Missense mutations in Na+:HCO3– cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular ac
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《美国生理学杂志》
Departments of Medicine and Surgery, University of Cincinnati, Cincinnati, Ohio
Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio
ABSTRACT
The kidney Na+:HCO3– cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH2 terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
acid-base balance; sodium hydrogen exchanger type 3; bicarbonate absorption
THE BASOLATERAL Na+:HCO3– cotransporter NBC1 functions in tandem with the apical Na+/H+ exchanger 3 (NHE3) in the proximal tubule and is essential for the reabsorption of majority of filtered bicarbonate load (3, 5, 7, 17, 22, 23, 29). Reduction in the Na+:HCO3– cotransporter activity interferes with the reabsorption of bicarbonate and results in proximal renal tubular acidosis (RTA). Recent studies reported several missense mutations in human NBC1 (SLC4A4) gene from families with proximal RTA (10, 11). Functional analysis in transfected nonpolarized cells or oocytes demonstrated that NBC activity was significantly reduced in R510H, R298S, and S427L mutants (2, 10, 11). Whereas the R298S and R510H mutants were thought to be targeted to the membrane (11), the possibility of abnormal trafficking in these two mutants has been raised (2). The S427L mutant shows membrane localization in oocytes (10).
Recent studies in nonepithelial cells showed that certain AE1 mutants associated with distal RTA were functionally active and trafficked into the plasma membrane in nonpolarized cells (6, 14, 21, 30). However, transfection of these AE1 mutants in polarized kidney cells showed mistargeting to the apical membrane, raising the possibility of renal bicarbonate wasting and generation of RTA in patients with these mutations (9, 25).
Our recent studies on the Na+:HCO3– cotransporter NBC1 identified the presence of a novel COOH-terminal motif (QQPFLS) that is spanning amino acid residues 1010 to 1015 and is essential for the targeting of NBC1 to the basolateral membrane (18). Deletion of this motif or substitution of the amino acid F within this motif resulted in the retargeting of NBC1 to the apical membrane in polarized kidney cells. The apically targeted NBC1 was functionally active (18).
The purpose of the current experiments was to examine the membrane domain targeting of NBC1 mutants that causes proximal RTA, as no studies have examined the trafficking of these mutants in polarized cells. Toward this end, NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in cultured polarized kidney epithelial cells. Parallel studies were performed in oocytes to examine the trafficking of NBC1 mutants along with their activities. The results suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly mistargeted to the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
MATERIALS AND METHODS
Construction of GFP-NBC1 full-length and point mutations. The full-length NBC1 was generated by PCR, using the human full-length kidney NBC1 DNA (3,257 bp and 1,035 amino acid residues) as a template (AF007216 [GenBank] ). The amplified wild-type NBC1 DNA was fused translationally in-frame to GFP by cloning into pcDNA3.1/NT-GFP-TOPO vector (Invitrogen, Carlsbad, CA).
Na+:HCO3– cotransporter NBC1 mutants R510H and S427L were generated with a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). We also generated the NBC1 mutant E91R, which is not a documented human mutation but has been reported to reside adjacent to the R298 and may display properties similar to R298S mutation (24). Sense and antisense primers of missense R510H, S427L, and E91R mutants were designed as follows: NBC1 R510H sense primer: 5'-GACTATTTGGAGTTTCACCTTTGGATTGGCCTG TGG-3', NBC1 R510H antisense primer: 5'-CCACAGGCCAATCCAAAGGTGAAA CTCCAAATAGTC; NBC1 E91R sense primer: 5'-GCCAGGTGGATCAAGTTTCG AGAAAAAGTGGAACAGGG-3', NBC1 E91R antisense primer: 5'-CCCTGTTCCA CTTTTTCTCGAAACTTGATCCACCTGGC-3'; NBC1 S427L sense primer: 5'-CAAG CTTTTGGCAATTCTCTTC-3', NBC1 S427L antisense primer: 5'-GAAGAGAATT GCCAAAAGAGCTTG-3'. We also generated the pancreatic NBC1 R342S mutant that is identical to the kidney NBC1 R298S mutant (11), as both pancreatic and kidney NBC1 variants are identical except for the fact that the first 41 amino acids of the NH2-terminal end of kidney NBC1 are replaced with 85 amino acids in pancreatic NBC1 (1). As a result, the amino acid 298 in kidney NBC1 corresponds to the amino acid 342 in pancreatic NBC1. Toward this end, the full-length human pancreatic NBC1 was generated by RT-PCR, using the human heart RNA. The sense and antisense primers for the pancreatic NBC1 (AF069510 [GenBank] ) were 5'-GAATTCAGGATGGAGGATGAAGCTGTCCTG-3' and5'-AGCGGCCGCCTCAGCATGATGTGTGGCGTTCAAGGAATGT-3', respectively. The amplified wild-type pNBC1 DNA was fused translationally in-frame to GFP by cloning into pcDNA3.1/NT-GFP-TOPO vector (Invitrogen). The sense primer of pNBC1 R342S is 5'-CCACGAGATTGGCAGCGCCATTGCCACCCTG-3' and the antisense primer of pNBC1 R342S is 5'-CAGGGTGGCAATGGCGCTGCCAATCTCGTGG-3'. The Na+:HCO3– cotransporter pNBC1 mutant R342S was generated with QuikChange Site-Directed Mutagenesis Kit (Stratagene) as above. Cycling parameters for the QuikChange site-directed mutagenesis method are the following: segment 1, 95°C, 30 s, 1 cycle; and segment 2, 95°C, 30 s, 55°C, 1 min, 68°C, 6 min, 18 cycles. So, GFP NBC1 R510H, GFP NBC1 E91R, and GFP NBC1 S427L mutants were generated in the same way. All the DNA sample sequences were confirmed by DNA Core Facility of University of Cincinnati and Children's Hospital. Scheme 1 depicts the constructs of wild-type GFP-kNBC1 and GFP-kNBC1 mutants.
Scheme 1
Transient expression of wild-type GFP-NBC1 and GFP-NBC1 mutants in Madin-Darby canine kidney cells. The plasmids of pcDNA3.1/NT-GFP-NBC1-TOPO and GFP NBC1 mutant constructs confirmed by DNA sequencing were transformed into TOP 10 competent cells, and a single colony was picked up for growing in 50 ml of TB culture medium for each sample. A Qiagen EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA) was used to prepare and purify endotoxin-free plasmid. Madin-Darby canine kidney (MDCK) cells were transiently transfected with the wild-type GFP-NBC1 and GFP-NBC1 mutants and studied 48 h later according to established methods (18). Briefly, cells were plated in 24-well plates and transfected at 80% confluence using 1 μg of DNA and 4 μl of Lipofectamine 2000 (Invitrogen). The transfection efficiency was monitored using PCMV-SPORT -gal plasmid as control. A change in cell color in response to X-gal addition was used as a marker for -gal expression. All cells were colabeled with peanut lectin or phalloidin as markers of apical membrane or cell membrane labeling. Several independent transient transfection experiments were performed for each mutant. MDCK cells show 5–10% positive transfection rate with endotoxin-free plasmids.
Immunofluorescence labeling and confocal microscopy. MDCK cells were washed three times with PBS, fixed for 15 min at 4% paraformaldehyde in PBS, and washed three more times with PBS. Afterward, cells were permeabilized for 4 min with 0.1% Triton X-100 in PBS, washed three times with PBS, and costained with PNA-lectin (Molecular Probes, Eugene, OR) or the selective F-actin dye TRITC-phalloidin (Sigma, St. Louis, MO). Afterward, cells were washed three times with PBS, dehydrated with 50, 70, and 100% ethanol, and mounted on glass slides in Slow Fade with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes). Images were taken on a Zeiss LSM510 confocal microscope. Both Z-line and Z-stack images were obtained using the LSM 5 Image software to analyze the membrane targeting of GFP-NBC1 full-length and mutant constructs (18). More than 10 transfected cells were observed and scanned with Zeiss LSM510 confocal microscope.
Expression of full-length GFP-NBC1 and GFP-NBC1 mutants in oocytes. Stage IV-V oocytes were isolated, as previously described, and used for expression studies according to established methods (7, 18, 32). The capped GFP-NBC1 (full-length), GFP-NBC1-R510H, GFP-NBC1-S427L, GFP-NBC1-E91R, and GFP alone (no NBC insert) cRNAs were generated using mMESSAGE mMACHINE T7 Kit (from Ambion, Austin, TX) according to the manufacturer's instruction. Fifty nanoliters of cRNA (0.5 μg/μl) were injected with a Drummond 510 microdispenser via a sterile glass pipette with a tip of 20–30 μm. After injection, the oocytes were maintained in a solution of the following composition (in mM): 96 NaCl, 2.0 KCl, 1.0 MgCl2, 1.8 CaCl2, 5.0 HEPES, 2.5 Na pyruvate, 0.5 theophylline, 100 U/ml penicillin, and 100 μg/ml streptomycin; pH 7.5. Injected oocytes were stored in an incubator at 17°C and were used for electrophysiological experiments after 2 to 4 days.
Electrophysiology studies. Oocytes were placed on a nylon mesh in a perfusion chamber and continuously perfused (3 ml/min perfusion rate). The perfusion solution had the following composition (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH 7.5). After a stabilization period, when the membrane potential (Vm) was constant, the perfusion solution was switched to a CO2/bicarbonate-containing solution of the following composition (in mM): 30 NaHCO3, 66 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 15 HEPES (pH 7.5 and gassed with 5% CO2). Experiments were performed at room temperature (22–25°C). Membrane potentials were recorded with conventional microelectrode techniques by glass microelectrodes (resistance 3–5 m) filled with 3 M KCl and connected to an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) (7, 18, 32). The digitized signals were stored and analyzed on a personal computer using Axotape (Axon Instruments).
Western blot analysis. To examine the abundance of membrane-bound NBC1 in oocytes, oocytes were homogenized in lysis buffer in the presence or absence of 2% Triton X-100. The homogenized mixture without Triton X-100 was centrifuged at 14,000 g and the resultant supernatant was saved. This fraction is designated as C (cytoplasmic) and is predominantly comprised of the cytoplasmic contents. The homogenized mixture with 2% Triton X-100 was centrifuged and designated as M (membrane), which is predominantly comprised of the membrane proteins, with little cytoplasmic component. The lysis buffer without detergent consists of 2 mMEDTA, 0.1% -mercaptoethanol, 0.025% PMSF, 2 μg/ml leupeptin, 20 μg/ml aprotinin, 250 mM sucrose, and 100 mM dithiothreitol; the lysis buffer with detergent was the same as above, with 2% Triton X-100 added to the mixture. The protein concentration was estimated by Lowry assay (Bio-Rad, Hercules, CA). Western blot analysis of cytoplasmic or membrane fractions was performed according to established methods (4, 16) using GFP-specific polyclonal antibody (Invitrogen) at 1:500 dilution. Donkey anti-rabbit IgG-horseradish peroxidase (HRP) was used as the secondary antibody (Pierce, Rockford, IL). For estimation of protein loading in Western blots, goat -actin polyclonal antibody (1:1,000) was used (Santa Cruz Biotechnology, Santa Cruz, CA) as control, with rabbit anti-goat IgG-HRP (1:1,000) as the secondary antibody (The Jackson Laboratory, Bar Harbor, ME). The antigen-antibody complex was detected by chemiluminescence method using SuperSignal West Pico Chemiluminescent Substrate kit (Pierce). The experiments were repeated multiple times and GFP protein expression was normalized by -actin labeling on the same blot.
Statistics. Results are given as means ± SE. Statistical comparisons between the groups were performed by ANOVA. The data were considered significant if P < 0.05.
Materials. A high-fidelity PCR Amplification Kit, GFP Fusion TOPO TA Expression Kit, and Lipofectamine 2000 were purchased from Invitrogen. A mMESSAGE mMACHINE T7 Kit was purchased from Ambion. A EndoFree Plasmid Maxi Kit was purchased from Qiagen. DMEM (high glucose) medium was purchased from Life Technologies. All other chemicals were purchased from Sigma.
RESULTS
In the first series of experiments, we examined the expression and targeting of GFP with or without the wild-type NBC1 insert in MDCK cells. Figure 1A, top and bottom, shows the expression of GFP vector alone (no NBC1 insert). As demonstrated, GFP was predominantly accumulated in the cytoplasm (Z-stack images in top) with no localization on the membrane (Z-line images in bottom). However, transfection with the GFP-NBC1 full-length cDNA showed exclusive localization in the membrane (as visualized by Z-stack, front view) in cells colabeled with phalloidin (Fig. 1B). The specific membrane labeling (apical vs. basolateral) was examined using the Z-line image (side view) analysis. The results are demonstrated in Fig. 1C. Similar studies were performed in cells colabeled with PNA-lectin, a very specific marker of apical membrane labeling in polarized cells (9, 18). The Z-line (side view) images of the results (NBC1-GFP and PNA-lectin double labeling) are depicted in Fig. 1D. As demonstrated, the full-length NBC1 was targeted exclusively to the basolateral membrane domain (Fig. 1, C and D). These results are consistent with published reports on the basolateral localization of NBC1 in epithelial cells (7, 13, 17, 20, 22, 23, 26–29). Not all MDCK cells were labeled with PNA-lectin, presumably due to GFP labeling.
In the next series of experiments, we examined the expression of GFP-NBC1 mutants R510H, E91R, and S427L (see MATERIALS AND METHODS). Figure 2A demonstrated Z-stack (front view) image analysis of the expression of GFP-NBC1 mutant R510H colabeled with phalloidin in MDCK cell. The results demonstrate that GFP-NBC1-R510H mutant is predominantly retained in the cytoplasm, with some GFP signal detected on the membrane. The Z-line (side view) image analysis of the expression studies labeled with phalloidin or PNA-lectin demonstrated that in addition to the cytoplasm (Fig. 2A), GFP-NBC1-R510H mutant showed basolateral membrane localization with faint labeling on the apical membrane (Fig. 2, B and C). These results indicate that missense mutation from Arg to His in amino acid residue 510 of kidney NBC1 results in the predominant retention of the mutant protein in the cytoplasm. Figure 3 depicts results with GFP-kNBC1-E91R mutant. The Z-stack (front view) image analysis of GFP-kNBC1-E91R mutant colabeled with phalloidin (Fig. 3A) shows membrane as well as cytoplasmic localization. The Z-line image analysis (side view) of the expression studies demonstrated that GFP-kNBC1-E91R mutant colabeled with phalloidin or PNA-lectin showed basolateral localization with faint labeling on apical membrane (Fig. 3, B and C).
Next, we examined the expression of GFP-kNBC1-S427L mutant in MDCK cells according to the above method. The Z-stack (front view) expression of GFP-kNBC1-S427L mutant colabeled with phalloidin demonstrated the predominant localization of this mutant on the membrane with some cytoplasmic retention (Fig. 4A). The Z-line (side view) image analysis of the results showed predominant localization of the mutant protein on the apical as well as basolateral membrane, with some cytoplasmic retention (Fig. 4B). Colabeling with PNA-lectin confirmed the apical and basolateral membrane localization of GFP-kNBC1–427L mutant (Fig. 4C).
Some of the NBC1 missense mutations have been examined in oocytes (10). In the next series of experiments, we examined the functional activity and expression of wild-type GFP-kNBC1 and GFP-kNBC1 mutants (R510H, E91R, and S427L) in frog oocytes. Functional activity was measured by membrane potential recording, and membrane expression was determined by Western blot analysis. To correlate the function of NBC1 (wild-type or mutants) with membrane expression directly, Western blotting was performed on the same oocytes used for functional assay. Toward this end, membrane potential was first measured in oocytes injected with GFP, full-length GFP-kNBC1, or GFP-kNBC1 NBC1 mutant (R510H, E91R, or S427L) cRNA as described. Thereafter, oocytes were homogenized in the lysis buffer with or without 2% Triton X-100 (see MATERIALS AND METHODS) and subjected to Western blot analysis.
Membrane potential was measured by conventional intracellular microelectrodes in oocytes 2 to 4 days after injection with cRNA for GFP alone, full-length GFP-kNBC1, or GFP-kNBC1 NBC1 mutants (R510H, E91R, S427L). After an equilibrating period, the perfusion solution was switched to a solution containing 30 mM HCO3– and gassed with 5% CO2-95% O2 at pH 7.5. This solution allows for the inward movement of NBC1 down the Na and bicarbonate gradients. Figure 5A shows representative membrane potential tracings and Fig. 5B demonstrates the results of several experiments. A continuous line (Fig. 5A) indicates the time of exposure to CO2/HCO3–. Exposure of control (GFP) oocytes to CO2/HCO3– did not alter the membrane potential (Fig. 5A, panel 1). Exposure of oocytes that were injected with the full-length GFP-kNBC1 cRNA to CO2/HCO3– resulted in an immediate and sustained hyperpolarization (Fig. 5A, panel 2). Exposure of oocytes that were injected with R510H or E91R mutant to CO2/HCO3– resulted in a hyperpolarization that was significantly reduced (Fig. 5A, panels 3 and 4). Interestingly, exposure of oocytes that were injected with the GFP-kNBC1-S427L mutant cRNA to CO2/HCO3– resulted in no hyperpolarization at all (Fig. 5A, panel 5). The hyperpolarizations in Fig. 5A were reversible on removal of CO2/HCO3– from the perfusion medium. Figure 5B depicts results summation. As indicated, the resting membrane potential (Vm) was not different in wild-type or mutant NBCs (P > 0.05). As demonstrated, a pronounced hyperpolarization on introduction of CO2/HCO3– was observed only in GFP-kNBC-1-injected oocytes. The changes in Vm for GFP-kNBC1-injected oocytes (65.7 ± 2.6) were significantly different vs. other mutants (R510H: 12.6 ± 1.7; E91R: 8.5 ± 1.9; S427L: 1.6 ± 1.0; P < 0.0001 vs. each mutant, n = 6 for each mutant).
To correlate the functional results with NBC1 membrane expression in oocytes, Western blot analysis of membrane and cytoplasmic fractions was performed in oocytes injected with cRNA for wild-type or NBC1 mutants. As demonstrated, oocytes injected with the GFP-kNBC1 cRNA showed abundant expression in the membrane fraction (M) with little expression in the cytoplasm (C) (Fig. 6, left). -Actin abundance is shown as the loading control. In contrast to wild-type NBC1, GFP-NBC1-R510H and GFP-NBC1-E91R mutants showed abundant cytoplasmic expression, with lower expression in the membrane (Fig. 6, panels 2 and 3 from left). -Actin blots are shown for control of loading. Interestingly, GFP-NBC1-S427L showed significant expression in membrane fraction when results were adjusted for protein loading (-actin), with 78% of the NBC1 detected in the membrane and the other 22% in the cytoplasmic fraction (Fig. 6).
In the last series of experiments, we examined the expression of GFP pancreatic NBC1 mutant R342S, which is the homolog of kidney NBC1 mutant R298S. Our attempts to generate the kidney NBC1 R298S mutant construct were not successful but for the purpose of tracking its trafficking, the pancreatic NBC1-R342S mutant was generated, fused in frame to GFP (see MATERIALS AND METHODS), transiently expressed in MDCK cells, and examined. For comparison, the wild-type pancreatic NBC1 cDNA was fused to GFP (see MATERIALS AND METHODS) and examined. As demonstrated in Fig. 7A (Z-stack or front view), wild-type pancreatic NBC1 was localized predominantly on the membrane with very low cytoplasmic retention. The Z-line images (side view) demonstrated the predominant localization of wild-type pancreatic NBC1 on the basolateral membrane (Fig. 7B). Figure 7, C and D, demonstrates the expression and localization of the GFP-pNBC1-R342S mutant in MDCK cells. The Z-stack (front view) images of GFP-pNBC1-R342S mutant colabeled with phalloidin demonstrated the predominant localization of this mutant on the membrane with some cytoplasmic labeling (Fig. 7C). The Z-line (side view) image analysis of the results showed predominant localization of the mutant protein on the apical as well as the basolateral membrane, with some cytoplasmic retention (Fig. 7D).
DISCUSSION
NBC1, also referred to as SLC4A4, is a member of the AE/NBC superfamily and is located basolaterally in various epithelia. NBC1 mediates vectorial transport of bicarbonate in epithelial tissues. In the kidney, NBC1 is located on the basolateral membrane of proximal tubules and mediates the exit of bicarbonate from cell to blood. In the pancreas, NBC1 is located on the basolateral membrane of ducts and mediates the entry of bicarbonate from blood to cell (1, 4, 8, 13, 17, 19, 20, 23, 26, 27, 29). The kidney NBC1 variant (kNBC1) has a stoichiometry of three equivalent of bicarbonate per sodium, whereas the pancreatic variant (pNBC1) has a stoichiometry of two bicarbonate per sodium. In both the pancreatic duct as well as proximal tubule, NBC1 is the main mechanism for the transport of bicarbonate across the basolateral membrane.
Several missense mutations in NBC1 have been identified in patients with RTA, a condition manifested by decreased reabsorption of bicarbonate in the kidney proximal tubule with consequent renal bicarbonate wasting and systemic metabolic acidosis (10–12). Aside from the Q29X mutation that results in loss of NBC1 function due to abrupt truncation of kidney NBC1 protein, the molecular basis of RTA in other reported NBC1 missense mutations is less well understood. Functional studies in cultured nonepithelial cells or oocytes indicate that NBC1 activity is significantly decreased in missense mutations R510H, S427L, and R298S (2, 10, 11). However, the basis of NBC1 dysregulation in these missense mutations is less well understood, as no study has examined the expression of these mutants in polarized epithelial cells. Furthermore, one of these mutants (R510H) was examined in cultured nonepithelial cells, whereas the other mutant (S427L) was studied in oocytes (10). As such, in addition to the lack of knowledge on membrane targeting (basolateral vs. apical) of these mutants, the question of possible impact of the host expression system (mammalian vs. non mammalian) remains unanswered.
To address these question, wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH2 terminally tagged GFP, and transiently expressed in MDCK cells, a well-known mammalian epithelial cell line. Confocal microscopy was used to determine the apical, cytoplasmic, or basolateral localization of wild-type and mutant NBC1s. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and function of NBC1s. Our results demonstrate that wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain, confirming published reports on NBC1 localization in epithelial cells (Fig. 1). However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm, with residual labeling on the apical membrane (Fig. 2). Interestingly, GFP-NBC1 mutant S427L was predominantly detected on the apical membrane with residual cytoplasmic retention as well as basolateral membrane labeling (Fig. 4). GFP-kNBC1-E91R mutant was predominantly mistargeted to the cytoplasm with residual membrane labeling in MDCK cells (Fig. 3).
In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction (Fig. 6), confirming the studies in MDCK cells and consistent with the expected membrane expression of this cotransporter. However, NBC1-R510H and -E91R mutants were predominantly expressed in the cytoplasm, whereas NBC1-S427L mutant was predominantly targeted to the membrane (Fig. 6). Functional analysis of NBC1 activity by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R, E91R, and S427L exhibited significant reduction in their activity in response to exposure to CO2/HCO3– (Fig. 5).
The current studies demonstrate abnormal trafficking of NBC1 mutants in polarized epithelial cells, with some well-known missense mutations causing mistargeting of NBC1 to the apical membrane and some other mutations causing cytoplasmic retention of the cotransporter. Recent studies from our laboratories identified a COOH-terminal motif (QQPFLS) on kidney NBC1, which is essential for the basolateral targeting of NBC1 (18). Our studies demonstrated that the deletion of this motif or mutagenesis of the amino acid F within this motif causes the retargeting of NBC1 to the apical membrane (18). Distinct from the present studies, the apically targeted NBC1 mutants in those studies were functionally active (18). The amino acid residues that are mutated in NBC1 are not in proximity to the COOH-terminal motif QQPFLS (18) based on primary structure. However, whether any of these residues is juxtaposed with the QQPFLS in three-dimensional structure remains unknown.
Recent studies identified mutations in the Cl–/HCO3– exchanger AE1 in patients with distal RTA, a bicarbonate wasting condition involving distal nephron. AE1, which is normally located on the basolateral membrane of (A) intercalated cells in cortical and outer medullary collecting duct, works in tandem with the apical H+-ATPase (12, 29). Bicarbonate wasting in patients with distal RTA raised the possibility that AE1 mutants became functionally inactive. However, studies with AE1 mutants in nonpolarized cells were less conclusive (6, 14, 21, 30). Very recent studies by Denovald et al. and Rungroj et al. (9, 25) showed that AE1 mutants are mistargeted to the apical membrane. Given the functionality of AE1 mutants in nonepithelial cells, Denovald et al. and Rungroj et al. concluded that the mistargeting of AE1 to the apical membrane was responsible for bicarbonate wasting, as the apically located AE1 secretes bicarbonate into the lumen in exchange for luminal chloride. Coupled to the current studies, we suggest that a number of mutations in bicarbonate-absorbing transporters in the kidney (NBC1 or AE1) cause the mistargeting of the bicarbonate transporter to the apical membrane. It is worth mentioning that other AE1 mutations cause the retention of this exchanger in the endoplasmic reticulum or in late endosomes/lysosomes, therefore causing distal RTA by reducing bicarbonate absorption rather than secretion in the collecting duct (21, 31).
Functional studies in oocytes demonstrated that despite considerable abundance in the membrane (Fig. 6), all NBC1 mutants showed significant reduction in their activity (Fig. 5). The reduction in NBC1 activity was most striking in S427L mutant, which showed almost no activity (Fig. 5), despite considerable expression level in the membrane (Fig. 6). S427L mutant showed significant targeting to the membrane in MDCK cells as well, although a good portion of this labeling was retargeted to the apical membrane (Fig. 4). Taken together, these studies demonstrate that the missense mutation S427L is an inactivating mutation that in addition causes the mistargeting of NBC1 to the apical membrane. In contrast to the apically targeted AE1 mutations that cause RTA by secreting bicarbonate into the distal nephron, the NBC1 S427L missense mutation causes RTA by decreasing bicarbonate reabsorption in the proximal nephron as this mutation renders the cotransporter inactive. The H510R mutation similarly causes proximal RTA by decreasing bicarbonate reabsorption in the proximal tubule. Whether we can draw any conclusions from the pancreatic NBC1-R342S mutation to its kidney NBC1 mutant homolog (NBC1-R298S) remains to be seen. Current ongoing studies are attempting to address this question.
In conclusion, our findings suggest that the permanent isolated proximal RTA in patients with the H510R or S427L missense mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
GRANTS
These studies were supported by National Institutes of Health Grants DK-62809 (to M. Soleimani), DK-51630 (to J. B. Matthews), and CA-095286 (to L. Conforti), a Merit Review Award, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic, Inc. (to M. Soleimani).
FOOTNOTES
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.
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Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio
ABSTRACT
The kidney Na+:HCO3– cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH2 terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
acid-base balance; sodium hydrogen exchanger type 3; bicarbonate absorption
THE BASOLATERAL Na+:HCO3– cotransporter NBC1 functions in tandem with the apical Na+/H+ exchanger 3 (NHE3) in the proximal tubule and is essential for the reabsorption of majority of filtered bicarbonate load (3, 5, 7, 17, 22, 23, 29). Reduction in the Na+:HCO3– cotransporter activity interferes with the reabsorption of bicarbonate and results in proximal renal tubular acidosis (RTA). Recent studies reported several missense mutations in human NBC1 (SLC4A4) gene from families with proximal RTA (10, 11). Functional analysis in transfected nonpolarized cells or oocytes demonstrated that NBC activity was significantly reduced in R510H, R298S, and S427L mutants (2, 10, 11). Whereas the R298S and R510H mutants were thought to be targeted to the membrane (11), the possibility of abnormal trafficking in these two mutants has been raised (2). The S427L mutant shows membrane localization in oocytes (10).
Recent studies in nonepithelial cells showed that certain AE1 mutants associated with distal RTA were functionally active and trafficked into the plasma membrane in nonpolarized cells (6, 14, 21, 30). However, transfection of these AE1 mutants in polarized kidney cells showed mistargeting to the apical membrane, raising the possibility of renal bicarbonate wasting and generation of RTA in patients with these mutations (9, 25).
Our recent studies on the Na+:HCO3– cotransporter NBC1 identified the presence of a novel COOH-terminal motif (QQPFLS) that is spanning amino acid residues 1010 to 1015 and is essential for the targeting of NBC1 to the basolateral membrane (18). Deletion of this motif or substitution of the amino acid F within this motif resulted in the retargeting of NBC1 to the apical membrane in polarized kidney cells. The apically targeted NBC1 was functionally active (18).
The purpose of the current experiments was to examine the membrane domain targeting of NBC1 mutants that causes proximal RTA, as no studies have examined the trafficking of these mutants in polarized cells. Toward this end, NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in cultured polarized kidney epithelial cells. Parallel studies were performed in oocytes to examine the trafficking of NBC1 mutants along with their activities. The results suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly mistargeted to the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
MATERIALS AND METHODS
Construction of GFP-NBC1 full-length and point mutations. The full-length NBC1 was generated by PCR, using the human full-length kidney NBC1 DNA (3,257 bp and 1,035 amino acid residues) as a template (AF007216 [GenBank] ). The amplified wild-type NBC1 DNA was fused translationally in-frame to GFP by cloning into pcDNA3.1/NT-GFP-TOPO vector (Invitrogen, Carlsbad, CA).
Na+:HCO3– cotransporter NBC1 mutants R510H and S427L were generated with a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). We also generated the NBC1 mutant E91R, which is not a documented human mutation but has been reported to reside adjacent to the R298 and may display properties similar to R298S mutation (24). Sense and antisense primers of missense R510H, S427L, and E91R mutants were designed as follows: NBC1 R510H sense primer: 5'-GACTATTTGGAGTTTCACCTTTGGATTGGCCTG TGG-3', NBC1 R510H antisense primer: 5'-CCACAGGCCAATCCAAAGGTGAAA CTCCAAATAGTC; NBC1 E91R sense primer: 5'-GCCAGGTGGATCAAGTTTCG AGAAAAAGTGGAACAGGG-3', NBC1 E91R antisense primer: 5'-CCCTGTTCCA CTTTTTCTCGAAACTTGATCCACCTGGC-3'; NBC1 S427L sense primer: 5'-CAAG CTTTTGGCAATTCTCTTC-3', NBC1 S427L antisense primer: 5'-GAAGAGAATT GCCAAAAGAGCTTG-3'. We also generated the pancreatic NBC1 R342S mutant that is identical to the kidney NBC1 R298S mutant (11), as both pancreatic and kidney NBC1 variants are identical except for the fact that the first 41 amino acids of the NH2-terminal end of kidney NBC1 are replaced with 85 amino acids in pancreatic NBC1 (1). As a result, the amino acid 298 in kidney NBC1 corresponds to the amino acid 342 in pancreatic NBC1. Toward this end, the full-length human pancreatic NBC1 was generated by RT-PCR, using the human heart RNA. The sense and antisense primers for the pancreatic NBC1 (AF069510 [GenBank] ) were 5'-GAATTCAGGATGGAGGATGAAGCTGTCCTG-3' and5'-AGCGGCCGCCTCAGCATGATGTGTGGCGTTCAAGGAATGT-3', respectively. The amplified wild-type pNBC1 DNA was fused translationally in-frame to GFP by cloning into pcDNA3.1/NT-GFP-TOPO vector (Invitrogen). The sense primer of pNBC1 R342S is 5'-CCACGAGATTGGCAGCGCCATTGCCACCCTG-3' and the antisense primer of pNBC1 R342S is 5'-CAGGGTGGCAATGGCGCTGCCAATCTCGTGG-3'. The Na+:HCO3– cotransporter pNBC1 mutant R342S was generated with QuikChange Site-Directed Mutagenesis Kit (Stratagene) as above. Cycling parameters for the QuikChange site-directed mutagenesis method are the following: segment 1, 95°C, 30 s, 1 cycle; and segment 2, 95°C, 30 s, 55°C, 1 min, 68°C, 6 min, 18 cycles. So, GFP NBC1 R510H, GFP NBC1 E91R, and GFP NBC1 S427L mutants were generated in the same way. All the DNA sample sequences were confirmed by DNA Core Facility of University of Cincinnati and Children's Hospital. Scheme 1 depicts the constructs of wild-type GFP-kNBC1 and GFP-kNBC1 mutants.
Scheme 1
Transient expression of wild-type GFP-NBC1 and GFP-NBC1 mutants in Madin-Darby canine kidney cells. The plasmids of pcDNA3.1/NT-GFP-NBC1-TOPO and GFP NBC1 mutant constructs confirmed by DNA sequencing were transformed into TOP 10 competent cells, and a single colony was picked up for growing in 50 ml of TB culture medium for each sample. A Qiagen EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA) was used to prepare and purify endotoxin-free plasmid. Madin-Darby canine kidney (MDCK) cells were transiently transfected with the wild-type GFP-NBC1 and GFP-NBC1 mutants and studied 48 h later according to established methods (18). Briefly, cells were plated in 24-well plates and transfected at 80% confluence using 1 μg of DNA and 4 μl of Lipofectamine 2000 (Invitrogen). The transfection efficiency was monitored using PCMV-SPORT -gal plasmid as control. A change in cell color in response to X-gal addition was used as a marker for -gal expression. All cells were colabeled with peanut lectin or phalloidin as markers of apical membrane or cell membrane labeling. Several independent transient transfection experiments were performed for each mutant. MDCK cells show 5–10% positive transfection rate with endotoxin-free plasmids.
Immunofluorescence labeling and confocal microscopy. MDCK cells were washed three times with PBS, fixed for 15 min at 4% paraformaldehyde in PBS, and washed three more times with PBS. Afterward, cells were permeabilized for 4 min with 0.1% Triton X-100 in PBS, washed three times with PBS, and costained with PNA-lectin (Molecular Probes, Eugene, OR) or the selective F-actin dye TRITC-phalloidin (Sigma, St. Louis, MO). Afterward, cells were washed three times with PBS, dehydrated with 50, 70, and 100% ethanol, and mounted on glass slides in Slow Fade with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes). Images were taken on a Zeiss LSM510 confocal microscope. Both Z-line and Z-stack images were obtained using the LSM 5 Image software to analyze the membrane targeting of GFP-NBC1 full-length and mutant constructs (18). More than 10 transfected cells were observed and scanned with Zeiss LSM510 confocal microscope.
Expression of full-length GFP-NBC1 and GFP-NBC1 mutants in oocytes. Stage IV-V oocytes were isolated, as previously described, and used for expression studies according to established methods (7, 18, 32). The capped GFP-NBC1 (full-length), GFP-NBC1-R510H, GFP-NBC1-S427L, GFP-NBC1-E91R, and GFP alone (no NBC insert) cRNAs were generated using mMESSAGE mMACHINE T7 Kit (from Ambion, Austin, TX) according to the manufacturer's instruction. Fifty nanoliters of cRNA (0.5 μg/μl) were injected with a Drummond 510 microdispenser via a sterile glass pipette with a tip of 20–30 μm. After injection, the oocytes were maintained in a solution of the following composition (in mM): 96 NaCl, 2.0 KCl, 1.0 MgCl2, 1.8 CaCl2, 5.0 HEPES, 2.5 Na pyruvate, 0.5 theophylline, 100 U/ml penicillin, and 100 μg/ml streptomycin; pH 7.5. Injected oocytes were stored in an incubator at 17°C and were used for electrophysiological experiments after 2 to 4 days.
Electrophysiology studies. Oocytes were placed on a nylon mesh in a perfusion chamber and continuously perfused (3 ml/min perfusion rate). The perfusion solution had the following composition (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH 7.5). After a stabilization period, when the membrane potential (Vm) was constant, the perfusion solution was switched to a CO2/bicarbonate-containing solution of the following composition (in mM): 30 NaHCO3, 66 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 15 HEPES (pH 7.5 and gassed with 5% CO2). Experiments were performed at room temperature (22–25°C). Membrane potentials were recorded with conventional microelectrode techniques by glass microelectrodes (resistance 3–5 m) filled with 3 M KCl and connected to an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) (7, 18, 32). The digitized signals were stored and analyzed on a personal computer using Axotape (Axon Instruments).
Western blot analysis. To examine the abundance of membrane-bound NBC1 in oocytes, oocytes were homogenized in lysis buffer in the presence or absence of 2% Triton X-100. The homogenized mixture without Triton X-100 was centrifuged at 14,000 g and the resultant supernatant was saved. This fraction is designated as C (cytoplasmic) and is predominantly comprised of the cytoplasmic contents. The homogenized mixture with 2% Triton X-100 was centrifuged and designated as M (membrane), which is predominantly comprised of the membrane proteins, with little cytoplasmic component. The lysis buffer without detergent consists of 2 mMEDTA, 0.1% -mercaptoethanol, 0.025% PMSF, 2 μg/ml leupeptin, 20 μg/ml aprotinin, 250 mM sucrose, and 100 mM dithiothreitol; the lysis buffer with detergent was the same as above, with 2% Triton X-100 added to the mixture. The protein concentration was estimated by Lowry assay (Bio-Rad, Hercules, CA). Western blot analysis of cytoplasmic or membrane fractions was performed according to established methods (4, 16) using GFP-specific polyclonal antibody (Invitrogen) at 1:500 dilution. Donkey anti-rabbit IgG-horseradish peroxidase (HRP) was used as the secondary antibody (Pierce, Rockford, IL). For estimation of protein loading in Western blots, goat -actin polyclonal antibody (1:1,000) was used (Santa Cruz Biotechnology, Santa Cruz, CA) as control, with rabbit anti-goat IgG-HRP (1:1,000) as the secondary antibody (The Jackson Laboratory, Bar Harbor, ME). The antigen-antibody complex was detected by chemiluminescence method using SuperSignal West Pico Chemiluminescent Substrate kit (Pierce). The experiments were repeated multiple times and GFP protein expression was normalized by -actin labeling on the same blot.
Statistics. Results are given as means ± SE. Statistical comparisons between the groups were performed by ANOVA. The data were considered significant if P < 0.05.
Materials. A high-fidelity PCR Amplification Kit, GFP Fusion TOPO TA Expression Kit, and Lipofectamine 2000 were purchased from Invitrogen. A mMESSAGE mMACHINE T7 Kit was purchased from Ambion. A EndoFree Plasmid Maxi Kit was purchased from Qiagen. DMEM (high glucose) medium was purchased from Life Technologies. All other chemicals were purchased from Sigma.
RESULTS
In the first series of experiments, we examined the expression and targeting of GFP with or without the wild-type NBC1 insert in MDCK cells. Figure 1A, top and bottom, shows the expression of GFP vector alone (no NBC1 insert). As demonstrated, GFP was predominantly accumulated in the cytoplasm (Z-stack images in top) with no localization on the membrane (Z-line images in bottom). However, transfection with the GFP-NBC1 full-length cDNA showed exclusive localization in the membrane (as visualized by Z-stack, front view) in cells colabeled with phalloidin (Fig. 1B). The specific membrane labeling (apical vs. basolateral) was examined using the Z-line image (side view) analysis. The results are demonstrated in Fig. 1C. Similar studies were performed in cells colabeled with PNA-lectin, a very specific marker of apical membrane labeling in polarized cells (9, 18). The Z-line (side view) images of the results (NBC1-GFP and PNA-lectin double labeling) are depicted in Fig. 1D. As demonstrated, the full-length NBC1 was targeted exclusively to the basolateral membrane domain (Fig. 1, C and D). These results are consistent with published reports on the basolateral localization of NBC1 in epithelial cells (7, 13, 17, 20, 22, 23, 26–29). Not all MDCK cells were labeled with PNA-lectin, presumably due to GFP labeling.
In the next series of experiments, we examined the expression of GFP-NBC1 mutants R510H, E91R, and S427L (see MATERIALS AND METHODS). Figure 2A demonstrated Z-stack (front view) image analysis of the expression of GFP-NBC1 mutant R510H colabeled with phalloidin in MDCK cell. The results demonstrate that GFP-NBC1-R510H mutant is predominantly retained in the cytoplasm, with some GFP signal detected on the membrane. The Z-line (side view) image analysis of the expression studies labeled with phalloidin or PNA-lectin demonstrated that in addition to the cytoplasm (Fig. 2A), GFP-NBC1-R510H mutant showed basolateral membrane localization with faint labeling on the apical membrane (Fig. 2, B and C). These results indicate that missense mutation from Arg to His in amino acid residue 510 of kidney NBC1 results in the predominant retention of the mutant protein in the cytoplasm. Figure 3 depicts results with GFP-kNBC1-E91R mutant. The Z-stack (front view) image analysis of GFP-kNBC1-E91R mutant colabeled with phalloidin (Fig. 3A) shows membrane as well as cytoplasmic localization. The Z-line image analysis (side view) of the expression studies demonstrated that GFP-kNBC1-E91R mutant colabeled with phalloidin or PNA-lectin showed basolateral localization with faint labeling on apical membrane (Fig. 3, B and C).
Next, we examined the expression of GFP-kNBC1-S427L mutant in MDCK cells according to the above method. The Z-stack (front view) expression of GFP-kNBC1-S427L mutant colabeled with phalloidin demonstrated the predominant localization of this mutant on the membrane with some cytoplasmic retention (Fig. 4A). The Z-line (side view) image analysis of the results showed predominant localization of the mutant protein on the apical as well as basolateral membrane, with some cytoplasmic retention (Fig. 4B). Colabeling with PNA-lectin confirmed the apical and basolateral membrane localization of GFP-kNBC1–427L mutant (Fig. 4C).
Some of the NBC1 missense mutations have been examined in oocytes (10). In the next series of experiments, we examined the functional activity and expression of wild-type GFP-kNBC1 and GFP-kNBC1 mutants (R510H, E91R, and S427L) in frog oocytes. Functional activity was measured by membrane potential recording, and membrane expression was determined by Western blot analysis. To correlate the function of NBC1 (wild-type or mutants) with membrane expression directly, Western blotting was performed on the same oocytes used for functional assay. Toward this end, membrane potential was first measured in oocytes injected with GFP, full-length GFP-kNBC1, or GFP-kNBC1 NBC1 mutant (R510H, E91R, or S427L) cRNA as described. Thereafter, oocytes were homogenized in the lysis buffer with or without 2% Triton X-100 (see MATERIALS AND METHODS) and subjected to Western blot analysis.
Membrane potential was measured by conventional intracellular microelectrodes in oocytes 2 to 4 days after injection with cRNA for GFP alone, full-length GFP-kNBC1, or GFP-kNBC1 NBC1 mutants (R510H, E91R, S427L). After an equilibrating period, the perfusion solution was switched to a solution containing 30 mM HCO3– and gassed with 5% CO2-95% O2 at pH 7.5. This solution allows for the inward movement of NBC1 down the Na and bicarbonate gradients. Figure 5A shows representative membrane potential tracings and Fig. 5B demonstrates the results of several experiments. A continuous line (Fig. 5A) indicates the time of exposure to CO2/HCO3–. Exposure of control (GFP) oocytes to CO2/HCO3– did not alter the membrane potential (Fig. 5A, panel 1). Exposure of oocytes that were injected with the full-length GFP-kNBC1 cRNA to CO2/HCO3– resulted in an immediate and sustained hyperpolarization (Fig. 5A, panel 2). Exposure of oocytes that were injected with R510H or E91R mutant to CO2/HCO3– resulted in a hyperpolarization that was significantly reduced (Fig. 5A, panels 3 and 4). Interestingly, exposure of oocytes that were injected with the GFP-kNBC1-S427L mutant cRNA to CO2/HCO3– resulted in no hyperpolarization at all (Fig. 5A, panel 5). The hyperpolarizations in Fig. 5A were reversible on removal of CO2/HCO3– from the perfusion medium. Figure 5B depicts results summation. As indicated, the resting membrane potential (Vm) was not different in wild-type or mutant NBCs (P > 0.05). As demonstrated, a pronounced hyperpolarization on introduction of CO2/HCO3– was observed only in GFP-kNBC-1-injected oocytes. The changes in Vm for GFP-kNBC1-injected oocytes (65.7 ± 2.6) were significantly different vs. other mutants (R510H: 12.6 ± 1.7; E91R: 8.5 ± 1.9; S427L: 1.6 ± 1.0; P < 0.0001 vs. each mutant, n = 6 for each mutant).
To correlate the functional results with NBC1 membrane expression in oocytes, Western blot analysis of membrane and cytoplasmic fractions was performed in oocytes injected with cRNA for wild-type or NBC1 mutants. As demonstrated, oocytes injected with the GFP-kNBC1 cRNA showed abundant expression in the membrane fraction (M) with little expression in the cytoplasm (C) (Fig. 6, left). -Actin abundance is shown as the loading control. In contrast to wild-type NBC1, GFP-NBC1-R510H and GFP-NBC1-E91R mutants showed abundant cytoplasmic expression, with lower expression in the membrane (Fig. 6, panels 2 and 3 from left). -Actin blots are shown for control of loading. Interestingly, GFP-NBC1-S427L showed significant expression in membrane fraction when results were adjusted for protein loading (-actin), with 78% of the NBC1 detected in the membrane and the other 22% in the cytoplasmic fraction (Fig. 6).
In the last series of experiments, we examined the expression of GFP pancreatic NBC1 mutant R342S, which is the homolog of kidney NBC1 mutant R298S. Our attempts to generate the kidney NBC1 R298S mutant construct were not successful but for the purpose of tracking its trafficking, the pancreatic NBC1-R342S mutant was generated, fused in frame to GFP (see MATERIALS AND METHODS), transiently expressed in MDCK cells, and examined. For comparison, the wild-type pancreatic NBC1 cDNA was fused to GFP (see MATERIALS AND METHODS) and examined. As demonstrated in Fig. 7A (Z-stack or front view), wild-type pancreatic NBC1 was localized predominantly on the membrane with very low cytoplasmic retention. The Z-line images (side view) demonstrated the predominant localization of wild-type pancreatic NBC1 on the basolateral membrane (Fig. 7B). Figure 7, C and D, demonstrates the expression and localization of the GFP-pNBC1-R342S mutant in MDCK cells. The Z-stack (front view) images of GFP-pNBC1-R342S mutant colabeled with phalloidin demonstrated the predominant localization of this mutant on the membrane with some cytoplasmic labeling (Fig. 7C). The Z-line (side view) image analysis of the results showed predominant localization of the mutant protein on the apical as well as the basolateral membrane, with some cytoplasmic retention (Fig. 7D).
DISCUSSION
NBC1, also referred to as SLC4A4, is a member of the AE/NBC superfamily and is located basolaterally in various epithelia. NBC1 mediates vectorial transport of bicarbonate in epithelial tissues. In the kidney, NBC1 is located on the basolateral membrane of proximal tubules and mediates the exit of bicarbonate from cell to blood. In the pancreas, NBC1 is located on the basolateral membrane of ducts and mediates the entry of bicarbonate from blood to cell (1, 4, 8, 13, 17, 19, 20, 23, 26, 27, 29). The kidney NBC1 variant (kNBC1) has a stoichiometry of three equivalent of bicarbonate per sodium, whereas the pancreatic variant (pNBC1) has a stoichiometry of two bicarbonate per sodium. In both the pancreatic duct as well as proximal tubule, NBC1 is the main mechanism for the transport of bicarbonate across the basolateral membrane.
Several missense mutations in NBC1 have been identified in patients with RTA, a condition manifested by decreased reabsorption of bicarbonate in the kidney proximal tubule with consequent renal bicarbonate wasting and systemic metabolic acidosis (10–12). Aside from the Q29X mutation that results in loss of NBC1 function due to abrupt truncation of kidney NBC1 protein, the molecular basis of RTA in other reported NBC1 missense mutations is less well understood. Functional studies in cultured nonepithelial cells or oocytes indicate that NBC1 activity is significantly decreased in missense mutations R510H, S427L, and R298S (2, 10, 11). However, the basis of NBC1 dysregulation in these missense mutations is less well understood, as no study has examined the expression of these mutants in polarized epithelial cells. Furthermore, one of these mutants (R510H) was examined in cultured nonepithelial cells, whereas the other mutant (S427L) was studied in oocytes (10). As such, in addition to the lack of knowledge on membrane targeting (basolateral vs. apical) of these mutants, the question of possible impact of the host expression system (mammalian vs. non mammalian) remains unanswered.
To address these question, wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH2 terminally tagged GFP, and transiently expressed in MDCK cells, a well-known mammalian epithelial cell line. Confocal microscopy was used to determine the apical, cytoplasmic, or basolateral localization of wild-type and mutant NBC1s. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and function of NBC1s. Our results demonstrate that wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain, confirming published reports on NBC1 localization in epithelial cells (Fig. 1). However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm, with residual labeling on the apical membrane (Fig. 2). Interestingly, GFP-NBC1 mutant S427L was predominantly detected on the apical membrane with residual cytoplasmic retention as well as basolateral membrane labeling (Fig. 4). GFP-kNBC1-E91R mutant was predominantly mistargeted to the cytoplasm with residual membrane labeling in MDCK cells (Fig. 3).
In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction (Fig. 6), confirming the studies in MDCK cells and consistent with the expected membrane expression of this cotransporter. However, NBC1-R510H and -E91R mutants were predominantly expressed in the cytoplasm, whereas NBC1-S427L mutant was predominantly targeted to the membrane (Fig. 6). Functional analysis of NBC1 activity by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R, E91R, and S427L exhibited significant reduction in their activity in response to exposure to CO2/HCO3– (Fig. 5).
The current studies demonstrate abnormal trafficking of NBC1 mutants in polarized epithelial cells, with some well-known missense mutations causing mistargeting of NBC1 to the apical membrane and some other mutations causing cytoplasmic retention of the cotransporter. Recent studies from our laboratories identified a COOH-terminal motif (QQPFLS) on kidney NBC1, which is essential for the basolateral targeting of NBC1 (18). Our studies demonstrated that the deletion of this motif or mutagenesis of the amino acid F within this motif causes the retargeting of NBC1 to the apical membrane (18). Distinct from the present studies, the apically targeted NBC1 mutants in those studies were functionally active (18). The amino acid residues that are mutated in NBC1 are not in proximity to the COOH-terminal motif QQPFLS (18) based on primary structure. However, whether any of these residues is juxtaposed with the QQPFLS in three-dimensional structure remains unknown.
Recent studies identified mutations in the Cl–/HCO3– exchanger AE1 in patients with distal RTA, a bicarbonate wasting condition involving distal nephron. AE1, which is normally located on the basolateral membrane of (A) intercalated cells in cortical and outer medullary collecting duct, works in tandem with the apical H+-ATPase (12, 29). Bicarbonate wasting in patients with distal RTA raised the possibility that AE1 mutants became functionally inactive. However, studies with AE1 mutants in nonpolarized cells were less conclusive (6, 14, 21, 30). Very recent studies by Denovald et al. and Rungroj et al. (9, 25) showed that AE1 mutants are mistargeted to the apical membrane. Given the functionality of AE1 mutants in nonepithelial cells, Denovald et al. and Rungroj et al. concluded that the mistargeting of AE1 to the apical membrane was responsible for bicarbonate wasting, as the apically located AE1 secretes bicarbonate into the lumen in exchange for luminal chloride. Coupled to the current studies, we suggest that a number of mutations in bicarbonate-absorbing transporters in the kidney (NBC1 or AE1) cause the mistargeting of the bicarbonate transporter to the apical membrane. It is worth mentioning that other AE1 mutations cause the retention of this exchanger in the endoplasmic reticulum or in late endosomes/lysosomes, therefore causing distal RTA by reducing bicarbonate absorption rather than secretion in the collecting duct (21, 31).
Functional studies in oocytes demonstrated that despite considerable abundance in the membrane (Fig. 6), all NBC1 mutants showed significant reduction in their activity (Fig. 5). The reduction in NBC1 activity was most striking in S427L mutant, which showed almost no activity (Fig. 5), despite considerable expression level in the membrane (Fig. 6). S427L mutant showed significant targeting to the membrane in MDCK cells as well, although a good portion of this labeling was retargeted to the apical membrane (Fig. 4). Taken together, these studies demonstrate that the missense mutation S427L is an inactivating mutation that in addition causes the mistargeting of NBC1 to the apical membrane. In contrast to the apically targeted AE1 mutations that cause RTA by secreting bicarbonate into the distal nephron, the NBC1 S427L missense mutation causes RTA by decreasing bicarbonate reabsorption in the proximal nephron as this mutation renders the cotransporter inactive. The H510R mutation similarly causes proximal RTA by decreasing bicarbonate reabsorption in the proximal tubule. Whether we can draw any conclusions from the pancreatic NBC1-R342S mutation to its kidney NBC1 mutant homolog (NBC1-R298S) remains to be seen. Current ongoing studies are attempting to address this question.
In conclusion, our findings suggest that the permanent isolated proximal RTA in patients with the H510R or S427L missense mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
GRANTS
These studies were supported by National Institutes of Health Grants DK-62809 (to M. Soleimani), DK-51630 (to J. B. Matthews), and CA-095286 (to L. Conforti), a Merit Review Award, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic, Inc. (to M. Soleimani).
FOOTNOTES
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.
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