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1 Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
2 Department of Physics, Case Western Reserve University, Cleveland, OH 44106, USA
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(Received 22 March 2004;
accepted after revision 23 June 2004;
first published online 24 June 2004)
Corresponding authors I. Kurtz and A. Pushkin: UCLA Division of Nephrology, 10833 Le Conte Avenue, Room 7-155 Factor Building, Los Angeles, CA 90095-1689, USA. Email: ikurtz{at}mednet.ucla.edu and apushkin{at}mednet.ucla.edu
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Introduction |
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65% when the transporter functions with a HCO3–: Na+ stoichiometry of 3: 1 (Gross et al. 2002). No inhibition of kNBC1-mediated flux was found when the stoichiometry of the cotransporter was switched to 2: 1 following a protein kinase A (PKA)-dependent phosphorylation of kNBC1-Ser982. In addition, we demonstrated that the C-terminus of kNBC1 (kNBC1-ct) binds CAII in vitro with high (Kd < 0.2 µM) affinity (Gross et al. 2002) suggesting that CAII and kNBC1 may physically interact in vivo. Binding of CAII and kNBC1 would be predicted to replace cytoplasmic diffusion of bicarbonate from CAII to kNBC1 with more efficient intermolecular transfer of bicarbonate between the two proteins thereby potentially enhancing the transport rate of kNBC1. Reithmeier and colleagues were first to show that another member of the SLC4 bicarbonate cotransporter superfamily, the anion exchanger AE1 also binds CAII (Vince & Reithmeier, 1998; Reithmeier, 2001). AE1 interacts electrostatically via acidic L886DADD motif in its C-terminus with basic residues in the N-terminus of CAII (Vince & Reithmeier, 2000). In addition to acidic aspartate residues, a non-polar leucine has been shown to be required for CAII binding (Vince et al. 2000). Functional studies have demonstrated that CAII stimulates the transport function of AE1, and the anion exchangers AE2 and AE3 (Sterling et al. 2001a). As a model for the CAII-mediated enhancement of HCO3– transport through AE1, it was hypothesized that a complex of AE1 and CAII functions in red blood cells as a transport metabolon. In this model, the efficiency of bicarbonate transport via AE1 is enhanced by the intermolecular transfer of bicarbonate between CAII and AE1, thereby eliminating the slower cytoplasmic diffusion of HCO3– between the two proteins (Vince & Reithmeier, 1998, 2000; Vince et al. 2000; Reithmeier, 2001; Sterling et al. 2001a,b). A similar mechanism had been originally hypothesized to exist in multienzyme complexes catalysing sequential metabolic reactions termed metabolons allowing metabolites to transfer directly between the active sites of the enzymes (Srere, 1985; Velot et al. 1997; Miles et al. 1999).
Our previous finding that the kNBC1-ct binds with high affinity to CAII and that inhibition of CA activity decreases kNBC1-mediated flux (Gross et al. 2002) suggests that these proteins may also form a transport metabolon on the basolateral membrane of proximal tubule cells. Unlike AE1, which transports Cl– and HCO3– with a 1: 1 stoichiometry, kNBC1-mediated transport is electrogenic (Gross & Kurtz, 2002; Kurtz et al. 2004). Whether the electrogenicity of kNBC1 is affected by its interaction with CAII is currently unknown. We have shown that in mPCT cells transfected with exogenous wild-type kNBC1 (wt-kNBC1), the stoichiometry of kNBC1 is 3: 1 (Gross et al. 2001b), similar to the stoichiometry of the electrogenic sodium bicarbonate cotransport in the basolateral membrane of the renal proximal tubule (Yoshitomi et al. 1987; Muller-Berger et al. 1997a,b; Kunimi et al. 2000; Gross & Kurtz, 2002). Importantly, the stoichiometry was shifted to 2: 1 following treatment of mPCT cells expressing kNBC1 with cAMP (Gross et al. 2001b). This process involved a PKA-mediated phosphorylation of Ser982 in the C-terminus of the cotransporter (Gross et al. 2001b). A similar mechanism of regulation of the cotransporter stoichiometry was shown recently with the pancreatic variant of NBC1, pNBC1 (Gross & Kurtz, 2002; Gross et al. 2003). We have previously shown that Asp986 and Asp988 required for the cAMP induced stoichiometry shift of kNBC1 are located in close proximity to the PKA phosphorylation site, K979KGS (Gross et al. 2002). These aspartate residues are part of a putative D986NDD motif of acidic amino acids that in addition to another putative acidic kNBC1 motif, L958DDV, could be involved in CAII binding. Based on these considerations we hypothesized that a potential mechanism for the cAMP-induced shift in stoichiometry of kNBC1 via phosphorylation of Ser982 may require binding/dissociation of CAII (Gross et al. 2002; Gross & Kurtz, 2002). Whether phosphorylation of Ser982 affected the binding of CAII to kNBC1 or whether binding of CAII interferes with phosphorylation of Ser982 was not determined.
Therefore in the present paper, we studied how binding of CAII to kNBC1 and the activity of the enzyme affect both the flux through the cotransporter and its transport stoichiometry. In addition, we examined the mechanism of the interaction of these proteins by mapping the amino acid residues in kNBC1 responsible for binding of CAII. ACTZ only inhibits kNBC1-mediated flux when the PKA phosphorylation site at Ser982 is not phosphorylated (Gross et al. 2002). Therefore we determined whether the PKA-dependent phosphorylation of kNBC1 affects the binding of CAII. Finally, we examined whether a complex of kNBC1 with CAII functions as a transport metabolon.
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Methods |
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We have previously shown that the C-terminus of kNBC1 binds CAII with high affinity and with the molar ratio 1: 1 (Gross et al. 2002). Analysis of the C-terminus of kNBC1 has revealed two putative acidic motifs, L958DDV and D986NDD, which could potentially be involved in the binding of CAII (Fig. 1). The first motif is similar to the AE1-LDADD sequence involved in binding of CAII (Vince & Reithmeyer, 2000). In the second putative kNBC1 motif, Asp986 and Asp988 are known to be required for the cAMP-mediated shift of kNBC1 stoichiometry from 3: 1 to 2: 1 in proximal tubule cells (Gross et al. 2002).
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We have recently shown that both the N- and C-termini of the human electrogenic sodium bicarbonate cotransporter NBC1 are localized in the cytoplasm (Tatishchev et al. 2003). The coding sequence of the cytoplasmic C-terminal 85 amino acids of the wild-type or mutant kNBC1 (Fig. 1) was inserted into XhoI–EcoRI site of pRSETB vector (Invitrogen, Carlsbad, CA, USA) and expressed in Escherichia coli BL21(DE3)pLysS cells as an N-terminally His6-tagged fusion protein (His6–kNBC1-ct). Approximately 25 g of cells were collected by centrifugation at 6000 g for 20 min, washed 3 times with phosphate-buffered saline (PBS; 10 mM sodium phosphate, pH 7.4, containing 140 mM NaCl), and suspended in 250 ml of BugBusterHT reagent (Novagen, Madison, WI, USA) containing protease inhibitors: 1 mM phenylmethylsulphonylfluoride (PMSF), 1 mM EDTA, 1 µg ml–1 pepstatin, 1 µg ml–1 leupeptin, and 1 µg ml–1 aprotinin (all protease inhibitors were from Roche, Indianapolis, IN, USA). After incubation for 20 min at room temperature, the homogenate was centrifuged at 18 000 g for 20 min at 4°C. The supernatant was dialysed for 16 h at 4°C against PBS, spun at 18 000 g for 20 min, and purified on a Ni-Superflow resin (Novagen) column (2 cm x 8 cm) according to the manufacturer's protocol. Fractions containing His6–kNBC1-ct were combined, dialysed against 20 mM Tris-HCl, pH 7.5, and loaded into a 3 cm x 6 cm column of DEAE-cellulose DE52 (Whatman, Maidstone, Kent, UK) equilibrated with the same buffer. The proteins were eluted with a 0–100 mM gradient of NaCl in 50 mM Tris-HCl, pH 7.5. Fractions containing His6–kNBC1-ct were combined, and then concentrated and transferred to PBS using a Centriplus YM-3 centrifugal filter device (Millipore, Bedford, MA, USA). All purification procedures were performed at 4°C. A polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (SDS-PAGE) and Western blotting (Fig. 2) showed one protein band, which reacted with a C-terminal NBC1-specific antibody, NBC1-4b2 (Tatishchev et al. 2003), and a penta-His antibody (Qiagen, Valencia, CA, USA). The size of the purified fusion protein was 15 kDa, in agreement with the size predicted based on the amino acid composition of the construct.
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The 36 amino acid His6-containing vector sequence of 3 kDa could potentially affect the binding characteristics of the C-terminus of kNBC1. Therefore the His6–kNBC1-ct construct was treated with a recombinant enterokinase (Novagen) to hydrolyse the enterokinase site between the vector and C-terminal coding region of kNBC1. After digestion was completed, which was determined by SDS-PAGE and Western blotting with NBC1-4b2 and penta-His (Qiagen) antibodies (Fig. 2), the enterokinase was absorbed on enterokinase-capture beads (Novagen). The vector sequence containing His6-tag was purified on a Ni-Superflow resin (Novagen) column (2 cm x 8 cm). The binding of the un-tagged kNBC1-ct and His6–kNBC1-ct to CAII was measured and compared using Western blotting with the NBC1-4b2 antibody. The results indicated that the binding of His6–kNBC1-ct was not significantly different from the untagged kNBC1-ct. Silver staining of the SDS gels with a kit from Bio-Rad (Hercules, CA, USA) confirmed the results of Western blotting. Therefore, the His6-tagged wild-type or mutant kNBC1 constructs were used in binding experiments.
Mutagenesis of the full-length kNBC1 and kNBC1-ct
All mutations in the full-length human kNBC1 and the C-terminus of human kNBC1 were performed using a QuikChange site-directed mutagenesis kit (Invitrogen, Carlsbad, CA, USA). Sequences of all constructs were confirmed by bidirectional sequencing using a ABI 310 sequencer (Perkin Elmer, Foster City, CA, USA).
Expression and purification of GST-tagged human CAII
The coding sequence of human CAII was inserted into the XhoI–NotI site of pGEX-4T-3 vector (Amersham Biosciences, Piscataway, NJ, USA) and expressed in E. coli BL21 cells as a fusion glutathione transferase (GST) construct (GST-CAII). Approximately 50 g of cells were collected by centrifugation at 6000 g for 20 min, washed 3 times with PBS, and suspended in 250 ml of BugBusterHT reagent (Novagen) containing protease inhibitors. After incubation for 20 min at room temperature, the homogenate was centrifuged at 18 000 g for 20 min at 4°C. The supernatant was purified on a 2 cm x 6 cm Glutathione Sepharose Fast Flow column (Amersham Biosciences) using the manufacturer's protocol. The purity of the GST-CAII construct was confirmed by SDS-PAGE and Western blotting using anti-GST antibody (Amersham Biosciences). Part of the purified construct was dialysed against deionized water, lyophilized and used for CAII quantification in binding experiments using the Western blotting technique. The rest of the purified GST-CAII was incubated with 20 ml of Glutathione Sepharose for 16 h at 4°C. The unbound GST-CAII was washed with PBS, and the amount of bound GST-CAII per 1 ml of the beads was detected using Western blotting with the anti-GST antibody.
Binding of His6–kNBC1-ct to GST-CAII
Glutathione Sepharose beads (20 µl) with coupled GST-CAII (see previous protocol) were incubated with 1 ml of 1 µM wild-type or mutant His6–kNBC1-ct in PBS for 16 h at 4°C. After centrifugation at 12 000 g for 15 s in an Eppendorf 5415C microcentrifuge, the supernatant was collected and combined with supernatants derived from three washes of the beads with 1 ml of PBS each. The amount of the His6–kNBC1-ct in the supernatant and bound to the beads was measured using Western blotting with both NBC-4b2 and penta-His (Qiagen) antibodies. Each blot was calibrated with the known amounts of His6–kNBC1-ct. The measurements were only performed in the linear range of each calibration curve. Each binding experiment was repeated 4–5 times. For quantification of His6–kNBC1-ct, different amounts of His6–kNBC1-ct and lysozyme (Sigma, St Louis, MO, USA) were resolved on SDS-PAGE. The gels were stained with Coomassie blue R (Sigma), scanned on an UMAX PowerLook III scanner and analysed using Adobe Photoshop 7 software (Adobe Systems). The intensity of the His6–kNBC1-ct bands was determined by comparison of their intensity with the intensity of lysozyme bands of known amounts.
In vitro phosphorylation of the His6–kNBC1-ct
The His6–kNBC1-ct contains one putative PKA phosphorylation site at Ser982. We have previously shown that Ser982 is the only kNBC1 amino acid that can be phosphorylated by PKA (Abuladze et al. 1998; Gross et al. 2001b). Approximately 100 µg of the purified His6–kNBC1-ct was incubated in 1 ml of 50 mM Tris-HCl, pH 7.5, containing 10 mM ATP, 2 mM MgCl2 and 200 U of bovine heart PKA (Promega, Madison, WI, USA). After incubation for 20 h at 20°C, the solution was passed consecutively through a Centriplus YM-30 and a Centriplus YM-3 centrifugal filter devices (Millipore). The concentrate was transferred into PBS and analysed by isoelectrofocusing on Criterion IEF ready gels (Bio-Rad), transferred to polyvinylidene difluoride (PVDF) membrane, and analysed by Western blotting with both NBC1-4b2 and penta-His (Qiagen) antibodies. A single band with an isoelectric point more acidic than the non-phosphorylated His6–kNBC1-ct was detected (Fig. 2). The concentrate did not have PKA activity.
Binding of kNBC1 to CAII in mouse kidney extracts
Mice were killed on the day of study by exposure to CO2 and the kidneys removed and used for the binding studies. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California, Los Angeles. Approximately 1 g of mouse kidney was disrupted in 20 ml BugBusterHT reagent (Novagen) containing protease inhibitors. After incubation for 2 h at 4°C, the homogenate was centrifuged at 18 000 g for 20 min at 4°C. The supernatant was dialysed for 16 h at 4°C against PBS containing 0.05% Triton X-100, spun at 18 000 g for 20 min, and the resulting supernatant was passed through a Glutathione Sepharose column (1 cm x 1 cm) with bound full-length CAII. After washing with 500 ml PBS containing 0.05% Triton X-100, proteins bound to the column were eluted with 1 x SDS buffer and analysed by SDS-PAGE and Western blotting with the NBC1-4b2 antibody.
SDS-PAGE and Western blotting
SDS-PAGE was performed using 15% polyacrylamide ready gels from Bio-Rad. Proteins separated by SDS-PAGE were electrotransferred onto PVDF membrane (Amersham Biosciences). Non-specific binding was blocked by incubation for 1 h in Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.5, 140 mM NaCl) containing 5% dry milk and 0.05% Tween 20 (Bio-Rad). The NBC-4b2 NBC1-ct-specific antibody and mouse penta-His antibody (Qiagen) were used at dilutions 1: 1000 and 1: 5000, respectively. Secondary horseradish peroxidase-conjugated species-specific antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used at a dilution 1: 20 000. Bands were visualized using ECL kit and ECL hyperfilm (Amersham Biosciences).
Cell culture and transfection
The experiments were performed with the mouse proximal tubule mPCT cell line, which lacks endogenous electrogenic sodium bicarbonate cotransport (Gross et al. 2001a). Cells were studied between passages 15 and 25. Cells were transiently transfected with kNBC1constructs as previously described (Gross et al. 2001a). For transfection, cells were grown in mouse renal tubular epithelium (mRTE) medium containing a 1: 1 mixture of DMEM and Ham's F12 medium and the following additives: 10 ng ml–1 EGF, 5 µg ml–1 insulin, 5 µg ml–1 transferrin, 4 µg ml–1 dexamethasone, 10 units ml–1 interferon-
, 2 mM glutamine and 5% fetal bovine serum, on filters. Cells were transfected with the corresponding plasmid using Lipofectamine (Gibco, Grand Island, NY, USA) as per the manufacturer's protocol. Mock-transfected cells were transfected with the vector only. All plasmids were purified with EndofreeTM plasmid purification kit (Qiagen) prior to their use.
HCO3–: Na+ stoichiometry and cotransporter flux
mPCT cells were grown on permeable filter supports, mounted in an Ussing chamber, and permeabilized with amphotericin B as previously described (Gross et al. 2001a,b). The stoichiometry of the cotransporter was determined from Erev and eqn (1) (Gross et al. 2001a, b):
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Statistics
Experiments were performed at least 4 times. The results for the reversal potential and stoichiometry are presented as the mean ±S.E.M. Student's unpaired t test was used for statistical analysis, with P < 0.05 considered significant. Dunnet's t test was used to compare multiple experimental groups with controls.
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Results |
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Binding of human wt-kNBC1-ct to the human CAII is illustrated in Fig. 3. The data indicate that non-specific binding to Glutathione Sepharose is 5% of the total binding at 1 µM concentration of the binding peptide. This concentration was utilized in the binding studies with the wild-type or mutant kNBC1-ct variants. Binding of the N986NNN mutant of kNBC1-ct to CAII at this concentration was 10% of the wild-type kNBC1-ct (Fig. 3).
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90% supporting the hypothesis of the involvement of acidic amino acid residues in this process. Replacement of Asp986 with asparagine decreased the binding by 70%. Asp988 and Asp989 were responsible for 20% and 15% of total binding, respectively. Therefore Asp988 and Asp989 play a significantly less important role in the interaction between the kNBC1-ct and CAII than Asp986.
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75% indicating that the acidic amino acid residues in this motif are also important for CAII binding. Comparison of the single residue mutants L958DNV and L958NDV indicated that Asp959 in the L958DDV motif is significantly more important for CAII binding than Asp960 (80%versus 25%). The replacement of Leu958 with asparagine decreased the binding by 20%, contrary to the lack of binding shown for replacement of leucine in the LDADD motif of AE1 (Vince et al. 2000). The data indicate that Asp959 plays a key role in binding of CAII to the L958DDV motif of kNBC1 and that Leu958 and Asp960 play a minor role in binding to CAII. The data shown in Fig. 4 indicate that two clusters of acidic amino acids in the C-terminus of kNBC1 are involved in binding of CAII. Furthermore, the data suggest that the first aspartates in these clusters, Asp959 and Asp986, are very important for the strength of the interaction, whereas Asp988 and Asp989 in the D986NDD motif, and Asp960 and also Leu958 in the L958DDV motif play significantly smaller roles.
Binding of the full-length kNBC1 to CAII in mouse kidney extracts
Additional studies were performed to determine whether the full-length wt-kNBC1 could bind CAII. The wt-kNBC1 extracted from mouse kidney, which is highly homologous (97% identity) to the full-length human kNBC1 with identical L958DDV and D986NDD motifs in the C-terminus, was passed through a Glutathione Sepharose column with the bound wt-CAII. The results of this experiment, presented in Fig. 5, indicate that CAII binds to the full-length wt-kNBC1 confirming results of the in vitro binding of the recombinant kNBC1-ct to CAII.
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We have previously shown that ACTZ inhibits flux through kNBC1 in mPCT cells when it functions with a transport stoichiometry of 3: 1 and does not affect the flux in the 2: 1 mode (Gross et al. 2002). The stoichiometry of the cotransporter was shifted from 3: 1 to 2: 1 following the PKA-dependent phosphorylation of kNBC1-Ser982 (Gross et al. 2001b). Furthermore, we hypothesized (Gross & Kurtz, 2002) that the phosphorylation of kNBC1-Ser982 might prevent binding of CAII to the cotransporter. Therefore in the present study we determined how phosphorylation of Ser982 affects the binding of CAII. For these experiments, the C-terminal His6-tagged kNBC1 construct was in vitro phosphorylated by PKA and used in the CAII binding assay. The results (Fig. 6) show that phosphorylation of Ser982 increases the binding of CAII. The replacement of Ser982 with acidic aspartate residue known to mimic negatively charged phosphate at physiological pH (Hoeffler et al. 1994; Kwak et al. 1999; Lin et al. 2000) did not significantly change the binding of kNBC1-ct to CAII, indicating that the interaction is not only charge but also size specific.
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Lack of effect of CAII binding on the baseline HCO3–: Na+ stoichiometry of kNBC1
The functional significance of the binding data was evaluated in electrophysiological studies using mPCT cells transfected with the wild-type and mutant kNBC1 constructs. Two functional parameters were determined in these studies: the HCO3–: Na+ stoichiometry of kNBC1 and the flux through the cotransporter. Figure 7A summarizes the effect of CAII binding on kNBC1 stoichiometry. All the L958DDV and D986NDD motif mutants retain a stoichiometry of 3: 1 as in wt-kNBC1, indicating that binding of CAII to wt-kNBC1 does not affect the baseline cotransporter stoichiometry. In order to determine whether loss of CAII activity without perturbing its binding to kNBC1 affected the cotransporter stoichiometry, additional experiments were done using mPCT cells expressing wt-kNBC1. Following inhibition of CAII activity with 0.1 mM ACTZ, the stoichiometry of kNBC1 remained 3: 1. In mutants with decreased binding ACTZ also did not affect the kNBC1 stoichiometry indicating inhibition of CA activity not associated with kNBC1 also does not affect the cotransporter stoichiometry. Therefore neither binding of CAII to kNBC1 nor inhibition of its enzymatic activity affects the baseline stoichiometry of the cotransporter.
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We have previously shown (Gross et al. 2001b) that the PKA-mediated phosphorylation of Ser982 in kNBC1 shifts the cotransporter stoichiometry from 3: 1 to 2: 1. The stoichiometry shift is dependent on two aspartate residues in the C-terminus of the cotransporter, Asp986 and Asp988 (Gross et al. 2002), which are components of the D986NDD motif also involved in CAII binding (Fig. 4A). These results suggest that CAII binding to the D986NDD motif is associated with the PKA-mediated shift of kNBC1 stoichiometry. Nevertheless, two mutants with similar binding of CAII were identified, D986NND and D986NDN (Fig. 7A), of which only one (D986NDN) was able to shift the stoichiometry from 3: 1 to 2: 1. Both mutants were associated with a 20% decrease in CAII binding (Fig. 7A). Therefore, the inability of cAMP treatment to shift the HCO3–: Na+ stoichiometry in the D986NND mutant was not associated with decreased CAII binding. These findings suggest that Asp988 and Asp989 play similar roles in CAII binding, but that factor(s) other than CAII binding are also likely to be involved in the PKA-mediated shift in kNBC1 transport stoichiometry.
Effect of CAII on kNBC1-mediated flux: evidence that the complex of kNBC1 and CAII functions as a transport metabolon
Inhibition of CAII activity decreased the flux through kNBC1 by 65% when the cotransporter functions with a 3: 1 transport stoichiometry (Fig. 8A). Further studies were done to determine whether the ability of the enzymatic activity of CAII to modulate the flux through the cotransporter requires that the proteins bind normally. In these experiments, the ability of ACTZ to inhibit the flux through kNBC1 was assessed in mPCT cells expressing kNBC1 mutants with different ability to bind CAII in vitro. As shown in Fig. 8A, in the kNBC1 mutants, which had impaired CAII binding, the ability of ACTZ to inhibit kNBC1-mediated flux was also significantly diminished. Moreover, mutation of the L958DDV and D986NDD motifs showed that there was a significant positive linear correlation (r= 0.95) between CAII binding and flux inhibition by ACTZ (Fig. 8B). The flux through the kNBC1 N986NNN and N986NND mutants with significantly impaired binding to CAII was not affected by ACTZ (Figs 8A and B). The data indicate that when CAII is bound to kNBC1, its enzymatic activity can efficiently enhance the current through the cotransporter, whereas the activity of unbound (cytoplasmic) CAII does not appear to play a role in kNBC1-mediated transport. Based on these findings, we propose that a complex of kNBC1 and CAII exists on the basolateral membrane of the proximal tubule cells that functions as a transport metabolon.
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Discussion |
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We identified in the C-terminus of kNBC1 two acidic motifs, L958DDV and D986NDD involved in binding to CAII. Since kNBC1 binds CAII in a 1: 1 molar ratio (Gross et al. 2002) and therefore has one CAII binding site, our data suggest that the CAII binding site in kNBC1 consists of at least two acidic motifs (Fig. 9). In contrast, only one acidic motif, LDADD, in the C-terminus of AE1 was shown to be responsible for the binding to CAII (Vince et al. 2000). The reason for this difference is not clear but could arise from the different experimental techniques used for CAII binding assay in our study and by Vince et al. (2000). Specifically, in the current study, immobilization techniques were not used, which could potentially modify the CAII binding sites on the cotransporter. Of interest, Dahl et al. (2003) have shown that additional C-terminal amino acid residues in the kidney variant of AE1 (kAE1) are required for the CAII-enhanced kAE1-mediated bicarbonate transport. In contrast to AE1, the leucine residue in the L958DDV motif in kNBC1 is not absolutely required for the CAII binding since its replacement with asparagine decreased the binding only by 20%. The difference between L958DDV (kNBC1) and LDADD (AE1) motifs in CAII binding suggested that additional amino acid residues in the C-terminus of kNBC1 might be involved in the enzyme binding. This hypothesis was further supported in the experiments demonstrating the role of a second kNBC1 acidic motif, D986NDD, in CAII binding.
Analysis of the homology between members of the SLC4 bicarbonate transporter superfamily suggests that they might bind CAII through their C-termini. Recent data indicate that CAII binds not only to AE1, but also in addition to AE2 and AE3 (Sterling et al. 2002), NBC3 (Loiselle et al. 2004), and NBC4 (Kurtz et al. unpublished observations). In addition, Alvarez et al. (2003) have shown that carbonic anhydrase IV interacts with pNBC1. It is interesting to note that a bicarbonate transporter down-regulated in adenoma (DRA), which belongs to the SLC26 gene family, binds CAII to a significantly lesser extent than kNBC1 and AE1 (Sterling et al. 2003). It was therefore suggested that the transport activity of DRA is not modulated by CAII binding. Importantly, the 42 amino acid C-terminus of DRA used by Sterling et al. (2003) in their binding experiments did not contain clusters of acidic amino acids that could potentially bind CAII. The membrane topology prediction analysis (http://www.ch.embnet.org/software/TMPRED_form.html) indicates that the C-terminal domain of DRA is longer than 42 residues and contains acidic amino acid clusters that might be involved in CAII binding. Importantly, treatment of the DRA-expressing HEK293 cells with 0.1 mM ACTZ decreased their bicarbonate transport activity by 53% indicating that CA activity is important for the DRA-mediated bicarbonate transport. Therefore, additional experiments are required to determine whether CAII binds to DRA, and to evaluate the importance of this potential interaction for the DRA-mediated bicarbonate transport as well as other bicarbonate transporting members of the SLC26 family.
In the proximal tubule, intracelluluar CAII enhances the rate of hydration of CO2 to form H2CO3, which dissociates to H+ that is recycled across the apical membrane via the Na+–H+ exchanger isoform NHE3 (Biemesderfer et al. 1997, 2001), and HCO3–, which is transported across the basolateral membrane via kNBC1. Recently, it has been shown that CAII binds another member of the Na+–H+ exchanger gene family, NHE1 (Li et al. 2002), and the two proteins may function as a transport metabolon. (Li et al. 2002). Whether NHE3 is capable of interacting with CAII has not been addressed. Ion flux through NHE3 and kNBC1 is currently thought to be coupled via various factors including intracellular Na+ and pH/HCO3–, and PKA-dependent phosphorylation of each transporter (Kurashima et al. 1977; Gross et al. 2001b). Whether changes in intracellular Na+ and pH/HCO3– can alter the extent of binding of CAII to kNBC1 and provide an additional mechanism for modulating its transport properties remains to be determined. Furthermore, the concept that not only kNBC1 but in addition NHE3 can bind CAII and function as a transport metabolon is an attractive hypothesis that requires further study.
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References |
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Alvarez BV, Loiselle FB, Supuran CT, Schuartz GJ & Casey JR (2003). Direct extracellular interaction between carbonic anhydrase IV and the human NBC1 sodium/bicarbonate co-transporter. Biochemistry 42, 12321–12329.[CrossRef][Medline]
Biemesderfer D, DeGray B & Aronson PS (2001). Active (9.6 S) and inactive (21, S0 oligomers of NHE3 in microdomains of the renal brush border. J Biol Chem 276, 10161–10167.
Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK & Aronson PS (1997). Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol 273, F289–F299.[Medline]
Burckhardt BC, Cassola AC & Fromter E (1984). Electrophysiological analysis of bicarbonate permeation across the peritubular cell membrane of rat kidney proximal tubule. II. Exclusion of HCO3– effects on other ion permeabilities and of coupled electroneutral HCO3– transport. Pflugers Arch 401, 43–51.[CrossRef][Medline]
Burg M & Green N (1977). Bicarbonate transport by isolated perfused rabbit proximal tubules. Am J Physiol 233, F307–F314.[Medline]
Burnham C, Amlal H, Wang Z, Shull GE & Soleimani M (1997). Cloning and functional expression of a human kidney Na+-HCO3– cotransporter. J Biol Chem 72, 19111–19114.
Dahl NK, Jiang L, Chernova MN, Stuart-Tilley AK, Shmukler BE & Alper SL (2003). Deficient HCO3– transport in an AE1 mutant with normal Cl– transport can be rescued by carbonic anhydrase II presented on an adjacent AE1 protomer. J Biol Chem 278, 44949–44958.
Gross E, Fedotoff O, Pushkin A, Abuladze N, Newman D & Kurtz I (2003). Phosphorylation-induced modulation of pNBC1 function. distinct roles for the amino- and carboxy-termini. J Physiol 549, 673–682.
Gross E, Hawkins K, Abuladze N, Pushkin A, Cotton CU, Hopfer U & Kurtz I (2001a). The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent. J Physiol 531, 597–603.
Gross E, Hawkins K, Pushkin A, Abuladze N, Hopfer U, Sassani P, Dukkipati R & Kurzt I (2001b). Phosphorylation of Ser982 in kNBC1 shifts the HCO3–: Na+ stoichiometry from 3: 1 to 2: 1 in proximal tubule cells. J Physiol 537, 659–665.
Gross E & Kurtz I (2002). Structural determinants and significance of regulation of electrogenic Na+-HCO3– cotransporter stoichiometry. Am J Physiol 283, F876–F587.
Gross E, Pushkin A, Abuladze N, Fedotoff O & Kurtz I (2002). Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp986, Asp988 and kNBC1-carbonic anhydrase II binding. J Physiol 544, 679–685.
Hoeffler WK, Levinson AD & Bauer EA (1994). Activation of c-Jun transcription factor by substitution of a charged residue in its N-terminal domain. Nucl Acid Res 22, 1305–1312.
Kunimi M, Muller-Berger S, Hara C, Samarzija I, Seki G & Fromter E (2000). Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3– absorption to norepinephrine. Pflugers Arch 440, 908–917.[CrossRef][Medline]
Kurashima K, Yu FH, Cabado AG, Szabo EZ, Grinstein S & Orlowski J (1977). Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms. J Biol Chem 272, 28672–28679.[CrossRef]
Kurtz I, Petrasek D & Tatishchev S (2004). Molecular mechanisms of electrogenic sodium bicarbonate cotransport: structural and equilibrium thermodynamic considerations. J Membr Biol 197, 77–90.[CrossRef][Medline]
Kwak YG, Hu N, Wei J, George, AL Jr, Grobaski TD, Tamkun MM & Murray KT (1999). Protein kinase A phosphorylation alters Kvß1.3 subunit-mediated inactivation of the Kv1.5 potassium channel. J Biol Chem 274, 13928–13932.
Li X, Alvarez B, Casey JR, Reithmeier RAF & Fleigel L (2002). Carbonic anhydrase II binds to and enhances activity of Na+/H+ exchanger. J Biol Chem 277, 36085–36091.
Lin YF, Jan YN & Jan LY (2000). Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells. EMBO J 19, 942–955.[CrossRef][Medline]
Loiselle FB, Molgan PE, Alvarez BV & Casey JR (2004). Regulation of the human NBC3 Na+/HCO3– cotransporter by Carbonic anhydrase II & PKA. Am J Physiol 286, C1423–C1433.[CrossRef]
McKinney TD & Burg M (1977). Bicarbonate and fluid absorption by renal proximal straight tubules. Kidney Int 12, 1–8.[Medline]
Marino CR, Jeanes V, Boron WF & Schmitt BM (1999). Expression and distribution of the Na+-HCO3– cotransporter in human pancreas. Am J Physiol 277, G487–G494.[Medline]
Miles EW, Rhee S & Devies DR (1999). The molecular basis of substrate channeling. J Biol Chem 274, 12193–12196.
Muller-Berger S, Coppola S, Samarzija I, Seki G & Fromter E (1997b). Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. I. Change of amiloride-inhibitable K+ conductance. Pflugers Arch 434, 373–382.[CrossRef][Medline]
Muller-Berger S, Nesterov VV & Fromter E (1997a). Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide. Pflugers Arch 434, 383–391.[CrossRef][Medline]
Reithmeier RAF (2001). A membrane metabolon linking carbonic anhydrase with chloride/bicarbonate anion exchangers. Blood Cells Mol Dis 27, 85–89.[CrossRef][Medline]
Romero MF, Hediger MA, Boulpaep EL & Boron WF (1997). Expression cloning and characterization of a renal electrogenic Na+/HCO3– cotransporter. Nature 387, 409–413.
Sasaki S & Marumo F (1989). Effects of carbonic anhydrase inhibitors on basolateral base transport of rabbit proximal straight tubule. Am J Physiol 257, F947–F952.[Medline]
Schmitt BM, Biemesderfer D, Romero MF, Boulpaep EL & Boron WF (1999). Immunolocalization of the electrogenic Na+-HCO3– cotransporter in mammalian and amphibian kidney. Am J Physiol 276, F27–F38.[Medline]
Seki G & Fromter E (1992). Acetazolamide inhibition of basolateral base exit in rabbit renal proximal tubule S2 segment. Pflugers Arch 422, 60–65.[CrossRef][Medline]
Srere PA (1985). The metabolon. Trends Biochem Sci 10, 109–110.[CrossRef]
Sterling D, Alvarez BV & Casey JR (2002). The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl–/HCO3– exchanger binds carbonic anhydrase IV. J Biol Chem 277, 25239–25246.
Sterling D, Brown NJ, Supuran C & Casey JR (2003). The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II. Am J Physiol 283, C1522–C1529.
Sterling D, Reithmeier RAF & Casey JR (2001a). A transport metabolon. Functional interaction of carbonic anhydrase II and chroride/bicarbonate exchangers. J Biol Chem 276, 47886–47894.
Sterling D, Reithmeier RAF & Casey JR (2001b). Carbonic anhydrase: in the driver's seat for bicarbonate transport. J Pancreas 2, 165–170.
Tatishchev S, Abuladze N, Pushkin A, Newman D, Lu W, Weeks D, Sachs G & Kurtz I (2003). Identification of membrane topography of the electrogenic sodium bicarbonate cotransporter pNBC1 by in vitro transcription/translation. Biochemistry 42, 755–765.[CrossRef][Medline]
Tsuruoka S, Swenson ER, Petrovic S, Fujimura A & Schwartz GJ (2001). Role of basolateral carbonic anhydrase in proximal tubular fluid and bicarbonate absorption. Am J Physiol 280, F146–F154.
Velot C, Mixon MB, Tiege M & Srere PA (1997). Model of the quinary structure between Krebs TCA cycle enzymes. a model for the metabolon. Biochemistry 36, 14271–14276.[CrossRef][Medline]
Vince JW, Carlsson U & Reithmeier RA (2000). Localization of the Cl–/HCO3– anion exchanger binding site to the amino-terminal region of carbonic anhydrase II. Biochemistry 39, 13344–13349.[CrossRef][Medline]
Vince JW & Reithmeier RAF (1998). Carbonic anhydrase II binds to the carboxyl-terminus of human band 3, the erythrocyte Cl–/HCO3– exchanger. J Biol Chem 273, 28430–28437.
Vince JW & Reithmeier RAF (2000). Identification of the carbonic anhydrase II binding site in the Cl–/HCO3– anion exchanger AE1. Biochemistry 39, 5527–5533.[CrossRef][Medline]
Yoshitomi K, Burckhardt BC & Fromter E (1987). Rheogenic sodium-bicarbonate transport in the peritubular cell membrane of rat renal proximal tubule. Pflugers Arch 405, 360–366.[CrossRef]
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) and the N986NNN mutant His6–kNBC1-ct (
) constructs to GST-CAII bound to Glutathione Sepharose beads was determined. In control experiments binding of the purified human His6-tagged wild-type kNBC1-ct to Glutathione Sepharose beads alone was studied (•). The data are shown as a percentage of maximum wild-type kNBC1-ct binding.
