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Am J Physiol Cell Physiol 293: C738-C748, 2007. First published July 25, 2007; doi:10.1152/ajpcell.00157.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters

Patricio E. Morgan,1 Silvia Pastoreková,2 Alan K. Stuart-Tilley,3 Seth L. Alper,3 and Joseph R. Casey1

1Membrane Protein Research Group, Department of Physiology and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; 2Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic; and 3Molecular and Vascular Medicine and Renal Units, Beth Israel Deaconess Medical Center, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts

Submitted 16 April 2007 ; accepted in final form 29 May 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3– conversions and coupled HCO3– transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl–/HCO3– exchanger AE2 or the putative Cl–/HCO3– exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl–/HCO3– exchangers to form a bicarbonate transport metabolon.

SLC4; SLC26; bicarbonate transport metabolon

SECRETION OF HYDROCHLORIC acid is central to digestion in the stomach. Parietal cells of the gastric mucosal glands actively secrete acid upon stimulation. The gastric H+-K+-ATPase located in the parietal cell apicovesicular membrane pumps H+ into the gastric lumen, accompanied by Cl– movement through Cl– channels. To sustain acid secretion, parietal cells must replace the HCl lost at the apical surface. Two coupled processes provide the required H+ and Cl–. 1) At the parietal cell basolateral surface, CO2 diffuses into the cell from adjacent capillaries. Cytosolic carbonic anhydrase II (CAII) catalyzes the conversion, CO2 + H2O {leftrightarrow} HCO3– + H+, thus supplying a H+. 2) In the basolateral membrane, Cl–/HCO3– exchangers concomitantly extrude HCO3– to regulate intracellular pH while loading the cell with Cl–. CAII activity is thus functionally coupled to Cl–/HCO3– exchange activity to load parietal cells with HCl for secretion (29, 32, 50).

The carbonic anhydrase (CA) gene family of zinc metalloproteins includes 16 isoforms in mammals differing in tissue and subcellular distribution (14, 42, 48). CAIX is anchored to the extracellular surface by a transmembrane segment, placing its highly active catalytic domain on the extracellular side of the plasma membrane (35). In normal tissues, CAIX is abundant only in mucosa of stomach and gallbladder (34), with lower expression in intestinal epithelia (41), pancreatic ducts (20), and epididymis (18). Greatly increased CAIX expression is, however, associated with local tissue hypoxia in many types of proliferating carcinomas, including clear cell adenocarcinoma of the kidney, squamous cell carcinoma of the cervix, and ovarian, colorectal, and esophageal carcinomas (35).

Gastric parietal cells are but one example illustrating the functional link between CAs and bicarbonate transport proteins in facilitation of transepithelial bicarbonate transport, in some cases through apparently direct interactions (4, 21, 41). Among these latter are the binding of intracellular CAII to the cytosolic COOH-terminal tails of the Cl–/HCO3– exchangers AE1 (53), AE2, AE3 (47), and SLC26A6 (6), and the Na+-HCO3– cotransporters NBCe1 (5, 7, 38) and NBC3/NBCn1 (24), and the binding of the glycosylphosphatidylinositol-linked exo-enzyme CAIV to extracellular loop four of AE1 (43). Localization of CAs immediately adjacent to bicarbonate transporters in bicarbonate transport metabolons may maximize the transmembrane bicarbonate concentration gradient in the immediate locale of the transporter polypeptide, thus increasing bicarbonate transport rate (47). Evidence arguing against the bicarbonate transport metabolon model has, however, recently been presented for AE1 (37) and for NBCe1 (25).

Bicarbonate transporters are widely expressed and involved in the regulation of intracellular pH and [Cl–], cell volume, cell migration, and trans-epithelial acid/base and Cl– secretion (2, 28, 39, 45). The widely expressed Cl–/HCO3– exchanger SLC4A2/AE2 (2, 39) is most abundant in stomach and choroids (3, 4). AE2 has in the past been considered responsible for most basolateral parietal cell uptake of Cl– destined for HCl secretion and most extrusion of HCO3– generated intracellularly during acid secretion (29, 32, 50). Indeed, an AE2 knockout mouse model exhibits greatly reduced gastric HCl secretion (12). More recently, the parietal cell basolateral membrane anion transporter SLC26A7 has been proposed to contribute to gastric HCl secretion (36). The transport mechanism of SLC26A7 remains controversial, with reports of function as a Cl– channel (19, 22) and as a Cl–/HCO3– exchanger (36).

Basolateral HCO3– efflux from parietal cells is required for apical acid secretion. Thus CAIX localization to the parietal cell basolateral membrane suggests a role in HCl secretion. Interaction of CAIX with basolateral Cl–/HCO3– exchangers of the parietal cell might serve physiologically to augment maximum achievable rates of acid secretion. Direct interaction of CAIX with Cl–/HCO3– exchangers would localize CAIX to the sites of HCO3– efflux on the basolateral cell surface, potentially minimizing diffusion barriers for CO2/HCO3– during acid secretion. This study addresses the possible functional and physical interaction of CAIX with the Cl–/HCO3– exchanger polypeptides of the parietal cell basolateral surface, AE2 and SLC26A7. We found that CAIX directly interacts with AE2 and increases its apparent transport activity, whereas CAIX interacts neither functionally nor physically with SLC26A7. The extracellular catalytic domain of CAIX mediates its interaction not only with AE2, but also with the related anion exchangers AE1 and AE3. CAIX is the first transmembrane CA shown directly to bind bicarbonate transporters. The CAIX/AE2 interaction may functionally contribute to the parietal cell HCl secretion apparatus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. ECL chemiluminescent reagent was from Perkin Elmer Life Sciences. Rabbit anti-mouse IgG conjugated to horseradish peroxidase was from Amersham Life Sciences. Anti-GST (Z-5) rabbit IgG, anti-HA (Y-11) rabbit IgG, and goat anti-rabbit IgG conjugated to horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-green fluorescence protein monoclonal antibody (A11121) was from Molecular Probes. Affinity-purified rabbit polyclonal antibody to mouse AE2 COOH-terminal amino acids 1224-1237 has been previously described. Acetazolamide (ACTZ), poly-L-lysine, and BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, acetoxymethyl ester] were from Sigma Canada (Oakville, Canada). DMEM, calf serum, and fetal bovine serum were from GIBCO-Invitrogen (Burlington, ON, Canada).

Molecular biology. Plasmid DNA for transfection was prepared using Qiagen columns (Qiagen, Mississauga, Canada). Mammalian cDNA expression constructs described previously include human erythroid AE1 (pJRC9) (8), catalytically inactive CAII mutant V143Y (47), and mammalian expression constructs for CAIX: fl-CAIX, {Delta}C-CAIX, {Delta}PG-CAIX have been described previously (33, 55). COOH-terminally hemagglutinin epitope (HA)-tagged rat AE2a was created using PCR mutagenesis with the forward primer 5'-GAT TCA GGA AGT CAA GGA G-3' and the mutagenic reverse oligonucleotide 5'-TCT GGA CAG CAG AAG CTT CTA GGC GTA GTC GGG CAC GTC GTA GGG GTA CAC AGG CAT GGG CAT-3' which introduces a new stop codon, following the HA tag and a HindIII site. BstEII/HindIII fragment was cloned into the same sites of pJF6 plasmid (10), and called pJF7. COOH-terminally HA-tagged rat full-length AE3 was constructed by PCR mutagenesis using the forward primer 5'-CTC CTC TGG GTG GTC AAG TC-3' and the mutagenic reverse oligonucleotide 5'-CCA TCT TGA GGG GAA TTC TCA GGC GTA GTC GGG CAC GTC GTA GGG GTA CAC AGG CAT GTG GAG-3', which introduces a new stop codon, following the HA-tag and an EcoRI site. Sfi1/EcoR1 fragment was cloned into the same sites of pJRC32 plasmid (46) and called pJF8. SLC26A7 cDNA was kindly provided by Dr. M. Soleimani in pGEM-T-Easy (Promega). cDNA encoding NH2-terminally HA-tagged SLC26A7 polypeptide was constructed by PCR. The forward primer (5'-CTA GCT AGC TAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TAC AGG AGC AAA GAG GAA AAA G-3') included an NheI site followed by a start codon, the HA tag sequence (YPDYDVPDYP) and the first seven codons of SLC26A7. The reverse primer (5'-CCG CTC GAG TCA GAC TTC ACT GTG GTC ACT G-3') encoded the COOH-terminal seven codons followed by an XhoI site. The cDNA sequence encoding the complete open reading frame was generated by PCR (30 cycles of 94°C for 15 s, 55°C for 30 s, 68°C for 150 s) and inserted into the NheI and XhoI sites of the mammalian expression vector pcDNA3.1(+) (Invitrogen Life Technologies) to generate pPM1. Yellow fluorescence protein (YFP) EYFP-V163S cDNA was expressed in mammalian expression vector pcDNA3.1(+) (11).

Tissue culture. Human AE1, rat AE2, and rat AE3 protein and CAIX constructs were expressed by transient transfection of HEK293 cells (13), using the calcium phosphate method (40). Cells were grown at 37°C in 5% CO2 in DMEM, supplemented with 5% (vol/vol) fetal bovine serum and 5% (vol/vol) calf serum.

Anion exchange activity assay. Two days posttransfection HEK293 cells on coverslips were rinsed in serum-free medium and incubated in 4 ml of serum-free medium, containing 2 µM BCECF-AM (37°C, 20 min). Coverslips were mounted in a fluorescence cuvette and perfused at 3.5 ml/min alternately with 5% CO2-bubbled Ringer buffer, containing either 140 mM sodium chloride or 140 mM sodium gluconate, along with (in mM) 5 glucose, 5 potassium gluconate, 1 calcium gluconate, 1 MgSO4, 2.5 NaH2PO4, 25 NaHCO3, 10 HEPES, pH 7.4. Fluorescence changes were monitored in a Photon Technologies International RCR fluorimeter at excitation wavelengths 440 and 503 nm and emission wavelength 529 nm. Fluorescence data were converted to pHi by calibration using the nigericin/high potassium method (51), with pH values of 6.5, 7.0, and 7.5. Transport rates were obtained from the first 100 s of alkalinization and acidification and determined as the slope (dpH/dt) of the line fitted by the least squares method.

Immunodetection. Two days posttransfection, cells were washed with PBS (140 mM NaCl, 3 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and lysates of the whole tissue culture cells were prepared by addition of SDS-PAGE sample buffer [20% (vol/vol) glycerol, 2% (vol/vol) 2-mercaptoethanol, 4% (wt/vol) SDS, 1% (wt/vol) bromophenol blue, 150 mM Tris, pH 6.8]. Before analysis, samples were sheared through a 26-gauge needle (Becton Dickinson) and heated to 65°C for 5 min. Samples were resolved by SDS-PAGE on 7.5% acrylamide gels (23). Proteins were transferred to PVDF membranes by electrophoresis for 1 h at 100 V at room temperature, in buffer composed of 20% (vol/vol) methanol, 25 mM Tris, and 192 mM glycine (52). PVDF membranes were blocked by incubation for 1 h in TBST-M buffer [TBST buffer 0.1% (vol/vol) Tween 20, 137 mM NaCl, 20 mM Tris, pH 7.5], containing 10% (wt/vol) nonfat dry milk and then incubated overnight in 10 ml TBST-M [5% (wt/vol) nonfat dry milk], containing 3 µl mouse anti-AE1 monoclonal antibody IVF12 (15). Blots were incubated for 1 h with 10 ml of TBST-M containing 1:3,000 diluted donkey anti-mouse IgG conjugated to horseradish peroxidase. Blots were visualized and quantified, using ECL reagent and a Kodak Image Station 440CF.

Coimmunoprecipitation. Two days posttransfection, cells were washed with PBS 4°C and detergent solubilized by addition of 250 µl of IPB buffer (1% Igepal, 5 mM EDTA, 0.15 M NaCl, 0.15% deoxycholate, 10 mM Tris, pH 7.5), supplemented with protease inhibitors (Mini Complete, Roche Molecular Biochemical) (49). Lysates were incubated with anti-CAIX monoclonal antibodies that recognize either the proteoglycan-like attachment domain (M75) or the catalytic domain (M10) (55), and protein A Sepharose resin at 4°C for overnight. Resin was washed and then resuspended in SDS-PAGE sample buffer (49). Samples were electrophoresed on 7.5% acrylamide gels. Immunoblots were probed with an anti-HA tag for antibody AE2, and AE3 and SLC26A7, and monoclonal antibody anti-AE1 (IVF 12) for AE1.

Cell surface processing. Cell surface processing assays were performed, as described previously (49). Briefly, HEK293 cells grown in 100-mm dishes were transiently transfected with AE and CAIX cDNAs, as described above. Two days posttransfection, cells were washed first with phosphate-buffered saline and then with borate buffer (154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl2, 10 mM boric acid, pH 9.0), and then incubated at 4°C for 30 min with 5 ml borate buffer containing 0.5 mg/ml Sulfo-NHS-SS-Biotin (Pierce, IL). After being washed three times with cold quenching buffer (192 mM glycine, 25 mM Tris, pH 8.3), cells were solubilized at 4°C in 500 µl IPB buffer supplemented with protease inhibitors. Cell lysates were centrifuged for 20 min at 16,000 g, and the resulting supernatant was divided equally. Half was saved for subsequent SDS-PAGE analysis total protein. To the other half was added immobilized streptavidin resin (50 µl of 1–3 mg of streptavidin/ml of settled gel as a 50% slurry in PBS, containing 2 mM NaN3), with subsequent overnight incubation at 4°C with gentle rocking. Samples were centrifuged for 2 min at 8,000 g, and supernatants were collected and retained for SDS-PAGE analysis (unbound fraction). The resin pellet was washed five times with IPB, and proteins were then eluted from the resin by the addition of 250 µl of SDS-PAGE sample buffer and incubation at 65°C for 5 min. The amount of AE was quantified in these samples (total protein, unbound fraction, and the fraction eluted from resin) by SDS-PAGE and immunoblotting.

GST pulldown. Bacterial expression constructs encoding CAIX variants GST-flCAIX, GST-PG, GST-PGCA, and GST-CA were expressed and purified as previously described (43, 55). GST fusion protein (2 nmol of each) was bound to 100 µl of glutathione Sepharose 4B resin (50% slurry equilibrated with PBS; Amershan Biosciences). AE2-transfected HEK293 cells were grown in 100-mm Petri dishes and lysed with 500 µl IPB buffer. Cell lysate (250 µl) was incubated with the GST-CAIX resin overnight, washed with 0.1% Igepal, 1 mM EDTA, 0.15 M NaCl, 10 mM Tris, pH 7.5, then 0.05% SDS, 2 mM EDTA, 10 mM Tris, pH 7.5 and finally 2 mM EDTA, 10 mM Tris, pH 7.5. Samples were eluted with SDS-PAGE sample buffer and incubated at 65°C for 5 min (43).

Immunocytochemistry. Fragments of human gastric mucosal biopsies (obtained per protocol approved by the Committee on Clinical Investigations of Beth Israel Deaconess Medical Center) were fixed overnight at 4°C in 2% paraformaldehyde, quenched in PBS containing 50 mM glycine, then stored until use at 4°C in PBS containing 0.02% NaN3. Semithin cryosections of fixed biopsy tissue were cut on a Leica CM1850 cryotome. Mounted cryosections were immunostained with anti-CAIX antibodies M75 (1:100 for 2 h) or M10 (1:10 overnight), rinsed, and incubated 1 h with Cy3-conjugated anti-mouse Ig, then rinsed and postfixed for 30 min in 3% paraformaldehyde. Glycine-quenched, postfixed sections then underwent epitope unmasking with 1% SDS for 5 min. After being rinsed, sections were incubated overnight with affinity-purified rabbit polyclonal anti-mouse AE2 amino acids 1224–1237 (1:200) (3) followed by 2-h incubation with Alexa 488-conjugated anti-Ig. Costained sections were imaged with a Bio-Rad MRC1024 laser confocal fluorescence microscope. Images were compiled in Adobe Photoshop 5.0.

Statistical analysis. Values are expressed ± SE. Statistical significance was determined using an unpaired t-test (Microsoft Excel), with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CAIX coexpression increases Cl–/HCO3– exchange activity of SLC4 Cl–/HCO3– exchangers but not SLC26A7. We examined the effect of CAIX on Cl–/HCO3– exchange activity. HEK293 cells were transiently transfected with individual cDNAs encoding AE1, AE2, AE3, or SLC26A7, with or without cotransfected CAIX. The HEK293 cell line was used in these experiments because these cells express low endogenous Cl–/HCO3– exchange activity (21). Cells loaded with the pH-sensitive dye, BCECF-AM, were perfused alternately with Ringer buffer containing 140 mM NaCl and with chloride-free Ringer buffer. The resulting changes of the transmembrane [Cl–] gradient drove HCO3– movement, which was monitored by measurements of intracellular pH (pHi). Transport rates were determined by linear regression of the initial rate of change of pHi upon changes of perfusion buffer (Table 1). Figure 1A presents representative bicarbonate transport data for AE2 and AE2-CAIX cotransfected HEK293 cells. Coexpression of AE proteins with CAIX significantly increased the AE-mediated bicarbonate transport activity for AE1, AE2, and AE3 by 32 ± 10, 28 ± 7, and 37 ± 9%, respectively, but did not affect SLC26A7 activity (Fig. 1B). Transport rates were all corrected for the background rate of vector-alone transfected cells.


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  		Table 1. Anion exchanger transport activity

 

Figure 1
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  		Fig. 1. Effect of carbonic anhydrase IX (CAIX) on Cl–/HCO3– transport activity. HEK293 cells were transiently cotransfected with AE cDNA, with or without CAIX cDNA. A: cells were perfused alternately with Ringer buffer containing 140 mM NaCl (open bar at top) and chloride-free Ringer buffer containing 140 mM Na gluconate (filled bar). Representative experiment measuring transport activity for AE2-transfected cells (black trace, top) and cells cotransfected with AE2 and CAIX (gray trace, bottom). B: summary of transport activity for CAIX-transfected cells coexpressing AE1 (n = 8), AE2 (n = 8), or AE3 (n = 5; gray bars) normalized to transport activities in the absence of CAIX (filled bars). *Significant difference (P < 0.05).

 
Observations of SLC26A7 transport activity are of particular interest in light of the current controversy about SLC26A7 transport mechanism, alternately reported as a pH-sensitive Cl– channel of low HCO3– permeability (19) or as a Cl–/HCO3– exchanger (36). We showed that the transporter SLC26A7 exhibits apparent Cl–/HCO3– exchanger activity, but CAIX has no effect on this activity (Fig. 1B). Transport activity was measured during alkalinization after chloride withdrawal, or during acidification after reintroducing chloride in the perfusion Ringer buffer (Fig. 2). Cells transfected with AE2 or with SLC26A7 and assayed in parallel exhibited upon removal of Cl– bicarbonate fluxes of 22 and 2.5 mM/min, respectively (Fig. 2, A and B). SLC26A7 transport activity proved to be significantly faster during acidification (HCO3– efflux) than during alkalinization (HCO3– influx). Relative polypeptide levels of AE2 and SLC26A7 (normalized to beta-actin) in the same cells in which transport was measured were 0.29 and 0.23, respectively (Fig. 2C). The small difference in AE2 and SLC26A7 polypeptide abundance was insufficient to explain the large difference in their apparent Cl–/HCO3– exchange activities.


Figure 2
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  		Fig. 2. Comparison of expression and bicarbonate transport activity of SLC26A7 and AE2. HEK293 cells were transiently transfected with vector alone (control) or HA epitope-tagged versions of AE2 or SLC26A7 cDNA. Cells were perfused alternately with Ringer buffer containing 140 mM NaCl (filled bar at top of A and B) and chloride-free Ringer buffer containing 140 mM Na gluconate (open bar). Representative transport activity assays for AE2-transfected cells (A) and SLC26A7-transfected cells (B). C: immunoblots of the cell samples used in bicarbonate transport assays measured in A and B, probed with anti-HA antibody to detect AE2 and SLC26A7 as indicated (arrows). Protein loading was normalized by beta-actin blot (bottom). D: summary of water-uncorrected bicarbonate transport activity during cell alkalinization (filled bar) and cell reacidification (gray bar) for SLC26A7-transfected cells (n = 12) and cells transfected with empty vector (n = 5). *Significant difference between SLC26A7 and vector-transfected cells (P < 0.05). #Significant difference between SLC26A7 acidification and alkalinization (P < 0.05).

 
Inhibition of CA activity reduces apparent Cl–/HCO3– exchange activity of SLC26A7. At least one SLC26 polypeptide, SLC26A6, interacts with cytosolic CAII to form a bicarbonate transport metabolon (6). Therefore, SLC26A7 was studied in absence and presence of 150 µM ACTZ, a CA inhibitor. ACTZ reduced Cl–/HCO3– exchange activity in SLC26A7-transfected HEK293 cells by 88 and 61% during alkalinization and acidification, respectively (not shown). This result is consistent with acceleration of apparent SLC26A7 transport activity by CA activity, perhaps the endogenous cytosolic CAII of HEK293 cells (47). SLC26A7 has two potential SLC26 consensus binding motifs for CAII [a hydrophobic amino acid followed by four amino acids at least two of which are acidic (6)], corresponding to the sequences 537 LDDIS and 623 LDSEK in the intracellular COOH-terminal STAS domain.

To test the possibility that CAII binds SLC26A7, SLC26A7 was coexpressed with the functionally inactive CAII mutant V143Y, which retains the ability to bind to SLC4 transporter COOH-terminal tail fusion proteins. Thus V143Y CAII [expressed in transfected cells at levels 20-fold higher than endogenous wild-type CAII (47)] is believed to act in a dominant negative manner, displacing wild-type CAII from binding sites on cytosolic portions of SLC4 polypeptides and so reducing their rates of Cl–/HCO3– exchange (47). V143Y CAII coexpression did not, however, significantly decrease the rate of Cl–/HCO3– exchange in SLC26A7-transfected cells [1.73 ± 0.24 mM/min (n = 5), compared with that of cells expressing SLC26A7 alone, 1.88 ± 0.15 mM/min (n = 4)]. Thus CAII activity accelerates the rate of SLC26A7-mediated apparent Cl–/HCO3– exchange activity; CAII/SLC26A7 binding is not required for the acceleration.

Increased activity of Cl–/HCO3– exchangers in the presence of coexpressed CAIX does not result from increased exchanger polypeptide abundance or surface expression. Differences in Cl–/HCO3– exchange activity may result from different levels of protein expression. To assess the possibility that CAIX increased AE activity through an increase of transporter expression, HEK293 cells were transiently cotransfected with CAIX and with individual cDNAs encoding AE1, AE2, AE3, or SLC26A7. Two days posttransfection, cells were solubilized, and samples were analyzed by SDS-PAGE and immunoblot to assess levels of Cl–/HCO3– exchanger expression. The amount of AE was normalized to the amount of beta-actin in the sample to normalize for protein load. HEK293 cells expressed similar levels of AE1, AE2, AE3, and SLC26A7 whether expressed alone (100%) or coexpressed with CAIX [98 ± 8, 100 ± 12, 104 ± 13, and 97 ± 12%, respectively (n = 6–8, not shown)]. Thus the increased Cl–/HCO3– exchange activity induced by CAIX did not result from altered AE expression.

A second way that CAIX expression could affect AE transport rate is to alter the efficiency of transporter processing to the plasma membrane. To examine the effect of CAIX on Cl–/HCO3– exchanger cell surface processing, we expressed transporters either alone or with CAIX in HEK293. The cells were labeled with membrane-impermeant Sulpho-NHS-SS-biotin and then solubilized in lysis buffer. Half of the cell lysate was incubated with streptavidin-Sepharose resin, which bound biotinylated proteins, representing proteins at the cell surface. Unbound, nonbiotinylated polypeptide remaining in the lysate represented intracellular anion exchanger polypeptide. The second half of the cell lysate (total fraction) was not treated with streptavidin-Sepharose and thus represented the total amount of protein in the sample. To assess the validity of this approach, we also determined whether a cytosolic marker protein, YFP, could be labeled by Sulpho-NHS-SS-biotin. Only 3.5 ± 6.8% of YFP was labeled indicating that the biotinylation protocol is reliable in labeling little if any cytosolic protein. Samples of the total and unbound fractions were subjected to SDS-PAGE, probed on immunoblots for the presence of AE1, AE2, or AE3 (Fig. 3), and quantified by densitometry. The assays revealed that CAIX expression did not significantly alter the efficiency of AE protein processing to the plasma membrane.


Figure 3
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  		Fig. 3. Effect of CAIX on AE surface processing. HEK293 cells transfected with AE cDNA, with or without CAIX cDNA, were labeled with membrane-impermeant Sulfo-NHS-SS-biotin. Half of the cell lysate was incubated with streptavidin-Sepharose resin to bind surface-biotinylated protein. Material remaining in the lysate (unbound) represented unbiotinylated protein from inside the cell. The second half of the cell lysate (total fraction) was not treated with streptavidin-Sepharose. Samples of the total and unbound fractions were subjected to SDS-PAGE and probed on immunoblots for the presence of AE1, AE2, or AE3. A: HA immunoblots detecting AE2 in total and unbound fractions from cells expressing AE2 in the absence and presence of coexpressed CAIX. B: summary of cell surface processing of AE1, AE2, and AE3 in presence and absence of CAIX. The amount of AE on blots was quantified by densitometry. % of AE at the cell surface = [(total AE – unbound AE)/total AE] x 100%.

 
The ability of CAIX to cooperate with anion exchangers to facilitate HCO3– transport across the plasma membrane was further evaluated in cells expressing AE2 and the functionally inactive CAII mutant, V143Y. AE2-mediated bicarbonate transport rate depends on the amount of AE2 expression in the plasma membrane. Therefore, transport activity values were normalized by the total cellular expression level of AE2 and the fraction of the protein expressed at the cell surface. The amount of AE2 expressed was 82 ± 22% (n = 6) and 79 ± 19% (n = 6) in cells cotransfected with AE2/CAII-V143Y and AE2/CAII-V143Y/CAIX, respectively, relative to expression in cells expressing AE2 alone. Similarly, the fraction of AE2 expressed at the cell surface was 100 ± 17% (n = 6) and 82 ± 22% (n = 6) for cells that coexpressed AE2/CAII-V143Y, AE2/CAII-V143Y/CAIX, respectively, relative to expression in cells expressing AE2 alone. Anion exchange assays of HEK293 cells expressing AE2 alone, AE2/CAII-V143Y, or AE2/CAII-V143Y/CAIX revealed that CAII-V143Y significantly reduced AE2-mediated bicarbonate transport by 23 ± 8%, relative to AE2 expressed alone (Fig. 4). Coexpression of CAIX not only rescued the AE2 inhibition caused by CAII-V143Y, but stimulated AE2 activity (Fig. 4).


Figure 4
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  		Fig. 4. Effect of carbonic anhydrases on AE2 Cl–/HCO3– transport activity. HEK293 cells were transiently transfected with cDNAs encoding either AE2 alone, AE2 and CAII-V143Y, or AE2, CAII-V143Y and CAIX. A: transfected HEK293 cells were perfused alternately with Ringer buffer containing 140 mM NaCl and chloride-free Ringer buffer containing 140 mM Na gluconate. Representative experiment measuring transport activity for AE2-transfected cells (black line), AE2/CAII-V143Y cotransfected cells (light gray line), and AE2/CAII-V143Y/CAIX cotransfected cells (dark gray line). B: summary of transport activity for AE2 (filled bar, n = 7)-, AE2/CAII-V143Y (light gray bar, n = 9)-, and AE2/CAII-V143Y/CAIX (dark gray bar, n = 8)-transfected cells expressed relative to AE2 alone transport rate. Data were corrected by the amount of AE protein at the cell surface and background activity. *Significant difference between AE2 and AE2/CAII-V143Y. #Significant difference between AE2 and AE2/CAII-V143Y-CAIX.

 
CAIX interacts in HEK293 cells with coexpressed AE2, but not with SLC26A7. Physical interactions between Cl–/HCO3– exchangers and CAIX were next investigated. HEK293 cells were cotransfected with CAIX and either AE1 or with HA-epitope-tagged constructs of AE2, AE3, or SLC26A7 cDNAs. The M75 CAIX antibody used for immunoprecipitation does not cross react with these anion transporters or with CAII (data not shown). Anti-CAIX antibody was able to precipitate AE2 and AE3 in a manner fully dependent on coexpression of CAIX (Fig. 5). In addition, AE1 precipitation was greatly enhanced by CAIX coexpression, but the small amount of AE1 precipitated in the absence of CAIX expression makes the result less certain. In contrast, SLC26A7 did not form a stable complex with CAIX as indicated by failure of the proteins to coimmunoprecipitate. Similar amounts of AE polypeptide were expressed in cell lysates transfected with AE alone or cotransfected with CAIX (Fig. 5). Similar amounts of CAIX were expressed in all lysates, including the lysate of cells coexpressing SLC26A7 and CAIX (not shown). These experiments revealed that CAIX forms a stable complex with AE1, AE2, and AE3, but not with SLC26A7.


Figure 5
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  		Fig. 5. Coimmunoprecipitation of anion exchangers and CAIX. HEK293 cells were transiently transfected with AE1, AE2, or AE3 cDNA alone or cotransfected with CAIX cDNA. Cell lysates were immunoprecipitated (IP) with anti-CAIX (M75), resolved by SDS-PAGE, blotted, and probed with an anti-HA antibody to detect AE2, AE3, and SLC26A7, and with anti-AE1 monoclonal antibody IVF12 for AE1 (right). Samples of the lysate (input lysate) were probed to indicate total amount of input CAIX and AE in each sample (left). The amount of material loaded into lysate is 4 times higher than for the immunoprecipitates. Migration of CAIX as 2 discrete bands has been noted previously and may result from differential glycosylation (33). Experiment is representative of 4 similar experiments.

 
Catalytic domain of CAIX is essential for interaction with AE2. To identify the region of CAIX that interacts with AE polypeptides, we examined the interaction of AE2 with domain-deleted CAIX variants (Fig. 6). CAIX is a transmembrane protein with an extracellular proteoglycan attachment-like domain (amino acids 53–111), an extracellular catalytic domain (amino acids 135–391), a single transmembrane domain (amino acids 415–436), and a short intracellular domain (amino acids 436–459) (Fig. 6A). To identify the AE-interacting region, constructs encoding different CAIX domains (55) were used: CAIX without the catalytic domain ({Delta}C-CAIX) and without proteoglycan attachment-like domain ({Delta}PG-CAIX). HEK293 cells were cotransfected with HA-AE2 and either CAIX or {Delta}C-CAIX or {Delta}PG-CAIX cDNA. Cell lysates were immunoprecipitated with antibodies against either the CAIX proteoglycan domain (M75) or the catalytic domain (M10), resolved by SDS-PAGE and immunoblotted with an anti-HA antibody. AE2 coimmunoprecipitated only with the CAIX constructs containing the catalytic domain. Conversely, there was no coimmunoprecipitation of AE2 in the absence of the CAIX catalytic domain (Fig. 6B).


Figure 6
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  		Fig. 6. Identification of an AE2-interacting domain of CAIX by coimmunoprecipitation of CAIX constructs. A: schematic model of CAIX protein. B: HEK293 cells were transiently cotransfected with AE2 and with one of three different CAIX construct cDNAs: full-length CAIX (CAIX), CAIX lacking its catalytic domain ({Delta}C-CAIX), and CAIX lacking its proteoglycan attachment-like domain ({Delta}PG-CAIX). Cell lysates were immunoprecipitated with anti-CAIX antibody M75 (lanes 1-3) or M10 (lane 4), resolved by SDS-PAGE, and blotted, and then probed with anti-HA antibody to detect AE2. Lane 4 is from a different blot than lanes 1-3. Experiment is representative of 3 similar experiments.

 
Interaction of AE2 with the CAIX catalytic domain was further examined with a GST pull-down approach. GST fusion proteins of the intact CAIX, proteoglycan-like attachment domain, CA catalytic domain, and CAIX without the transmembrane and intracellular domain (GST-CAIX, GST-PG, GST-C, and GST-PGCA, respectively) were expressed in bacteria and bound to glutathione resin (Fig. 7A). Cell lysates prepared from AE2 cDNA-transfected HEK293 were applied to the GST-CAIX variants immobilized on glutathione resin. After the resin was washed, proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE, blotted to a membrane, and probed for AE2. The three proteins containing the CAIX catalytic domain (GST-CAIX, GST-C, and GST-PGCA) bound similar amounts of AE2, whereas binding by GST alone or by GST-PG was negligible (Fig. 7B). Blots were stripped and probed to normalize for the amount of GST protein in each sample. Quantification confirmed that AE2 binding depends on the presence of the CAIX catalytic domain, since GST and GST-PG bound statistically indistinguishable amounts of CAIX (Fig. 7C). We conclude that AE2 binds the CAIX catalytic domain or that the presence of the catalytic domain is required to maintain an AE2-binding conformation of CAIX.


Figure 7
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  		Fig. 7. CAIX catalytic domain is sufficient to bind AE2 in a GST pull-down assay. A: schematic model of the GST-flCAIX fusion protein. GST-fusion proteins corresponding to 3 domain-deleted forms of CAIX were used: GST-PG, GST-PGCA, GST-C. B: equimolar amounts of GST or the indicated truncated CAIX-GST fusion proteins (see diagrams above lanes) were individually bound to glutathione-Sepharose resin. Cell lysates of HEK293 cells transfected with HA-tagged AE2 cDNA or with vector alone were applied to the GST fusion protein-resins and incubated overnight. After being washed, bound proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE electrophoresis, blotted, and probed with anti-HA antibody to detect AE2. C: immunoblots were stripped and probed with anti-GST antibody. The bar graph quantifies the amount of bound AE2 normalized to loaded GST fusion protein (n = 3). *Significant difference (P < 0.05) compared with GST alone.

 
CAIX and AE2 colocalize in gastric mucosa. To examine the CAIX-AE2 interaction in a physiological setting, the localization of CAIX relative to AE2 was studied in human stomach mucosa. Whereas AE2 was present in the basolateral membrane of parietal cells only, CAIX was detected by both M75 and M10 antibodies in basolateral membranes of parietal and chief cells (Fig. 8). Merged images of CAIX and AE2 showed that AE2 colocalized with CAIX in all AE2-expressing cells (parietal cells; Fig. 8). Colocalization of CAIX and AE2 in the basolateral membrane of human gastric parietal cells establishes the physiological plausibility of a physical interaction of CAIX with AE2 interaction that might facilitate bicarbonate transport across the parietal cells basolateral membrane to enhance maximal rates of acid secretion.


Figure 8
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  		Fig. 8. AE2 colocalizes with CAIX in parietal cell basolateral membranes of human gastric mucosal epithelium. Confocal immunofluorescence micrographs of human gastric mucosal biopsy specimens were fixed and immunostained with antibody to AE2 COOH terminus (green, left) and with anti-CAIX antibodies M75 and M10 (red, middle), as described in EXPERIMENTAL PROCEDURES. Right: merged images with colocalization in yellow. Transverse optical sections of gastric glands near the surface mucosa, in the mucous neck, and in the gastric pit are presented. Scale bar = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cl–/HCO3– exchangers at the basolateral surface of gastric parietal cells provide both the acid and Cl– essential for gastric HCl secretion. CAIX, the AE2 Cl–/HCO3– exchanger (SLC4A2), and the anion transporter SLC26A7 are expressed in the basolateral membrane of gastric parietal cells. The data presented here show that CAIX physically and functionally interacts with AE2, but not with SLC26A7. CAIX also physically interacts with and functionally couples to the other SLC4A polypeptides, AE1 and AE3. We further demonstrated that the extracellular catalytic domain of CAIX is responsible for interaction with AE2. Functional and physical interaction between a CA and a bicarbonate transporter has been described as a bicarbonate transport metabolon (47). Here, we showed for the first time that a transmembrane CA, CAIX, forms a bicarbonate transport metabolon with some SLC4A bicarbonate transporters. Coupling of CAIX and AE2 in a bicarbonate transport metabolon at the basolateral membrane of parietal cells may serve to maximize the acid secretory capacity of parietal cells.

Coexpression of CAIX with AE1, AE2, or AE3 increased Cl–/HCO3– exchange activity in HEK293 cells. The accelerated transport could not be attributed to effects of CAIX on transporter expression level or efficiency of cell surface processing. AE surface processing was similar among the AE isoforms, comparable to previous reports (47) and was not altered upon coexpression of CAIX for any of the AE isoform. Thus the effects of CAIX on Cl–/HCO3– exchange activity reflect increased apparent transport activity of a constant number of polypeptides in the plasma membrane. This conclusion was confirmed by the coexpression in HEK293 cells of CAIX, AE2, and the functionally inactive mutant of CAII V143Y. V143Y CAII will displace bound wild-type CAII from its binding site on AE2 and reduce AE2-mediated Cl–/HCO3– exchange activity (43, 47). Coexpression of CAIX together with CAII-V143Y CAII in cells that expressed AE2 prevented the reduction of Cl–/HCO3– exchange activity. This CAIX compensation for loss of CAII activity may reflect close physical interaction of CAIX with AE2.

AE1, AE2, and AE3 interact not only functionally but also physically with CAIX. Immunoprecipitation experiments demonstrated that AE1, AE2, and AE3 each bind CAIX, but SLC26A7 does not bind CAIX. Intracellular CAII and extracellular CAIV are the only CA isoforms previously described to participate in a bicarbonate transport metabolon. Both enzymes have a single globular domain (containing the catalytic site) that mediates interaction with bicarbonate transporters (30, 31). The more complex domain structure of CAIX includes an extracellular catalytic domain, a proteoglycan-like attachment domain (PG), a transmembrane domain (TM), and an intracellular domain (IC), any of which might have interacted with AE2. Immunoprecipitation with truncated versions of CAIX and GST pull-down experiments indicates specific AE2 interaction with the catalytic domain of CAIX but not with the PG, TM, or IC domains. Immunoprecipitation studies could not exclude the possibility that an intermediary scaffold protein mediated the CAIX/AE2 interaction. GST pull-down experiments, however, show that the interaction between CAIX and AE2 can be direct. In contrast, SLC26A7 did not bind CAIX and was not activated by CAIX expression.

To study whether AE2-CAIX interaction takes place in vivo, we examined stomach tissue, one of the few noncancerous tissues where CAIX is abundantly expressed. Immunohistochemistry revealed that AE2 and CAIX colocalize at the basolateral membranes of all parietal cells throughout the human gastric gland, consistent with a role of a CAIX/AE2 complex in human gastric acid secretion. Attempts to date to coimmunoprecipitate AE2 and CAIX from lysates of human or rat gastric mucosa have, however, not been definitively successful. The CAIX-AE2 interaction in gastric mucosa may be of too low an affinity to survive solubilization, perhaps due to the presence of distinct modulating proteins present in parietal cells and in HEK293 cells coexpressing AE2 and CAIX. The physiological role of CAIX in stomach is not clear, although the localization of CAIX to the basolateral surface of parietal cells and its physical interaction with AE2 strongly suggest a role in HCl secretion. Consistent with this interpretation, ca9(–/–) mice developed stomach hyperplasia with a 10–20% increase in the number of parietal cells and increased gastric levels of ca2 mRNA (31). Interestingly, the intraluminal gastric pH of ca9(–/–) mice remained the same as in wild-type mice, perhaps due to increased parietal cell number and increased CAII. Ca2(–/–) mice exhibit 2.5-fold upregulation of CAIX mRNA levels in gastric mucosa (36). Thus compensatory changes in ca9(–/–) mouse gastric mucosa may mask reduced intrinsic AE2-mediated Cl–/HCO3– exchange activity, reflecting disruption of the postulated CAIX/AE2 metabolon.

Recently, human SLC26A7 was found to function as a Cl–/HCO3– exchanger and proposed to participate in gastric HCl secretion (19, 22). The transport mechanism and physiological function of SLC26A7, however, remain controversial following a report that the protein functions exclusively as a Cl– channel (44). In the present work, we showed that HA-tagged SLC26A7 mediates apparent Cl–/HCO3– exchange activity at rates comparable to those of AE3, but ~10-fold slower than AE2 expressed in equivalent abundance at the surface of HEK293 cells. SLC26A7 resembled SLC26A3 in its ACTZ sensitivity in the absence of direct CAII binding (18). Some Cl–/HCO3– exchangers are directly inhibited by CA inhibitors (27). Since the sensitivity of SLC26A7 to direct inhibition by ACTZ has not been assessed, we cannot rule out the possibility that ACTZ inhibited SLC26A7 directly, rather than through CAII. Since ACTZ does not directly inhibit AE1 (9, 27), we consider this unlikely. SLC26A7 differed from SLC4 Cl–/HCO3– exchangers in its lack of CAIX binding, paralleled by its lack of increased activity upon CAIX coexpression.

A final observation concerning SLC26A7 relates to evidence for a rectifying behavior of the protein. We found a statistically significant higher rate of SLC26A7-mediated cytosolic acidification compared with cytosolic alkalinization. Although the standard Cl– removal/restoration assay used in this study is designed to elicit maximal transport rates rather than to mimic physiological conditions, a regulatory pattern favoring cytosolic PC acidification by a basolateral transporter also favors stimulated gastric acid secretion. The basis for this apparent rectification is not clear, although it might reflect alkaline pH-activated, and/or potential-sensitive, and/or electrogenic Cl–/HCO3– exchange, or alkalinization-induced increase in HCO3– permeability.

Interaction of CAIX and bicarbonate transporters in a bicarbonate transport metabolon may also occur in other tissues. AE2 (16) and CAIX (34) colocalize at the basolateral membrane of the excurrent ducts of the epididymis, where bicarbonate is reabsorbed. The acidic environment of the excurrent ducts maintains sperm in an immotile but viable state while they mature and are stored in the epididymis. In the digestive tract, CAIX is expressed (1, 26, 54) in basolateral membranes of gall bladder epithelial cells, hepatic biliary duct cells, and pancreatic duct cells, each of which secretes HCO3–-rich luminal fluid. Several candidate bicarbonate transporters are coexpressed with CAIX in these digestive tract epithelial cells (35).

CAIX is expressed in many cell types only upon transformation (35), and so is of great interest to tumor biology. CAIX may regulate extracellular pH during anaerobic tumor metabolism (17), enhancing extracellular acidification which may promote growth, tumor cell and endothelial remodeling, and activation of matrix metalloproteases predisposing to metastasis (7, 25). A CAIX bicarbonate transport metabolon could facilitate HCO3– influx into tumor cells, adding to enhanced proton/lactate symport in neutralizing the cytosolic acid load produced by enhanced anaerobic glycolysis. Cytosolic CAII within the same metabolon could convert imported HCO3– to CO2, which could then diffuse across the plasma membrane back into the extracellular space. This H+ shuttling facilitated by the bicarbonate transport metabolon could enhance tumor anaerobic metabolism. The major pathways of HCO3– entry in most cells are, however, Na+-dependent HCO3– cotransporters and Na+-dependent Cl–/HCO3– exchangers. As enhancement of NBCe1/SLC4A4 activity in Xenopus laevis oocytes by coexpressed CAII remains controversial (7, 25), a circumspect approach to the hypothesis of the bicarbonate transport metabolon remains appropriate.

We found that the catalytic domain of CAIX binds SLC4 Cl–/HCO3– exchangers and enhances transmembrane HCO3– flux. In gastric parietal cells, transport of bicarbonate to the blood by the basolateral AE2 Cl–/HCO3– exchanger acid-loads the cell. Rapid conversion of CO2 to HCO3– by cytosolic CAII and of HCO3– to CO2 by the extracellular CAIX, both bound to AE2, may increase local transmembrane bicarbonate gradients and so enhance rates of bicarbonate transport by AE2. Physiologically, the AE2-CAIX-CAII bicarbonate transport metabolon will serve to maximize achievable rates of stimulated acid secretion by gastric parietal cells.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
P. E. Morgan was supported by a postdoctoral award from the Canadian Institutes for Health Research (CIHR) Strategic Training Initiative in Cardiovascular Membrane Proteins and an Alberta Heritage Foundation for Medical Research (AHFMR) postdoctoral fellowship. J. R. Casey is a Scientist of the AHFMR. S. Pastoreková is supported by EU grant 502932 EUROXY. Support was provided by a CIHR operating grant to J. R. Casey, National Institutes of Health Grant DK-43495, and a Developmental Project Grant from the Harvard Cancer Center Renal Cancer SPORE CA101942 to S. L. Alper. The Morphology Core facility of the Harvard Digestive Diseases Center DK-34854 provided partial support for the confocal microscope.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Jennings for IVF12 antibody, Dr. M. Soleimani for the SLC26A7 clone, Dr. J. Fujinaga for preparing the pJF7 and pJF8 plasmids, Dr. C. Kelly for access to human gastric biopsy tissue, and Dr. M. Atkins for encouragement.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Casey, Dept. of Physiology, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7 (e-mail: joe.casey{at}ualberta.ca)

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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