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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
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
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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SLC4; SLC26; bicarbonate transport metabolon
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.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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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, 
C-CAIX, 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.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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-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.
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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 -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.
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C-CAIX) and without proteoglycan attachment-like domain (PG-CAIX). HEK293 cells were cotransfected with HA-AE2 and either CAIX or C-CAIX or 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).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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