western blot analysis of proteins from rat kidney. Effect of enzymatic deglycosylation. Membrane proteins from rat kidney cortex were isolated as described in Materials and Methods. Proteins were solubilized and submitted to Peptide:N-glycosidase F (0.25 U) digestion for 15 h at 37°C as described under Materials and Methods. Proteins were separated on SDS-PAGE (6% polyacrylamide gel), transferred to PVDF membranes, and incubated with affinity-purified polyclonal anti rNCC antibody (1:100). Lane 1, control untreated proteins; lane 2, proteins treated with N-glycanase for 15 h at 37°C; lane 3, control proteins after 15 h incubation at 37°C. The approximate molecular mass is given in kilodaltons and indicated on the left side of the figure.

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effect of tunicamycin and Endoglycosidase H treatment on rNCC protein. (A) Xenopus laevis oocytes were coinjected with rNCC cRNA and 50 µg/ml (final, 2.5 ng/oocyte) of tunicamycin (TM) (dissolved in DMSO). Control (Co) oocytes were coinjected with rNCC cRNA and DMSO alone. Proteins were isolated 4 d after injection and subjected to 6% polyacrylamide SDS gel electrophoresis, electroblotted onto PVDF membranes, and detected as indicated in Figure 1. Lane 1, protein from rNCC injected control oocytes; lane 2, protein from tunicamycin co-injected oocytes. (B) X. laevis oocytes were injected with rNCC cRNA and isolated protein was exposed to Endoglycosidase H or water (control) and incubated for 12 to 15 h at 37°C. The immunoblot was prepared as described above. Lane 1, control proteins after 12-h incubation at 37°C; lane 2, proteins treated with Endoglycosidase H for 12h at 37°C.

View larger version (55K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Western blot analysis of membranes from oocytes injected with water (labeled C for control), wild-type rNCC cRNA, N404Q cRNA, N424Q cRNA, or N404/N424Q cRNA. Oocytes were injected with water or 25 ng of cRNA transcribed from wild-type rNCC and mutants cDNA. Four days after injection, proteins were isolated and separated on a 7% SDS-PAGE, transferred to PVDF membranes, and incubated with anti-rNCC immune serum (1:500). Antibody-antigen complexes were detected as described in Materials and Methods.

To analyze the effect of N-linked glycosylation on the functional properties of rNCC, we assessed ²²Na⁺ uptake in WTrNCC, N404Q, N424Q and N424Q/404Q cRNA-injected oocytes. WTrNCC cRNA-injected oocytes exhibited the characteristic increased ²²Na⁺ uptake above that displayed by the water-injected oocytes (Figure 4). The increased uptake in rNCC cRNA oocytes was thiazide-sensitive. Xenopus oocytes injected with the same amount of cRNA transcribed from either N404Q or N424Q single glycosylation site mutant cDNAs exhibited significant ²²Na⁺ uptake that was metolazone inhibitable. However, as Figure 4 shows, the level of ²²Na⁺ uptake in N404Q and N424Q–injected oocytes was 50 to 60% lower than in paired groups of oocytes injected with WTrNCC cRNA (P < 0.01). The ²²Na⁺ uptake of the double glycosylation mutant was further decreased in what appears to be a synergistic manner, that is, the very small uptake of the double mutant is decreased more than would be expected from simple additive effects of the single mutants. Note, however, that oocytes injected with the double glycosylation mutant still exhibited some activity of the Na⁺:Cl^- cotransporter. Uptake in N404/424Q cRNA–injected oocytes was 518 ± 29 pmol · oocyte^-1 · h^-1. This value was significantly higher than the value observed in the water injected oocytes (210 ± 20 pmol · oocyte^-1 · h^-1 [P < 0.001]) and was reduced by addition of metolazone to the uptake medium (254 ± 74 pmol · oocyte^-1 · h^-1 [P < 0.001]). Thus, the activity of the cotransporter is decreased by eliminating glycosylation at one site and drastically diminished by elimination of glycosylation at both sites.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. 22Na+ uptake in wild-type rNCC and mutant-injected oocytes. Oocytes were injected with 25 ng of wild-type rNCC cRNA, N404Q cRNA, N424Q cRNA, or N404/N424Q cRNA and analyzed for 22Na+ uptake 4 days after injection as described in Materials and Methods. Water-injected oocytes were used as controls. The day before the assay, oocytes were incubated in Cl--free Na-isethionate solution. Uptake was assessed in the absence (white bars) or presence (solid bars) of 100 µM of metolazone in the uptake medium. Each bar represents the mean ± SE of 30 oocytes from two different experiments. * P < 0.01 versus wild-type and control water-injected oocytes.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. 22Na+ uptake in wild-type rNCC, EGFP-tagged rNCC, and EGFP-tagged mutant-injected oocytes. Oocytes were injected with 25 ng of wild-type rNCC cRNA, EGFP-tagged rNCC cRNA, EGFP-tagged N404Q cRNA, EGFP-tagged N424Q cRNA, or EGFP-tagged N404/N424Q double mutant (DM) cRNA and analyzed for 22Na+ uptake 4 d after injection as described in Materials and Methods. Water-injected oocytes were used as controls. The day before the assay, oocytes were incubated in Cl--free Na-isethionate solution. Uptake was assessed in the absence (white bars) or presence (solid bars) of 100 µM of metolazone in the uptake medium. Each bar represents the mean ± SE of 15 oocytes from two different experiments. * P < 0.05 versus EGFP-tagged rNCC and control water-injected oocytes.

View larger version (81K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Colocalization of EGFP-tagged cotransporter fluorescence and FM 4-64 membrane dye fluorescence. Images are of a representative oocyte that has been injected with 40 ng of EGFP-tagged rNCC cRNA, bathed in 4 µm FM of 4-64, excited at 488 nm, and visualized through the Zeiss LSM410 confocal microscope. (A) Fluorescence secondary to EGFP was measured with the 515- to 565-nm bandpass filter and recorded on the green channel. (B) FM 4-64 fluorescence of the same oocyte was measured using the 590- to 640-nm bandpass filter and recorded on the red channel. (C) Superimposition of A and B, demonstrating a yellow overlapping signal indicative of colocalization. (D) Planar surface plot of colocalization. (E) 3D surface plot of colocalization. These images were generated with Image-Pro Plus imaging software and demonstrate >99% surface colocalization of EGFP-WTrNCC and FM 4-64 fluorescence.

View larger version (48K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Representative confocal images of water-injected, rNCC-EGFP cRNA–injected, and N424Q-EGFP cRNA injected oocytes. Oocytes were injected with water or 40 ng of cRNA. Equatorial optical sections obtained through laser scanning confocal microscopy. (A) rNCC-EGFP cRNA–injected oocyte. (B) N424Q-EGFP cRNA–injected oocyte. (C) Water-injected oocyte.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Surface expression of EGFP-tagged rNCC and glycosylation mutants in oocytes by confocal microscopic fluorescence analysis. Oocytes were injected with water or 40 ng of rNCC-EGFP cRNA, N404Q-EGFP cRNA, N424Q-EGFP, or N404Q/N424Q-EGFP. Fluorescence of oocytes was visualized through a laser scanning confocal microscope and measured as described in Materials and Method. Fluorescence intensity is expressed as % of wild-type fluorescence. Graph of fluorescence of rNCC-EGFP cRNA-injected oocytes versus glycosylation mutants-EGFP cRNA-injected oocytes. P < 0.001 for all mutants versus wild-type and for all clones versus control (water-injected).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Na+ and Cl- transport kinetics of wild-type rNCC and mutants. (A) Extracellular Na+ concentration dependence of 22Na+ uptake in oocytes expressing WTrNCC (black circles), N404Q (black box), N424Q (open circle), and N404Q/N424Q (open box). (B) Extracellular Cl- concentration dependence of 22Na+ uptake in oocytes expressing WTrNCC (black circles), N404Q (black box), N424Q (open circle). Uptakes were performed with a fixed concentration of Na+ or Cl- at 40 mM, with changing concentrations of counterion from 0 to 20 mM for Na+ and 0 to 40 mM for Cl-. The experimental conditions otherwise correspond to those of Figure 4. The means of uptake in water-injected oocytes at the various Na+ and Cl- concentrations has been subtracted. Each point represents the mean ± SE of 15 oocytes. Lines were fit using the Michaelis-Menten equation.

Previous studies using [³H] metolazone binding (34) and more recently kinetic analysis of the cotransporter expressed in oocytes (12) indicated that increased extracellular chloride concentration decreased thiazide binding/sensitivity. This has led authors to propose that chloride and thiazides may share a common binding site. Given the alteration in chloride binding, we examined the thiazide sensitivity of the mutant cotransporters. These studies assessed the sensitivity of the cotransporters to metolazone by exposing groups of mutant and WTrNCC cRNA-injected oocytes to the diuretic at concentrations varying from 10^-9 to 10^-4 M. Given the lower baseline transport activity of the double glycosylation mutant, we injected 50 ng as well the standard 25 ng of cRNA to assess the thiazide sensitivity/affinity in the double mutant. Figure 10 demonstrates the changes in metolazone affinity with mutation of glycosylation sites. The lowest affinity was seen in WTrNCC cRNA-injected oocytes with an IC₅₀ of 2 x 10^-6 M. This value is identical to the previously reported IC₅₀ for metolazone in rNCC (12). The affinity in the mutant clones was shifted to the left. IC₅₀ for metolazone in mutant injected oocytes were 7 x 10^-7 M for N404Q, 9 x 10^-8 M for N424Q, and 1 x 10^-8 M for N404Q/N424Q. Thus, the observed affinity for thiazides was WTrNCC < N404Q < N424Q < N404/424Q. This stepwise decrease in IC₅₀ demonstrates an increase in metolazone sensitivity by at least two orders of magnitude with the elimination of glycosylation.

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   Fig. 10. Kinetics of thiazide sensitivity of WTrNCC and mutants. The sensitivity of the cotransporters to metolazone was determined by exposing groups of rNCC cRNA–injected oocytes to the diuretic at concentrations varying from 10-9 to 10-4 M. WTrNCC (black circles), N404Q (black box), N424Q (open circle), and N404Q/N424Q (open box). The diuretic was present in both the incubation and uptake periods. Data points represent means of percent changes in function, taking uptake of each clone in the absence of thiazide as 100%. Each point represents the mean ± SE of 25 oocytes from two different experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have also demonstrated that this glycosylation is essential for efficient surface expression. Given the close correlation between the magnitudes of decreased ²²Na⁺ uptake with decreased surface expression of the single mutants (50 to 60% decrease in both), it appears that the single mutants have decreased function predominantly secondary to decreased surface expression. However, the decrease in plasma membrane fluorescence signal (approximately 33% of WTrNCC) of the double mutant is out of proportion with the decrease in ²²Na⁺ uptake (approximately 8% of WTrNCC) of the double mutant. This suggests that the disruption of both sites may cause a dual effect of reducing surface expression and altering transport activity of individual cotransporters. However, given the decreased transport activity of the GFP-tagged cotransporters, we cannot rule out the possibility that the GFP is somehow affecting surface expression of the double mutant.

Kinetic analyses demonstrated a decrease in V_max with the glycosylation mutants and an even greater decrease in V_max with the double mutant. However, although the K_m for sodium was unchanged, there was a significant change in the apparent K_m for chloride. This increase in Cl^- affinity with the elimination of glycosylation suggests the Cl^- binding site may be near the glycosylation site or that glycosylation partially limits access to the chloride-binding site. The specificity of this interaction is seen by the unchanged K_m for sodium.

N-linked glycosylation of membrane proteins may play a role in protein folding and membrane targeting (35,36). The effect of glycosylation on the functional activity of different types of membrane transport proteins has been described previously. Glycosylation has been associated with alterations of substrate affinity before. In both the glucose transporter GLUT1 (37) and the opossum kidney cell line, organic cation transporter (38) prevention of glycosylation resulted in increased K_m for substrate, reflecting decreased substrate affinity. However, examples of increases in substrate affinity with inhibition of glycosylation appear to be quite rare. Alternatively, glycosylation has been shown to affect the maximal transport activity, but not the K_m in the glycine transporter GLYT1 (39), the Na⁺/H⁺ antiporter (40), the renal NaP_i-2 transporter (41), and the serotonin transporter (42). The mechanisms underlying these changes appear to be a defect in the protein targeting to the plasma membrane in non-glycosylated GLYT1 and probably in Na-P_i transporters, and an in situ reduction in the transport activity in the non-glycosylated Na⁺/H⁺ and serotonin transporters. The latter suggests that for some transporters, glycosylation might be important in maintaining the transporter in an active form in the membrane, perhaps by improving its stability or preventing it from proteolysis, as has been observed for misfolded proteins inside the lumen of the endoplasmic reticulum. Thus, the precise role of N-linked glycosylation may vary depending upon the specific protein of interest. Here we have a very interesting example in which glycosylation appears to alter surface expression and increase affinity for a substrate.

The remarkable increase in thiazide affinity by elimination of the glycosylation sites in rNCC is consistent with the effect on the apparent K_m for extracellular chloride. It has been proposed that the chloride and thiazide binding sites are either the same or at least partially shared (12,34). This effect on both chloride and thiazide affinity, but not on sodium affinity, is consistent with that theory. Additionally, recently published data (43) indicates that the flounder thiazide-sensitive cotransporter has decreased affinity for thiazides, and this transporter has three putative consensus glycosylation sites instead of two. This may represent an additional limitation on thiazide binding by increased glycosylation and is consistent with the important role of glycosylation in limiting the affinity of thiazide diuretics for the cotransporter.

This large alteration of inhibitor affinity by glycosylation is also a rarely reported phenomenon. An extensive literature search revealed no evidence of glycosylation altering inhibitor binding of transport proteins. In the Na,K-ATPase pump and the Na⁺ and Cl^--dependent serotonin transporter elimination of glycosylation had no effect on specific inhibitor binding (42,44). This appears to be the first report of a transport protein whose inhibitor binding is affected by glycosylation. Perhaps this is secondary to lack of investigation of glycosylation effects on inhibitor affinity, or this may indeed be a rare occurrence. The only instance of glycosylation affecting inhibitor binding of any membrane-bound protein was seen with the snake venom -neurotoxin binding to the nicotinic acetylcholine receptor (17). In this instance, the snake acetylcholine receptor is protected from the effects of the venom by glycosylation sites not present in mammalian acetylcholine receptors. These large alterations of binding affinity and inhibition for one of the most commonly used classes of anti-hypertensive agents by glycosylation have clear implications for hypertensive drug therapy. There are many other instances of glycosylated proteins whose inhibitors are used as medications. If this is indeed an under-investigated, more generalized effect of glycosylation, then the impact of these findings could be even greater.

In conclusion, we have demonstrated that the thiazide-sensitive Na-Cl cotransporter is glycosylated in vivo at two sites in the large putative extracellular loop between TM seven and eight by using a combination of site-directed mutagenesis and de-glycosylation experiments. This also proves for the first time that this putative extracellular loop predicted by hydropathy plots is indeed extracellular. ²²Na⁺ uptakes and confocal microscopic fluorescence measurements of EGFP fusion proteins were used to assess the functional significance of these sites. These studies demonstrated that these sites are essential for efficient surface expression, necessary for significant cotransport of sodium chloride, and involved in chloride binding. Importantly, glycosylation of the cotransporter has also been shown to have critical effects on the binding of the thiazide diuretic class of antihypertensive drugs.

	Acknowledgments

 
This work was supported by research grants No. 038545 from the Robert Wood Johnson Foundation to RSH, No. 97629m from the Mexican Council of Science and Technology (CONACYT) and No. 75197–553601 from Howard Hughes Medical Institute to GG, and DK36803 from the National Institutes of Health to SCH and GG. AM was supported by a scholarship grant from CONACYT and from the Dirección General del Personal Académico of the National University of Mexico. GG is an International Scholar of the Howard Hughes Medical Institute. EP was supported by a grant from the Hospital Clinic, Universitat de Barcelona. The work done on this project by EP was performed at the Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.

	Footnotes

 
Robert S. Hoover and Esteban Poch contributed equally to this work and are listed in alphabetical order.

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