1 Department of Medicine, University of Cincinnati, Cincinnati 45267 - 0585; and 2 Veterans Affairs Medical Center at Cincinnati, Ohio 45220
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ABSTRACT |
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HCO/base exchange;
however, the functional identity and distribution of PAT1 (SLC26A6) is
not known. In these studies, we investigated the functional identity,
tissue distribution, and membrane localization of PAT1. Expression
studies in Xenopus oocytes demonstrated that PAT1 functions
in Cl
/HCO
/HCO
duodenum; bicarbonate secretion; apical
Cl/HCO
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INTRODUCTION |
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THE DUODENAL EPITHELIUM
SECRETES an alkaline, HCO/HCO
absorption in exchange for HCO
/HCO
(16). The
molecular identity of apical Cl
/HCO
and secretion of base
(14, 21, 25).
Recent studies have identified a new class of anion exchangers,
including downregulated in adenoma (DRA), pendrin (PDS), and putative
anion transporter (PAT1) (10, 12, 14, 20, 28, 29, 32).
None of these transporters are structurally related to the AE (AE-1, 3 and 3) family. Indeed, the homology at the amino acid level between
DRA, PDS, or PAT1 and AE family members is <15% (the GenBank
accession nos. NP-000333, NP-003031, and NP-005061 were used for AE-1,
AE-2, and AE-3, respectively). DRA and PDS mediate
Cl/HCO
PAT1 was recently cloned from the pancreas based on homology to DRA and
pendrin (20). PAT1 maps to chromosome 3 and encodes a 738 amino-acid protein (20). Immunohistochemical studies
localized PAT1 to the apical membranes of the pancreatic duct cells and kidney tubules (20). A mouse ortholog of PAT1 was recently
cloned and found to be expressed on the apical membranes of the kidney proximal tubule (19). Little is known about the functional
identity of PAT1. Furthermore, the distribution and membrane
localization of PAT1 in other epithelial tissues have not been studied.
Accordingly, we examined the functional identity, tissue distribution,
and membrane localization of PAT1 in mouse. The results indicate that PAT1 is a Cl/HCO
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METHODS |
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PCR of mouse PAT1. Mouse EST database was blast searched against the human PAT1 sequence (GenBank accession no. AF279265). An expressed sequence tag (EST) (GenBank accession no. AI747461), which is highly similar to human PAT1, was identified. On the basis of the cDNA sequence of the EST, the following oligonucleotide primers (5'-GGG AGA TTG AAG TGG AAG TGT ACA TC, and 5'-AAG GCC AGA CTG ACT GCA ATA C) were designed and used for RT-PCR on RNA isolated from mouse kidney cortex. An ~200-bp PCR fragment was purified, which, on sequencing, was verified as mouse PAT1 and corresponded to the human nucleotides 2278-2468. The PCR product was used as a probe for Northern blot hybridization in mouse tissues.
RNA isolation and Northern blot hybridization. Total cellular RNA was extracted from various mouse tissues including gastrointestinal tract segments, kidney, liver, heart, brain, and lung using TriReagent (6). Hybridization was performed according to Church and Gilbert (7). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). 32P-labeled human and mouse probes were used for Northern experiments. For DRA hybridization, a 400-bp cDNA from the mouse DRA cDNA (EcoR I-EcoR I fragment) was used as a probe.
Enriched luminal membrane vesicle preparation.
For the duodenum, enriched luminal membrane vesicles were prepared by a
modification of the divalent cation precipitation technique (9,
17, 31). Briefly, mucosa was scraped with a glass slide from
rinsed intestinal segments and homogenized in 50 mM mannitol, 2 mM
Tris, pH 7.1, containing protease inhibitor cocktail (20 µg/ml
leupeptin, 10 µg/ml pepstatin, 8.7 µg/ml phenylmethylsulfonyl fluoride, and 1.25 µg/ml aprotinin). CaCl2 (1 M) was
added to a final concentration of 10 mM, and the homogenate was mixed
for 15 min at 4°C, then spun at 2,000 g. The pellet was
discarded, and the membrane vesicles present in the supernatant were
pelleted at 17,500 g for 20 min, washed once in 100 mM NaCl,
50 mM Tris, pH 7.4 containing protease inhibitor cocktail, resuspended
in the same buffer by passage through a 22-gauge needle, and frozen at
80°C. For isolation of membrane proteins from the proximal colon,
mucosa was scraped with a glass slide from rinsed proximal colon and
homogenized as above. Similar whole cell scrapings from the duodenum
were used for comparison.
Cloning of human and mouse PAT1. Full-length mouse PAT1 cDNA was cloned from the duodenum by RT-PCR. Briefly, total RNA was prepared from the duodenum, poly(A+)-selected using Oligotex latex beads (Qiagen), and then reverse transcribed at 42°C using SuperScript II RT (Life Technologies) and oligo(dT) primers. The following oligonucleotide primers were designed and used for RT-PCR: 5'-CGT CTG CAC TGC TCC CTC CTC CAT TG, and 5'-GAG TCC CAG GGC ATC CAT CCA TG (GenBank accession no. AY032863; Ref. 19). These primers encode nucleotides 45 to 2498 of mouse PAT1.
Full-length human PAT1 cDNA was cloned from cultured human pancreatic duct (CFPAC-1) cells by RT-PCR. Oligonucleotide primers (5'-ATG CCT TCA CTG TGT CTC TCT GGT CTT GCC and 5'-AAT ATG CAC CAG TTC CCT CCC TGT ACC GC) were designed based on human PAT1 sequence (GenBank accession no. AF279265). Amplification of the human or mouse PAT1 cDNA by the PCR was performed according to Clontech Advantage 2 PCR kit protocal. Briefly, each PCR contained 5 µl cDNA, 5 µl 10× PCR buffer, 1 µl 10 mM dNTP's, 10 pmol of each primer, and 1 µl Advantage 2 polymerase mix in a final volume of 50 µl. Cycling parameters were: 95°C, 1 min; 95°C, 30 s; and 68°C, 4 min. After PCR, the product was gel purified (revealing a single band of ~2.5 kb). Sequence analysis of the PCR products verified the sequence as human PAT1 or its mouse ortholog. The PCR products were ligated into pGEM-T easy vector for expression studies.Synthesis of mouse or human PAT1 cRNA. The capped PAT1 cRNAs were generated using mmessage mMACHINE kit (from Ambion) according to manufacturer's instruction. Briefly, the plasmids containing the full-length mouse or human PAT-1 cDNA were linearized, and the products were then in vitro transcribed to cRNAs, as described previously (34).
Expression of mouse or human PAT1 in Xenopus oocytes. X. oocytes were injected with mouse or human PAT1 cRNA, as used before (34). Fifty nanoliters of cRNA (0.2-1.3 µg/µl) were injected with a Drummond 510 microdispenser via a sterile glass pipette. Intracellular pH (pHi) in oocytes was measured with the pH-sensitive fluorescent probe 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) (Molecular Probes, Eugene, OR) as described previously (2, 5, 34). Oocytes were loaded with 10 µM BCECF-acetoxymethyl ester for 20 min at room temperature, transferred to a 1-ml perfusion chamber, and perfused at rate of 3 ml/min with the following solution (in mM): 63 NaCl, 33 NaHCO3, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES. Ratiometric fluorescence measurements were performed using Attofluor digital imaging system (Attofluor, Rockwell, MD). Excitation wavelengths were 450 and 490 nm, and fluorescence emission intensity was recorded at 520 nm. Data analyses were performed using Attograph and Attoview software packages provided with the imaging system. The ratios were obtained from the submembrane region of the oocytes that were visualized with a ×10 objective. Measured excitation ratios were converted to pHi by using a calibration curve that was constructed with the high-K+/nigericin method at the end of each experiment.
To examine the ClImmunoblotting and immunohistochemical staining of PAT1 in mouse duodenum. Duodenum from normal mice was cut into slices and mounted on holders to form tissue blocks. The tissues were fixed in a solution containing 0.1% glutaraldehyde plus 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.20, and stored in 0.1 M cacodylate buffer, pH 7.20, at 4 °C. For immunohistochemistry, the tissue blocks were sectioned into 5 µm section, placed on slides (Fisher brand-Superfrost/Plus), and incubated at 75°C for 1 h. The immunohistochemistry studies were performed according to standard protocols. A PAT1 specific antibody, raised against the amino terminal amino acid sequence RRDYHMERPLLNQE of human PAT1 (Genebank accession no. AF279265), was applied to the slides in 1/40 dilution in PBS + 1% BSA and in the presence of saponin and incubated in a humidified chamber for 2 h at room temperature. The specificity of PAT1 antibody was demonstrated in enriched luminal membrane vesicles isolated from the duodenum, where the immunoblot analysis identified a ~90-kDa band, which was blocked following preadsorption with the synthetic peptide (see RESULTS). The secondary antibody was applied to the slides in a dilution of 1:25. The peroxidase-antiperoxidase conjugate diluted in 1:100 in PBS + 1% BSA was applied to the slides. To develop a colored reaction product, the diaminobenzidine was used and the tissues were counterstained with Harris hematoxylin.
Materials. [32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes (Eugene, OR). mmessage mMACHINE kit was purchased from Ambion (Austin, Texas). The human multiple tissue blots were purchased from Clontech (Palo Alto, CA).
Statistical analyses. Values are expressed as means ± SE. The significance of difference between mean values were examined using ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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PAT1 mRNA expression.
To examine the distribution of PAT1 mRNA in mouse tissues, Northern
blots were prepared using total RNA from several tissues and then
probed with mouse PAT1 cDNA. The results were as shown in Fig.
1A: PAT1 mRNA levels are
highly expressed in heart, kidney, and stomach but are low in colon and
lung. A human multiple-tissue blot similarly indicates high expression
levels of PAT1 in the stomach (data not shown).
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Functional identity of PAT1. In these series of experiments, the functional identity of human (and mouse) PAT1 was examined using the oocyte expression system.
On the basis of structural similarity with DRA and pendrin (10, 21, 29, 33), we speculated that PAT1 could function in Cl
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DISCUSSION |
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The results of the above experiments indicate that the expression
of SLC26A6 (PAT1) is highly abundant in the small intestine but is low
in the colon (Fig. 2A). This pattern of expression is
opposite that for DRA, which is predominantly expressed in colon but is
minimally expressed in small intestine (Fig. 2B). Immunohistochemical staining studies in the duodenum localized PAT1 to
the brush border membrane domain of villus cells (Fig. 3). Expression
studies in oocytes demonstrated that PAT1 functions in
Cl/HCO
The upper gastrointestinal tract (and specifically duodenum) is exposed
to an acidic chyme delivered from stomach that can achieve a pH as low
as 1.5 (11, 13). One major defense mechanism for
protecting the duodenal mucosa against acid injury is via secretion of
bicarbonate (15, 16, 27). Several studies have examined
HCO/HCO
The apical Cl/HCO
in exchange for HCO
/HCO
(11, 16). The
molecular identity of apical Cl
/HCO
and secretion of
HCO
/HCO
/HCO
cDNA analysis indicates that PAT1 is closely related to a family of
anion transport proteins (SLC26A) that includes the rat sulfate-anion
transporter, the human diastrophic dysplasia sulfate transporter, the
DRA gene, and pendrin (3, 10, 12, 14, 20, 28, 32).
Sequence comparison revealed that PAT1 has ~34% homology with DRA
and pendrin, respectively, at the amino acid level (10, 20,
28). Both DRA and pendrin mediate
Cl/HCO
/HCO
/OH
(25), and a
sulfate/OH
exchanger (1).
PAT1 (or SLC26A6) is the first apical
Cl/HCO
/HCO
/base exchanger in
the small intestine, whereas DRA is a major apical
Cl
/base exchanger in the colon.
Whether PAT1 can function in other anion exchange modes is currently
under investigation. A recent study indicates that a mouse ortholog of
SLC26A6 or PAT1 is expressed in the kidney proximal tubule and can
function as a Cl/formate exchanger (19). For
this reason, the mouse ortholog of SLC26A6 is named
chloride/formate exchanger (CFEX) (19). With respect to
carrying formate, PAT (or CFEX) is very similar to pendrin. Pendrin,
which is another member of the SLC26A family (and is referred to as
SLC26A4), can function in both Cl
/HCO
/formate exchange modes (33). It would
be difficult to compare the rate of
Cl
/HCO
/formate exchange activity mediated via PAT1 (or
pendrin) in oocytes or other expression systems by radioflux assay or
other methods. Comparison of radiolabeled 36Cl flux in
exchange for formate (Cl
/formate exchange) or bicarbonate
(Cl
/HCO
In conclusion, PAT1 or SLC26A6 (or CFEX) is expressed in the upper
gastrointestinal tract, with mRNA expression predominantly limited to
the small intestine and stomach. Immunohistochemical staining studies
localized PAT1 to the apical domain of villus cells in the duodenum.
Functional studies demonstrated that PAT1 functions in
Cl/HCO
/base exchanger in the small
intestine and is mediating absorption of chloride and secretion of bicarbonate.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent technical assistance of Tracy Greely and Ljiljiana Pavelic.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54430 and DK-52821, a Merit review grant, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic Incorporated (to M. Soleimani).
Address for reprint requests and other correspondence: M. Soleimani, Div. of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00338.2001
Received 31 July 2001; accepted in final form 9 November 2001.
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