Role of glycosylation in the renal electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBCe1)

Inyeong Choi, Lihui Hu, José D. Rojas, Bernhard M. Schmitt, and Walter F. Boron

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBCe1 is important for the regulation of intracellular pH (pHi) and for epithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in many tissues, including kidney, pancreas, and brain. In the present study, we investigate glycosylation sites in NBCe1. Treatment of rat kidney membrane extracts with peptide N-glycosidase F (PNGase F) shifted the apparent molecular weight (MW) of NBCe1 from 130 to 116, the MW predicted from the deduced amino acid sequence. Treatment with endoglycosidase F2 or H or O-glycosidase did not affect the MW of NBCe1. Lectin-binding studies, together with the enzyme data, suggest that the N-linked carbohydrates are of tri- or tetra-antennary type. To localize glycosylation sites, we individually mutated the seven consensus N-glycosylation sites by replacing asparagine (N) with glutamine (Q) and assessing mutant transporters in Xenopus laevis oocytes. Immunoblotting of oocyte membrane extracts treated with PNGase F indicates that NBCe1 is normally glycosylated at N597 and N617 (both on the third extracellular loop). However, N592 (on the same loop) is glycosylated when the other two sites are mutated. The triple mutant (N592Q/N597Q/N617Q) is completely unglycosylated but, based on microelectrode measurements of membrane potential and pHi in oocytes, preserves the Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependence and electrogenicity of wild-type NBCe1.

transporter; pH measurement; acid-base mechanism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOST IMPORTANT pH buffer system in the body is CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Transporters that carry HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> not only have a tremendous impact on the regulation of intracellular pH (pHi), but they also play an important role in the movement of NaHCO3 and fluid across several epithelia. In particular, the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter plays key roles in both pHi regulation and epithelial transport. In astrocytes, for example, electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters are a major mechanism of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> uptake (5, 11, 16). In the kidney, electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters in the proximal tubule (7) are central in the reabsorption of ~85% of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> filtered in the glomeruli (4). In pancreatic ducts, electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters are key components of the mechanism responsible for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion by the exocrine pancreas (21).

The key step in understanding the molecular physiology of electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters was the expression cloning from the salamander kidney of the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC (35). Together with the previously cloned ClHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers or AEs (3, 12, 23), this first NBC defined a new bicarbonate transporter superfamily. Other closely related electrogenic NBCs were soon cloned from mammalian kidney (8, 34), pancreas (1), heart (10), and brain (6). Indeed, immunocytochemistry studies localize NBCe1 to the basolateral membrane of the proximal tubule (38), the basolateral membrane of pancreatic duct cells (27), and astrocytes and neurons in the brain (37). It is now clear that these NBC clones, which we will refer to as NBCe1, are alternative splice products of the same gene (2). NBCe1-A (also called kNBC1) is present mainly in the kidney, NBCe1-B (or pNBC1) is widely distributed but is present at particularly high levels in the pancreas, and NBCe1-C (or bNBC1) is present mainly in the brain.

The primary structure of rat NBCe1-A predicts a protein of 1,035 amino acids that is 30-40% identical to the AEs. Topological studies on AE1 suggest that the protein has cytoplasmic NH2 and COOH termini and as many as 14 transmembrane (TM) segments (14, 32). AE1 is N-glycosylated once in the fourth extracellular loop, between TM7 and TM8, whereas AE2 and AE3 have consensus glycosylation sites in the third extracellular loop, between TM5 and TM6. The oligosaccharide on human AE1 takes two forms, a long-chain polylactosaminyl structure or a shorter complex structure (24). Mutation of the N-glycosylation site in AE1 reduces targeting the protein to the cell surface in Xenopus oocyte studies, but the nonglycosylated protein is still functional (19). One role of N-glycosylation in AE1 may be to permit the interaction of AE1 with calnexin, an endoplasmic reticulum chaperone; altering the N-glycosylation site by mutagenesis eliminates the interaction of AE1 with calnexin (31). AE2 is also glycosylated, but it lacks sialic acids (44). The glycosylation status of AE3 is unknown.

The deduced amino acid sequence predicts that rat NBCe1 has seven putative consensus sites for N-linked glycosylation. On the basis of an AE1 topology model (14), three of the sites (N33, N199, and N208) are on the cytoplasmic NH2 terminus, one (N497) is within TM3, and the remaining three (N592, N597, and N617) are on the third extracellular loop. These last three sites are thus candidates for glycosylation. Western blots show that NBCe1 migrates at 130 kDa in the kidney (38), whereas the deduced amino acid sequence predicts a 116-kDa protein. Thus at least one of the consensus sites may be glycosylated. In the present study, we investigated the oligosaccharide composition of NBCe1-A using a glycosidase enzyme assay and a lectin-binding assay. To locate the N-linked glycosylation sites, we individually mutated asparagine (N) to glutamine (Q) at all seven putative glycosylation sites and also created double and triple mutants. We analyzed these mutants by expressing them in Xenopus oocytes and examining the molecular weight (MW) of each mutant transporter after treatment of peptide N-glycosidase F (PNGase F). We also performed microelectrode experiments to assess the function of mutant transporters. We found that NBCe1-A is normally glycosylated at two sites on the third extracellular loop and that the unglycosylated protein has normal function.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Extraction of crude membranes. A whole kidney from adult Sprague-Dawley rats or rabbits was removed under anesthesia (intraperitoneal injection, 100 mg/kg pentobarbital sodium). A frozen kidney from a cow was obtained commercially, and a kidney from the Ambistoma tigrium salamander was isolated as described previously (38). Slices of the kidney from each species were placed in an ice-cold homogenization buffer (HB)-I (250 mM sucrose, 20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin) and homogenized using a Polytron. For extraction of crude plasma membranes of oocytes expressing NBCe1, oocytes were homogenized in hypotonic HB-II (7.5 mM sodium phosphate, pH 7.4, 1 mM EDTA, 1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin) as described previously (10). Homogenates were centrifuged at 750 g in a microcentrifuge (Eppendorf, model 5415C) for 5 min at 4°C to remove cell debris; the supernatant was centrifuged for 30 min at 16,000 g at 4°C. The pellet, which contained most plasma and organellar membranes, was used for analysis.

Deglycosylation. Membrane fractions (200 µg) isolated from rat tissues were heat denatured at 70°C for 10 min in 0.2 M beta -mercaptoethanol, 0.5% SDS and protease-inhibitor cocktail. Some samples were then incubated overnight at 37°C in an incubation solution (40 mM Na2HPO4, pH = 7.4, 10 mM EDTA, 1% beta -mercaptoethanol, and 0.6% Triton X-100) containing PNGase F (8,000 mU/ml) or endoglycosidase (Endo) F2 (3.2 mU/ml). Other samples were treated overnight at 37°C in 200 mM sodium acetate buffer (pH = 5) containing either Endo H (40 mU/ml) or O-glycosidase (8 mU/ml). For membrane fractions extracted from oocytes, pellets were dissolved in 20 µl of Na2HPO4 (40 mM, pH 7.0), and SDS was added to a final concentration of 1%. After the membrane fractions were denatured at 70°C for 10 min, they were incubated at 37°C for 6 h in the presence of 5 U of N-glycosidase F in the incubation solution. The reaction was stopped by adding the SDS gel-loading buffer.

Lectin binding. We used the following lectins: concanavalin A (Con A), wheat germ agglutinin (WGA), glycine max agglutinin (soy bean agglutinin; SBA), jacalin, Galanthus nivalis (GNA), Lens culinaris (LCA), Ulex europaeus (UEA-I), and Tetragonobulys purpurea (LTA). Lectins coupled to agarose beads (Sigma, St. Louis, MO) were equilibrated at room temperature with the lectin-binding buffer, which contained (in mM) 150 NaCl, 20 Tris, pH 7.5, 1 MgCl2, 1 MnCl2, 1 CaCl2, and 1 ZnSO4, 0.5% Triton X-100, and protease-inhibitor cocktail. Agarose beads were then preabsorbed with 1% BSA in the lectin-binding buffer for 5 min at RT, followed by three 10-min washes in the lectin-binding buffer. Crude membrane proteins (50 µg) were then added to the lectin-binding buffer containing lectin-agarose beads to a final volume of 200 µl. For competition experiments, sugars (methyl-alpha -D-mannopyranoside for Con A and GNA; galactose for SBA and jacalin; N-acetyl-D-glucosamine for WGA; and alpha -mannose for LCA) were added to a final concentration of 0.28 M. For UEA-1 and LTA, alpha -L-fucose was used to a final concentration of 1 M. The reaction mixture, with or without competing sugar, was incubated overnight at 4°C in a rotating shaker. Afterward, the beads were washed three times with the lectin-binding buffer for 10 min.

Immunoblotting. Membrane extracts treated with glycosidases or material from lectin-binding assays were mixed with Laemmli loading buffer and heated for 10 min at 70°C. The proteins were then separated on a 7.5% SDS-polyacrylamide gel and transferred overnight to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). Blots were preincubated for 1 h in a blocking buffer containing 5% nonfat dry milk and 0.1% Tween 20 in Tris-saline (TBS; 50 mM Tris, pH 7.4; 150 mM NaCl) and then incubated with an antibody (1:500 dilution), either NBC-5 (38) or K1A (6), which recognizes the COOH terminus of NBCe1-A. After several washes with TBS containing 0.1% Tween 20, blots were incubated with anti-rabbit IgG conjugated to horseradish peroxidase (HRP; Chemicon) for 1 h (1:5,000 dilution). Blots were washed three times and developed by an HRP/hydrogen peroxide-catalyzed oxidation of luminol under alkaline conditions (Pierce, Rockford, IL).

Site-directed mutagenesis. The plasmid pTLN-NBCe1-A (35) served as a template for site-specific mutagenesis as described by Pusch et al. (33). Seven mutagenic primers were designed to replace the codons for Asn (N) at each of the putative glycosylation sites (N33, N199, N208, N497, N592, N597, and N617). We created a double (or triple) mutant with the same approach, but using a single (or double) mutant as the template. The final constructs were sequenced to verify the desired sequences.

Measurement of Vm and pHi. Wild-type and mutant cDNA were transcribed in vitro using a mMessage mMachine kit (Ambion, Austin, TX) with SP6 RNA polymerase. Defolliculated Xenopus oocytes (Stage V-VI) were prepared as described previously and injected with 20 ng of cRNA or 50 nl water and incubated in OR3 media (50% Leibovitz L-15 media with L-glutamine, 5 mM HEPES, pH 7.5) supplemented with 5 U/ml penicillin/streptomycin (35). Injected oocytes were maintained for 3-7 days at 18°C before use. Voltage and pH microelectrodes were prepared as described previously (17). The pH electrode tip was filled with proton ionophore 1 cocktail B (Fluka Chemical, Ronkonkoma, NY) and back filled with a pH 7 phosphate buffer. Electrodes were connected to high-impedance electrometers (model FD-223; WPI, Sarasota, FL), which in turn were connected to the analog-to-digital converter of a computer. In electrophysiological experiments, the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free ND-96 solution contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5); osmolality was adjusted to 196-200 mosmol/kgH2O. In solutions equilibrated with 1.5% CO2 (pH 7.5), we replaced 10 mM NaCl with 10 mM NaHCO3.

Statistics. Data are reported as means ± SE. Levels of significance were assessed using the unpaired, two-tailed Student's t-test. A P value of <0.05 was considered significant. Rates of pHi change (dpHi/dt) were fitted by a line using a least-square method.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

N-glycosylation of native NBCe1 in different species. To test whether the N-glycosylation of NBCe1 is a general phenomenon among various species, we first prepared membrane extracts prepared from the kidneys of rabbit, rat, bovine, and salamander and then treated these extracts with PNGase F. This enzyme digests the amide bond between the reducing end of N-acetylglucosamine and the beta -amide group of asparagine. We examined the effect of PNGase F treatment on the MW of the NBCe1 protein by immunoblot analysis, using an antibody specific to the COOH terminus of the protein. Figure 1 shows that PNGase F treatment reduced the apparent MW of NBCe1 in all species examined. The immunoreactive bands in rabbit, rat, and bovine decreased from ~130 to ~116, which is the size expected from the deduced amino acid sequence. Not shown are data demonstrating that PNGase F treatment also reduces the apparent MW of NBCe1 from ~130 to ~116 in mouse, human, and guinea pig kidneys. These results suggest that N-glycosylation of NBCe1 is a general phenomenon in mammals. As noted previously, salamander NBCe1 has an apparent MW of ~160 (38), which is 30 higher than in mammalian species. Because PNGase F treatment reduced the apparent MW of salamander NBCe1 to ~130, rather than the predicted MW of 116, it is possible that salamander kidney NBCe1 has an additional O-glycosylation site(s).


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1.   Immunoblot showing N-linked glycosylation of renal electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBCe1) from rabbit, rat, bovine, and salamander. The crude membranes extracted from kidneys were incubated in the absence (-) or presence (+) of peptide N-glycosidase F (PNGase F). Membrane proteins were separated on a 7.5% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with an antibody to the COOH terminus of rat NBCe1.

Previous work on preparations not treated with PNGase F showed that the antibody recognizes other lower MW bands in some preparations (37, 38) but not in others (22, 42). It is interesting to note that PNGase F treatment caused MWs of the faint 101 band of rabbit kidney to fall to 86, the 97 band of rat kidney to fall to 82, and the darker 103 band of bovine kidney to fall to 89, decreases of ~15 in each case. These results support the hypothesis that the lower MW bands represent COOH-terminal proteolytic products.

Glycosidase treatment of native rat NBCe1. To investigate carbohydrate components of the protein, we focused on rat kidney NBCe1, which has been characterized in terms of both its functional properties (18, 34, 39) and its cellular localization (6, 28, 37, 38). In Fig. 2, lane 1 (no enzyme treatment) and lane 2 (PNGase F) confirm the rat data of Fig. 1, which indicate that kidney NBCe1 has an N-glycosylated oligosaccharide. To verify directly the absence of an O-glycosylated carbohydrate-peptide linkage, we next treated rat kidney membrane extracts with an O-glycosidase, which digests the bond between the reducing end of N-acetyl-galactosamine (GalNAc) and the hydroxyl group of serine or threonine. We found that treatment with a combination of O-glycosidase and sialidase (separate experiments showed that sialidase had no effect by itself) did not change the MW of the protein, indicating absence of O-glycosylation (Fig. 2, compare lanes 2 and 3). To further characterize the carbohydrate component of NBCe1, we treated the protein with Endo F2, which cleaves biantennary complex-type oligosaccharides, or Endo H, which cleaves oligosaccharides of the high mannose and hybrid types. Because neither treatment shifted the MW of NBCe1 (Fig. 2, lanes 4 and 5), we can conclude that NBCe1 has an N-linked oligosaccharide that is probably of the tri- or tetra-antennary type.


View larger version (88K):
[in this window]
[in a new window]
 
Fig. 2.   Sensitivity of rat renal NBCe1 to various glycosidases. The enzymes used were PNGase F, O-glycosidase, endoglycosidase (Endo) F2, and Endo H. The O-glycosidase preparation also contained sialidase, which by itself had no effect on apparent molecular weight.

Lectin binding of native rat NBCe1. Because a lectin affinity profile can provide some insight into the nature of the linked oligosaccharide, we performed lectin affinity chromatography with a series of lectins immobilized to agarose beads. We incubated crude membrane extracts with lectin-agarose beads, allowed glycoprotein to bind to the lectin, added either nothing or a competing monosaccharide (see MATERIALS AND METHODS) to elute bound glycoprotein from the beads, isolated the beads, eluted bound glycoprotein (if present) from the beads, and then performed an immunoblot on the supernatant.

As shown in Fig. 3, the NBCe1 protein bound to Con A, glycine max agglutinin (i.e., SBA), and WGA. Con A has an affinity for D-glucopyranose or D-mannopyranose with unmodified hydroxyl groups at the C3, C4, and C6 positions. Elution with methyl-alpha -D-mannopyranoside markedly reduced the binding of NBC to Con A; the small residual signal probably reflects incomplete washing of the beads with the hapten sugar. In other experiments (not shown), we saw complete dissociation of the NBCe1 protein from the Con A-agarose beads. SBA preferentially binds to oligosaccharide structures with terminal GalNAc and, to a lesser extent, terminal galactose residues. Because NBCe1 did not bind to jacalin (Fig. 3), it is unlikely that SBA interacted with NBCe1 via a terminal nonreducing alpha -linked galactose. Therefore, SBA probably interacted with a terminal GalNAc residue on NBCe1. WGA has specificity for internal GlcNAc residues and, to a lesser extent, sialic acid residues.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Lectin binding of rat kidney NBCe1. Lectins coupled to agarose beads were incubated with crude membrane proteins from rat kidney. Afterward, either nothing was added (-), or a competing monosaccharide was added (+) to elute bound glycoprotein from the beads. Immunoblotting was performed with an antibody to the COOH terminus of NBCe1. Lectins used in the experiments are concanavalin A (Con A), jacalin, lectins from Galanthus nivalis (GNA), soy bean agglutinin (SBA), Lens culinaris (LCA), Tetragonobulys purpurea (LTA), wheat germ agglutinin (WGA), and Ulex europaeus (UEA-I).

In addition to jacalin, rat kidney NBCe1 does not bind to lectins from GNA, LCA, UEA-I, or LTA. The insensitivity to GNA indicates the absence of a terminal, nonreducing alpha -linked mannose, which suggests that the NBCe1 oligosaccharides are neither of the high mannose nor of the hybrid type. This interpretation is consistent with the insensitivity of NBCe1 to Endo F2 or Endo H. The absence of binding to LCA, LTA, and UEA-I indicates an absence of core and outer fucose residues. It is interesting to note that all three of these fucose-dependent lectins did interact with NBCe1 isolated from rat brain (not shown).

Mutational analysis of consensus N-glycosylation sites. As noted before, the amino acid sequence of rat kidney NBCe1-A predicts seven consensus N-glycosylation sites (N-X-S/T): N33, N199, N208, N497, N592, N597, and N617 (Fig. 4). To test whether these consensus sites are responsible for N-glycosylation, we individually mutated each asparagine (N) to glutamine (Q). We then heterologously expressed each mutant transporter in Xenopus oocytes, which are known to functionally express wild-type rat kidney NBCe1-A (34). After extracting crude oocyte plasma membranes, and treating some of them with PNGase F, we analyzed the mutant proteins by immunoblotting. As shown in Fig. 5, wild-type NBCe1-A expressed in oocytes and then either not treated or treated with PNGase F, had the same apparent MWs (i.e., ~130 and ~116, respectively) as native NBCe1 in mammalian tissues (see Figs. 1 and 2). The same is true for each of the first five N-to-Q mutants. It is interesting to note that N592 (predicted to be on the third extracellular loop, 28 amino acids downstream from the end of TM5) does not appear to be glycosylated significantly in the wild-type NBCe1 protein.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Consensus sites for N-glycosylation in rat NBCe1. The membrane topology is based on the topology of AE1 (14) and NBCe2 (41). The first 3 sites (N33, N199, and N208) are in the predicted cytoplasmic NH2 terminus, the fourth site (N497) is in the predicted transmembrane segment 3, and the last 3 sites (N592, N597, and N617) are in the third extracellular loop, between transmembrane segment 5 and 6.



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 5.   PNGase F treatment of the membrane extracts from oocytes expressing the mutant transporters. Mutant transporters were individually generated by replacing asparagine (N) with glutamine (Q) at each glycosylation site and then expressed in oocytes. The crude membranes extracted from oocytes were incubated in the absence (-) or presence (+) of PNGase F. WT, wild type.

For both N597Q (which expressed only modestly in this experiment) and N617Q, the MW in the absence of PNGase F treatment was only ~122, consistent with the hypothesis that N597 and N617 contribute about equally to glycosylation in the wild-type protein. Treatment with PNGase F reduced the MW to ~116 for each of these two mutants.

If N597 and N617 both normally contribute to the glycosylation of wild-type NBCe1-A, then one might predict that the double mutant N597Q/N617Q should have a MW of 116, and PNGase F should fail to produce a MW shift. However, as shown in Fig. 6, in the absence of PNGase F, N597Q/N617Q exhibited two bands, one at a MW of ~116 (presumably representing the predicted, nonglycosylated protein) and another at ~120 (presumably representing glycosylation at a residue other than N597 or N617). Treatment with PNGase F yielded only the band at ~116. The presence of the doublet in the absence of PNGase F implies that an alternative glycosylation site may be used when the two normal glycosylation sites are eliminated. To test the hypothesis that N592 is the alternative glycosylation site, we created the triple mutant N592Q/N597Q/N617. Indeed, this triple mutant yielded a single band at ~116, the expected MW of the nonglycosylated protein, both in the absence and the presence of PNGase F. Thus, in the absence of all three consensus N-glycosylation sites on the third extracellular loop, Xenopus oocytes failed to glycosylate NBCe1 measurably. We conclude that the oocyte can glycosylate N592 when bulky sugar chains are not present at positions 597 and 617. The oocyte may also glycosylate N592 to a lesser extent when glycosylation occurs at position 617 but not 597: on the original films for Fig. 5B, bottom, as well as for a similar experiment (not shown), it is possible to see a faint doublet at ~122 kDa.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6.   PNGase F treatment of the membrane extracts from oocytes expressing the double (N597Q/N617Q) or triple mutant (N592/N597Q/N617Q). The crude membranes extracted from oocytes were incubated in the absence (-) or presence (+) of PNGase F.

Functional expression of unglycosylated NBCe1. To test the functional significance of the glycosylation of rat kidney NBCe1, we expressed the triple mutant N592Q/N597Q/N617Q in Xenopus oocytes and then used microelectrodes to monitor pHi and membrane potential (Vm). Previous work showed that, as expressed in Xenopus oocytes, wild-type NBCe1-A is electrogenic, Na+ dependent, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependent (34). As shown in Fig. 7A, for oocytes expressing the triple mutant, introducing 1.5% CO2/10 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> into the extracellular solution elicited a rapid fall in pHi (due to CO2 influx), followed by an increase that reflects the uptake of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or a related ion such as CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>. The introduction of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> also caused an immediate hyperpolarization (Fig. 7B), indicative of the electrogenic influx of Na+ with two or more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions (or an equivalent species, such as CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>). The negative shift in Vm was followed by a decay, the time course of which approximately followed the time course of the pHi recovery.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 7.   Microelectrode measurements of the intracellular pH (pHi) and membrane potential (Vm) in an oocyte expressing the triple mutant (N592/N597Q/N617Q). The oocyte was initially bathed with CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free ND-96 solution. During the indicated period, the oocyte was superfused with 1.5% CO2/10 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The mean rate of pHi change (dpHi/dt) was 13.8 ± 1.7 × 10-5 pH U/s during the pHi recovery from the CO2-induced acid load and -25.3 ± 7.3 × 10-5 pH U/s during the acidification elicited by removing Na+ (n = 6). These values are not significantly different from those in oocytes expressing wild-type NBCe1. For these, the mean dpHi/dt was 14.8 ± 1.8 × 10-5 pH U/s during the pHi recovery (n = 5; P = 0.35 in unpaired, 2-tailed t-test with comparable value for the triple mutant) and -27.6 ± 4.9 × 10-5 pH U/s in the absence of Na+ (n = 5; P = 0.41). In oocytes expressing the mutant transporter, applying CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> caused a mean hyperpolarization of 63.4 ± 2.5 mV, whereas removing Na+ caused a mean depolarization of 53.4 ± 1.8 mV (n = 6). In oocytes expressing wild-type NBCe1 (n = 5), applying CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> caused a mean hyperpolarization of 45.2 ± 7.7 mV (n = 5; P = 0.02), whereas removing Na+ caused a mean depolarization of 31.8 ± 5.1 mV (n = 5; P = 0.001).

Removing extracellular Na+ reversibly converted the pHi recovery to a very slow acidification, reflecting reversal of the mutant transporter. Simultaneously, Na+ removal caused a rapid depolarization, consistent with the electrogenic efflux of Na+ plus two or more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions (or their equivalent). A statistical analysis (Fig. 7 legend) shows that the rates of pHi change, both during the pHi recovery from the CO2-induced acid and the pHi decline elicited by Na+ removal, were not significantly different between oocytes expressing the triple mutants and those expressing wild-type NBCe1-A. The magnitudes of the Vm changes elicited by applying CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or removing Na+ were significantly greater for oocytes expressing the triple mutant. However, this latter observation could reflect differences in several other parameters (e.g., membrane resistance) not related to the transport rate. We conclude that, similar to wild type, the triple mutant is electrogenic and Na+ dependent and activated by applying CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. These results indicate that glycosylation is not required for the basic function of the protein. We obtained similar results (not shown) in mutants lacking one or both of the two natural glycosylation sites (i.e., N597Q, N617Q, and N597Q/N617Q).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Carbohydrate structure of rat kidney NBCe1. In this study, we provide the first direct evidence for the glycosylation of any of the Na+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters. Specifically, the MW shift produced by PNGase F indicates that the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter from rat kidney NBCe1 is a glycoprotein in which the carbohydrate moiety is linked to the protein by an N-glycosidic bond. The insensitivity of rat kidney NBCe1 to either Endo F2 or Endo H suggests that oligosaccharide structure of rat kidney NBCe1 is neither of the biantennary nor of the high mannose/hybrid type. Thus the carbohydrate is probably of the tri- or tetra-antennary/complex type. The lectin-binding assays further suggest that the carbohydrate components of rat kidney NBCe1 probably do not include a terminal nonreducing alpha -linked galactose (insensitivity to jacalin); a terminal nonreducing alpha -linked mannose (insensitivity to GNA); or either a core or outer fucose (insensitivity to LCA, LTA, and UEA-I). On the other hand, the carbohydrate component may include a terminal GalNAc (sensitivity to SBA), either an internal GlcNAc or a terminal sialic acid (WGA sensitivity), and a D-glucopyranose or D-mannopyranose with unmodified C3, C4, and C6 hydroxyl groups (Con A sensitivity).

The MW of the glycosylated renal NBCe1 is ~130 in rabbit, rat, and bovine. In rat, NBCe1 bands of similar MW appear in other tissues, including brain (6) and parotid gland (36). On the other hand, the MW of NBCe1 is ~160 in rat epididymis (22), and our unpublished data show that NBCe1 is mostly present in an unglycosylated form in rat stomach. Therefore, the carbohydrate component of NBCe1 is organ specific.

Our data indicate that the N-glycosylation of NBCe1 is common among various species. A sequence comparison reveals that three consensus glycosylation sites on the third extracellular loop of NBCe1 (i.e., N592, N597, and N617) are conserved among bovine, rabbit, human, mouse, and salamander. Salamander NBCe1 also has a fourth N-glycosylation on this loop. In addition, salamander NBCe1 has an additional carbohydrate linkage, probably an O-glycosylation because PNGase F treatment reduced the apparent MW of salamander NBCe1 to ~130, rather than the predicted MW of 116. It is interesting to note that the third extracellular loop of salamander NBCe1 has a cluster of threonines that are flanked by strongly hydrophobic residues, reminiscent of a mucin-type O-glycosylation site (13).

Glycosylation sites. Our data indicate that the N-linked glycosylation in NBCe1 normally occurs at N597 and N617 and thus prove that both of these residues, which are located between putative TM5 and TM6, face the extracellular fluid.

Glycosylation at N597 and N617 probably contributes about equally to the total glycosylation of the wild-type protein because 1) disrupting either N597 or N617 reduces the apparent MW of the mutant protein from ~130 to ~122, about one-half of the way to the MW of the unglycosylated protein; and 2) treatment of either mutant with PNGase F reduces the MW to ~116, the other one-half of the way. The N597Q/N617Q double mutant exhibits a pair of bands (MW of 116 and 120 in Fig. 6) that presumably represent the protein with or without glycosylation of N592. However, in the absence of PNGase F treatment, the N617Q single mutant exhibited only a single band (MW of 122), suggesting that glycosylation at N597 prevents glycosylation at N592.

By steric hindrance, the bulky carbohydrate moiety at N597 may contribute to the absence of glycosylation at N592 in wild-type NBCe1. However, steric hindrance is not the entire explanation because, as noted above, glycosylation at N592 is optional even for single and double mutants in which we disrupt glycosylation at N597. One possibility is that N592, which is a 28-amino acid residue from the putative end of TM5, is too close to the membrane for efficient glycosylation. However, the N-glycosylation site on the fourth extracellular loop of AE1 is only 15 residues from the putative end of TM5. Alternatively, folding of the extracellular loop near N592 may make this asparagine an intrinsically poor substrate for the oligosaccharyl transferase in the endoplasmic reticulum.

Retention of function in unglycosylated NBCe1. The Xenopus oocyte expression system has been very useful for analyzing the effect of glycosylation on the function of membrane proteins (25). In most of the membrane proteins studied to date, such as the glutamate transporter GLT1 (40), the gamma -aminobutyric acid transporter GAT1 (26), and the nicotinic acetylcholine receptor AChR (15), the naturally occurring N-glycosylation is essential for functional expression of the glycoprotein. Nonetheless, in other cases, such as the Na+-dicarboxylate cotransporter NaDC-1 (29), the Na-Pi cotransporter NaPi-2 (20), the water channel aquaporin-1 (43), and the Na+ channel ENaC (9), the N-glycosylation is not essential for protein function. We found that mutations at one of the two natural sites (N597Q or N617Q), at both of the two natural sites (N597Q/N617Q), or at the two natural sites plus the one alternative site (N592Q/N597Q/N617Q) yield transporters, as expressed in Xenopus oocytes, functionally comparable to the wild-type NBCe1 in terms of electrogenicity as well as Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependence.

The unglycosylated AE1 mutant is also functional in the Xenopus expression system (30). However, the oocytes expressing the unglycosylated AE1 mutant exhibit a lower exchanger activity than those expressing wild-type AE1, possibly due to incorrect protein folding in the endoplasmic reticulum. It is also reported that removing oligosaccharides from AE2 increases the susceptibility to proteolytic degradation (44). We cannot rule out the possibility that lack of a natural glycosylation pattern in our NBCe1 mutants might have reduced their folding efficiency and/or stability. However, if so, these deleterious effects must have been overcome by a very high level of expression.


    ACKNOWLEDGEMENTS

We thank D. Wong for computer support.


    FOOTNOTES

The work was supported by National Institutes of Health Grant DK-30344.

Present addresses: L. Hu, Leids Universitair Medisch Centrum, De Bleek 14, 2312 LP Leiden, The Netherlands; B. Schmitt, Anatomie I, Koellikerstr. 6, 97070 Wurzburg, Germany.

Address for reprint requests and other correspondence: I. Choi, Dept. of Physiology, Emory Univ. School of Medicine, 615 Whitehead Research Bldg., Atlanta, GA 30322 (E-mail: ichoi{at}physio.emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 25, 2003;10.1152/ajprenal.00131.2002

Received 9 April 2002; accepted in final form 14 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abuladze, N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A, and Kurtz I. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem 273: 17689-17695, 1998[Abstract/Free Full Text].

2.   Abuladze, N, Song M, Pushkin A, Newman D, Lee I, Nicholas S, and Kurtz I. Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3. Gene 251: 109-122, 2000[ISI][Medline].

3.   Alper, SL, Kopito RR, Libresco SM, and Lodish HF. Cloning and characterization of a murine band 3-related cDNA from kidney and from a lymphoid cell line. J Biol Chem 263: 17092-17099, 1988[Abstract/Free Full Text].

4.   Alpern, RJ, Warnock DG, and Rector FC. Renal acidification mechanism. In: The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 1986, p. 206-249.

5.   Bevensee, MO, Apkon M, and Boron WF. Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO3 cotransport. J Gen Physiol 110: 467-483, 1997[Abstract/Free Full Text].

6.   Bevensee, MO, Schmitt BM, Choi I, Romero MF, and Boron WF. An electrogenic Na/HCO3 cotransporter (NBC) with a novel COOH terminus, cloned from rat brain. Am J Physiol Cell Physiol 278: C1200-C1211, 2000[Abstract/Free Full Text].

7.   Boron, WF, and Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. J Gen Physiol 81: 53-94, 1983[Abstract].

8.   Burnham, CE, Amlal H, Wang Z, Shull GE, and Soleimani M. Cloning and functional expression of a human kidney Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. J Biol Chem 272: 19111-19114, 1997[Abstract/Free Full Text].

9.   Canessa, CM, Merillat AM, and Rossier BC. Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol Cell Physiol 267: C1682-C1690, 1994[Abstract/Free Full Text].

10.   Choi, I, Romero MF, Khandoudi N, Bril A, and Boron WF. Cloning and characterization of a human electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter isoform (hhNBC). Am J Physiol Cell Physiol 276: C576-C584, 1999[Abstract/Free Full Text].

11.   Deitmer, JW, and Schlue WR. The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech. J Physiol 388: 261-283, 1987[Abstract].

12.   Demuth, DR, Showe LC, Ballantine M, Palumbo A, Frzser PJ, Cioe L, Rovera G, and Curtis PJ. Cloning and structural characterization of human non-erythroid band 3-like protein. EMBO J 5: 1205-1214, 1986[Abstract].

13.   Elhammer, AP, Kezdy FJ, and Kurosaka A. The acceptor specificity of UDP-GalNAc:polypeptide N- acetylgalactosaminyltransferases. Glycoconj J 16: 171-180, 1999[ISI][Medline].

14.   Fujinaga, J, Tang XB, and Casey JR. Topology of the membrane domain of human erythrocyte anion exchange protein, AE1. J Biol Chem 274: 6626-6633, 1999[Abstract/Free Full Text].

15.   Gehle, VM, Walcott EC, Nishizaki T, and Sumikawa K. N-glycosylation at the conserved sites ensures the expression of properly folded functional ACh receptors. Brain Res Mol Brain Res 45: 219-229, 1997[ISI][Medline].

16.   Grichtchenko, II, and Chesler M. Depolarization-induced acid secretion in gliotic hippocampal slices. Neuroscience 62: 1057-1070, 1994[ISI][Medline].

17.   Grichtchenko, II, Choi I, Zhong X, Bray-Ward P, Russell JM, and Boron WF. Cloning, characterization, and chromosomal mapping of a human electroneutral Na+-driven Cl-HCO3 exchanger. J Biol Chem 276: 8358-8363, 2001[Abstract/Free Full Text].

18.   Grichtchenko, II, Romero MF, and Boron WF. Extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependence of electrogenic Na/HCO3 cotransporters cloned from salamander and rat kidney. J Gen Physiol 115: 533-545, 2000[Abstract/Free Full Text].

19.   Groves, JD, and Tanner MJ. Role of N-glycosylation in the expression of human band 3-mediated anion transport. Mol Membr Biol 11: 31-38, 1994[ISI][Medline].

20.   Hayes, G, Busch A, Lotscher M, Waldegger S, Lang F, Verrey F, Biber J, and Murer H. Role of N-linked glycosylation in rat renal Na/Pi-cotransport. J Biol Chem 269: 24143-24149, 1994[Abstract/Free Full Text].

21.   Isiguro, H, Steward MC, Wilson RW, and Case RM. Bicarbonate secretion in interlobular ducts from guinea pig pancreas. J Physiol 495: 179-191, 1996[Abstract].

22.   Jensen, L, Schmitt BM, Brown D, Berger UV, Hediger MA, Boron WF, and Breton S. Localization of sodium bicarbonate co-transporter (NBC) protein and mRNA in rat epididymis. Biol Reprod 60: 573-579, 1999[Abstract/Free Full Text].

23.   Kopito, RR, and Lodish HF. Primary structure and transmembrane orientation of the murine anion exchange protein. Nature 316: 234-238, 1985[ISI][Medline].

24.   Landolt-Marticorena, C, Charuk JH, and Reithmeier RA. Two glycoprotein populations of band 3 dimers are present in human erythrocytes. Mol Membr Biol 15: 153-158, 1998[ISI][Medline].

25.   Li, P, Gao XG, Arellano RO, and Renugopalakrishnan V. Glycosylated and phosphorylated proteins-expression in yeast and oocytes of Xenopus: prospects and challenges-relevance to expression of thermostable proteins. Protein Expr Purif 22: 369-380, 2001[ISI][Medline].

26.   Liu, Y, Eckstein-Ludwig U, Fei J, and Schwarz W. Effect of mutation of glycosylation sites on the Na+ dependence of steady-state and transient currents generated by the neuronal GABA transporter. Biochim Biophys Acta 1415: 246-254, 1998[ISI][Medline].

27.   Marino, CR, Jeans V, Boron WF, and Schmitt BM. Expression and distribution of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in human pancreas. Am J Physiol Gastrointest Liver Physiol 277: G487-G494, 1999[Abstract/Free Full Text].

28.   Maunsbach, AB, Vorum H, Kwon TH, Nielsen S, Simonsen B, Choi I, Schmitt BM, Boron WF, and Aalkjaer C. Immunoelectron microscopic localization of the electrogenic Na/HCO3 cotransporter in rat and ambystoma kidney. J Am Soc Nephrol 11: 2179-2189, 2000[Abstract/Free Full Text].

29.   Pajor, AM, and Sun N. Characterization of the rabbit renal Na+-dicarboxylate cotransporter using antifusion protein antibodies. Am J Physiol Cell Physiol 271: C1808-C1816, 1996[Abstract/Free Full Text].

30.   Pineda, J, and Aghajanian GK. Carbon dioxide regulates the tonic activity of locus coeruleus neurons by modulating a proton- and polyamine-sensitive inward rectifier potassium current. Neuroscience 77: 723-743, 1997[ISI][Medline].

31.   Popov, M, and Reithmeier RA. Calnexin interaction with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger 1 (band 3). J Biol Chem 274: 17635-17642, 1999[Abstract/Free Full Text].

32.   Popov, M, Tam LY, Li J, and Reithmeier RA. Mapping the ends of transmembrane segments in a polytopic membrane protein. Scanning N-glycosylation mutagenesis of extracytosolic loops in the anion exchanger, band 3. J Biol Chem 272: 18325-18332, 1997[Abstract/Free Full Text].

33.   Pusch, M, Steinmeyer K, Koch MC, and Jentsch TJ. Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 chloride channel. Neuron 15: 1455-1463, 1995[ISI][Medline].

34.   Romero, MF, Fong P, Berger UV, Hediger MA, and Boron WF. Cloning and functional expression of rNBC, an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter from rat kidney. Am J Physiol Renal Physiol 274: F425-F432, 1998[Abstract/Free Full Text].

35.   Romero, MF, Hediger MA, Boulpaep EL, and Boron WF. Expression cloning and characterization of a renal electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. Nature 387: 409-413, 1997[ISI][Medline].

36.   Roussa, E, Romero MF, Schmitt BM, Boron WF, Alper SL, and Thevenod F. Immunolocalization of anion exchanger AE2 and Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in rat parotid and submandibular glands. Am J Physiol Gastrointest Liver Physiol 277: G1288-G1296, 1999[Abstract/Free Full Text].

37.   Schmitt, BM, Berger UV, Douglas R, Bevensee MO, Hediger MA, Haddad GG, and Boron WF. Na/HCO3 cotransporters in rat brain: expression in glia, neurons and choroid plexus. J Neurosci 20: 6839-6848, 2000[Abstract/Free Full Text].

38.   Schmitt, BM, Biemesderfer D, Romero MF, Boulpaep EL, and Boron WF. Immunolocalization of the electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in mammalian and amphibian kidney. Am J Physiol Renal Physiol 276: F27-F36, 1999[Abstract/Free Full Text].

39.   Sciortino, CM, and Romero MF. Cation and voltage dependence of rat kidney electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter, rkNBC, expressed in oocytes. Am J Physiol Renal Physiol 277: F611-F623, 1999[Abstract/Free Full Text].

40.   Trotti, D, Aoki M, Pasinelli P, Berger UV, Danbolt NC, Brown RH, Jr, and Hediger MA. Amyotrophic lateral sclerosis-linked glutamate transporter mutant has impaired glutamate clearance capacity. J Biol Chem 276: 576-582, 2001[Abstract/Free Full Text].

41.   Virkki, LV, Wilson DA, Vaughan-Jones RD, and Boron WF. Functional characterization of human NBC4 as an electrogenic Na+-HCO3 cotransporter (NBCe2). Am J Physiol Cell Physiol 282: C1278-C1289, 2002[Abstract/Free Full Text].

42.   Vorum, H, Kwon TH, Fulton C, Simonsen B, Choi I, Boron W, Maunsbach AB, Nielsen S, and Aalkjaer C. Immunolocalization of electroneutral Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in rat kidney. Am J Physiol Renal Physiol 279: F901-F909, 2000[Abstract/Free Full Text].

43.   Zhang, R, Van Hoek AN, Biwersi J, and Verkman AS. A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k. Biochemistry 32: 2938-2941, 1993[ISI][Medline].

44.   Zolotarev, AS, Townsend RR, Stuart-Tilley A, and Alper SL. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid. Am J Physiol Gastrointest Liver Physiol 271: G311-G321, 1996[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 284(6):F1199-F1206
0363-6127/03 $5.00 Copyright © 2003 the American Physiological Society