Artigo Acesso aberto Revisado por pares

Molecular Cloning, Chromosomal Localization, Tissue Distribution, and Functional Expression of the Human Pancreatic Sodium Bicarbonate Cotransporter

1998; Elsevier BV; Volume: 273; Issue: 28 Linguagem: Inglês

10.1074/jbc.273.28.17689

ISSN

1083-351X

Autores

Natalia Abuladze, Ivan Lee, Debra K. Newman, James Hwang, Kathryn J. Boorer, Alexander Pushkin, Ira Kurtz,

Tópico(s)

Pancreatitis Pathology and Treatment

Resumo

We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem.272, 19111–19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an ∼7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5′ region of kNBC detected an ∼7.6-kilobase transcript only in the kidney. In situhybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescentin situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney. We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem.272, 19111–19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an ∼7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5′ region of kNBC detected an ∼7.6-kilobase transcript only in the kidney. In situhybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescentin situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney. Sodium bicarbonate cotransport mediates the coupled movement of Na+ and HCO3− ions across the plasma membrane of many cells (1Boron W.F. Boulpaep E.L. Kidney Int. 1989; 36: 392-402Abstract Full Text PDF PubMed Scopus (96) Google Scholar). This transport process is involved in bicarbonate secretion/absorption and intracellular pH (pHi) 1The abbreviations used are: pHi, intracellular pH; NBC, sodium bicarbonate cotransport protein; kNBC, kidney NBC; pNBC, pancreatic NBC; kb, kilobases; bp, base pair(s); DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; PCR, polymerase chain reaction; EST, Expressed Sequence Tag; BCECF, 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein; TMA, tetramethylammonium; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; NS, not significant. regulation. Functional Na(HCO3)n cotransport was first identified in the salamander Ambystoma tigritum kidney (2Boron W.F. Boulpaep E.L. J. Gen. Physiol. 1983; 81: 53-94Crossref PubMed Scopus (391) Google Scholar) and has since been documented functionally in numerous other cell types including pancreas (3Muallem S. Loessberg P.A. J. Biol. Chem. 1990; 265: 12806-12812Abstract Full Text PDF PubMed Google Scholar, 4Zhao H. Katzumoto U. Star R.A. Muallem S. Am. J. 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Depending on the cell type, the stoichiometry of Na+ to HCO3− flux is 3:1, 2:1, or 1:1. As characterized in many cell types, several features distinguish the Na(HCO3)n cotransporter from other bicarbonate-dependant transporters.1) Na(HCO3)ncotransport is not dependent on the presence of Cl−, 2) transport is inhibited by stilbenes, and 3) transport is stimulated in the presence of HCO3−. In the kidney, Na(HCO3)n cotransport was initially localized by functional studies to the basolateral membrane of the proximal tubule where it plays an important role in mediating electrogenic basolateral bicarbonate efflux (2Boron W.F. Boulpaep E.L. J. Gen. Physiol. 1983; 81: 53-94Crossref PubMed Scopus (391) Google Scholar, 21Alpern R.J. J. Gen. Physiol. 1985; 86: 613-636Crossref PubMed Scopus (199) Google Scholar, 22Yoshitomi K. Burckhardt B.-C. Frömter E. Pfluegers Arch. Eur. J. Physiol. 1985; 405: 360-366Crossref PubMed Scopus (213) Google Scholar). Although Na+-dependent and -independent Cl−/base exchangers also contribute to basolateral bicarbonate transport in the proximal tubule (23Alpern R.J. Chambers M. J. Gen. Physiol. 1987; 89: 581-598Crossref PubMed Scopus (83) Google Scholar, 24Guggino W.B. London E.L. Boulpaep Giebisch G. J. Membr. Biol. 1983; 71: 227-240Crossref PubMed Scopus (91) Google Scholar, 25Kurtz I. J. Clin. Invest. 1989; 83: 616-622Crossref PubMed Scopus (55) Google Scholar, 26Nakhoul N.L. Chen L.K. Boron W.F. Am. J. Physiol. 1990; 258: F371-F381PubMed Google Scholar, 27Sasaki S. Yoshiyama N. J. Clin. Invest. 1988; 81: 1004-1011Crossref PubMed Scopus (53) Google Scholar), current evidence suggests that electrogenic Na(HCO3)n cotransport mediates the majority of bicarbonate efflux in this nephron segment (1Boron W.F. Boulpaep E.L. Kidney Int. 1989; 36: 392-402Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Romero et al. have recently cloned a renal electrogenic sodium bicarbonate cotransporter (NBC) from rat (28Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. J. Am. Soc. Nephrol. 1996; 7 (abstr.): 1259Google Scholar) and salamander kidney (29Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F Nature. 1997; 387: 409-413Crossref PubMed Scopus (383) Google Scholar). Burnham et al. have reported the cloning of a sodium bicarbonate cotransporter from human kidney (30Burnham C.E. Amlal H. Wang Z. Shull G.E. Soleimani M. J. Biol. Chem. 1997; 272: 19111-19114Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The NBC clone isolated from salamander kidney encoded a 4.2-kb mRNA transcript that was expressed predominantly in kidney, with less expression in small intestine, large intestine, brain, eye, and bladder (29Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F Nature. 1997; 387: 409-413Crossref PubMed Scopus (383) Google Scholar). Human NBC isolated from kidney encoded a ∼7.6-kb mRNA and was reportedly also expressed in pancreas and brain by Northern analysis using a probe to the 3′ region of kNBC (30Burnham C.E. Amlal H. Wang Z. Shull G.E. Soleimani M. J. Biol. Chem. 1997; 272: 19111-19114Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). NBC expression in the kidney has recently been shown to be highest in the S1 proximal tubule, with less expression in the proximal straight tubule (31Abuladze N. Lee I Newman D. Hwang J Pushkin A. Kurtz I. Am. J. Physiol. 1998; 43: 628-633Google Scholar). The high level of expression in S1 proximal tubules is in keeping with the high rate of transepithelial bicarbonate transport in this segment (32Jacobson H.R. Am. J. Physiol. 1981; 240: F54-F62PubMed Google Scholar,33Schwartz G.J. Evan A.P. Am. J. Physiol. 1983; 245: F382-F390PubMed Google Scholar). The pancreas secretes digestive enzymes dissolved in a HCO3−-rich fluid (34Kuijpers G.A.J. Depont J.J.H.H. Annu. Rev. Physiol. 1987; 49: 87-103Crossref PubMed Scopus (18) Google Scholar). Pancreatic bicarbonate secretion is mediated by principal cells lining the pancreatic ducts (34Kuijpers G.A.J. Depont J.J.H.H. Annu. Rev. Physiol. 1987; 49: 87-103Crossref PubMed Scopus (18) Google Scholar, 35Case M.R. Curr. Opin. Gastroenterol. 1989; 5: 665-681Crossref Google Scholar). Previous functional studies have led to a cell model that can account for transcellular bicarbonate secretion. Apical bicarbonate secretion is thought to be mediated by an apical Cl−/base exchanger acting in parallel with a small conductance cystic fibrosis transmembrane conductance regulator Cl− channel on the apical membrane (5Zhao H. Star R.A. Muallem S. J. Gen. Physiol. 1994; 104: 57-85Crossref PubMed Scopus (116) Google Scholar, 36Gray M.A. Harris A. Coleman L. Greenwell J.R. Argent B.E. Am. J. Physiol. 1993; 264: C591-C602Crossref PubMed Google Scholar). Influx of H+ equivalents during the process of apical bicarbonate secretion requires the efflux of H+ or the influx of bicarbonate in the steady state. Studies of pig, rat, and guinea pig pancreatic ducts have demonstrated the presence of a basolateral Na+/H+ antiporter, which serves an important housekeeping role (5Zhao H. Star R.A. Muallem S. J. Gen. Physiol. 1994; 104: 57-85Crossref PubMed Scopus (116) Google Scholar, 8de Ondarza J. Hootman S.R. Am. J. Physiol. 1997; 272: G124-G134PubMed Google Scholar, 37Stuenkel E.L. Machen T.E. Williams J.A. Am. J. Physiol. 1988; 254: G925-G930PubMed Google Scholar, 38Veel T.O. Villanger O. Holthe M.S. Cragoe E.J. Raeder M.G. Acta Physiol. Scand. 1992; 144: 239-246Crossref PubMed Scopus (27) Google Scholar). A basolateral vacuolar-type H+/ATPase and Na(HCO3)ncotransporter are thought to play an important role in agonist-mediated bicarbonate secretion (5Zhao H. Star R.A. Muallem S. J. Gen. Physiol. 1994; 104: 57-85Crossref PubMed Scopus (116) Google Scholar, 6Villanger O. Veel T. Raeder M.G. Gastroenterology. 1995; 108: 850-859Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 7Ishiguro H., M. Steward C. Lindsay A.R. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 169-178Crossref PubMed Scopus (158) Google Scholar, 8de Ondarza J. Hootman S.R. Am. J. Physiol. 1997; 272: G124-G134PubMed Google Scholar, 39Ishiguro H. Steward M.C. Wilson R.W. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 179-191Crossref PubMed Scopus (118) Google Scholar). The relative contribution of these transporters to basolateral H+/base transport and their respective stimulation by secretogues in different species is controversial. The importance of basolateral Na(HCO3)n cotransport has recently been documented in studies of isolated rat and guinea pig pancreatic ducts (5Zhao H. Star R.A. Muallem S. J. Gen. Physiol. 1994; 104: 57-85Crossref PubMed Scopus (116) Google Scholar, 6Villanger O. Veel T. Raeder M.G. Gastroenterology. 1995; 108: 850-859Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 7Ishiguro H., M. Steward C. Lindsay A.R. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 169-178Crossref PubMed Scopus (158) Google Scholar, 8de Ondarza J. Hootman S.R. Am. J. Physiol. 1997; 272: G124-G134PubMed Google Scholar, 39Ishiguro H. Steward M.C. Wilson R.W. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 179-191Crossref PubMed Scopus (118) Google Scholar). Ishiguro et al. (7Ishiguro H., M. Steward C. Lindsay A.R. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 169-178Crossref PubMed Scopus (158) Google Scholar) reported that basolateral Na(HCO3)n cotransport contributed up to 75% of basolateral bicarbonate uptake during stimulation of transepithelial bicarbonate transport by secretin. Furthermore, in isolated pancreatic acini, Na(HCO3)n cotransport has been shown to participate in the regulation of pHi after acid loads (3Muallem S. Loessberg P.A. J. Biol. Chem. 1990; 265: 12806-12812Abstract Full Text PDF PubMed Google Scholar). On the basis of HCO3− flux measurements and thermodynamic considerations, it was concluded that this transporter contributes to HCO3− efflux under unstimulated conditions (3Muallem S. Loessberg P.A. J. Biol. Chem. 1990; 265: 12806-12812Abstract Full Text PDF PubMed Google Scholar, 7Ishiguro H., M. Steward C. Lindsay A.R. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 169-178Crossref PubMed Scopus (158) Google Scholar) with a stoichiometry of 3:1 (3Muallem S. Loessberg P.A. J. Biol. Chem. 1990; 265: 12806-12812Abstract Full Text PDF PubMed Google Scholar), although direct measurements of the stoichiometry have thus far not been performed. After depolarization of the basolateral membrane by secretin (40Novak I. Pahl C. Pfluegers Arch. Eur. J. Physiol. 1993; 425: 272-279Crossref PubMed Scopus (31) Google Scholar), the electrochemical driving forces would favor basolateral bicarbonate influx (7Ishiguro H., M. Steward C. Lindsay A.R. Case R.M. J. Physiol. ( Lond. ). 1996; 495: 169-178Crossref PubMed Scopus (158) Google Scholar). Although the functional characteristics of pancreatic Na(HCO3)n cotransport have begun to be investigated, the protein responsible for this function has not been identified. We report here the cloning the human pancreatic Na(HCO3)n cotransporter (pNBC). The predicted pNBC polypeptide is 1079 amino acids in length, whereas the NBC variant expressed in kidney (kNBC) consists of 1035 amino acids. The C-terminal 994 amino acids of pNBC and kNBC are identical. pNBC has a unique N terminus of 85 amino acids that replaces the initial 41 amino acids in kNBC. Expression of the cDNA encoding pNBC in Xenopusoocytes results in sodium-dependent and chloride-independent HCO3− transport, which is inhibited by DIDS. A 159-bp PCR product (2795–2954 bp in human pNBC) was generated using the human pancreas NBC EST clone W39298 (I.M.A.G.E clone) as a template, random primer-labeled with 32P and used to screen a human pancreas λgt10 cDNA library (CLONTECH, Palo Alto, CA). A similar approach was utilized by Burnham et al. (30Burnham C.E. Amlal H. Wang Z. Shull G.E. Soleimani M. J. Biol. Chem. 1997; 272: 19111-19114Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) while the present studies were in progress to obtain an NBC clone from human pancreas. Standard hybridization conditions were employed (42 °C, 50% formamide, 5× standard saline phosphate EDTA (SSPE), 5× Denhardt's solution, 0.5% SDS, 0.2 mg/ml prehybridization herring sperm DNA). The filters were washed three times with 1× SSC/0.1%SDS (42 °C) and once with 0.1× SSC/0.1%SDS (25 °C) (1×SSC = 0.15 m NaCl and 0.015 m sodium citrate). Positive clones were verified by sequencing. Two overlapping clones (7.1) and (9.2.1) were obtained that contained the entire coding region. To obtain a full-length clone containing the complete open reading frame, these two clones were spliced together using a common SpeI restriction site and subcloned into pPCR-Script SK(+) (Stratagene, La Jolla, CA). To increase the stability of the capped RNA transcribed from this clone, a poly(A) (70-mer) oligonucleotide was added to the 3′ end of the clone between a SalI and KpnI restriction site. The 5′ end of the coding sequence for pNBC was confirmed by 5′ rapid amplification of cDNA ends PCR amplification and primer extension analysis. Furthermore, to confirm that the pNBC amino acid sequence was derived from a bona fide transcript, we amplified the entire open reading frame of human pNBC by reverse transcription-PCR using Marathon Ready cDNA prepared from human pancreas (CLONTECH, Palo Alto, CA) as a template. Nucleotide sequences were determined bidirectionally by automated sequencing (ABI 310 Perkin-Elmer) using Taq polymerase (Ampli-Taq FS, Perkin-Elmer). Sequence assembly and analysis was carried out using Geneworks software (Oxford, UK). Northern blots with various human tissues were obtained fromCLONTECH. The various probes were random prime-labeled with [32P]dCTP to a specific activity of about 1.5 × 109 dpm/μg. The filters were prehybridized at 42 °C for 2 h using 50% formamide, 6× SSPE, 0.5% SDS, Denhardt's solution, and 0.1 mg/ml of sheared herring testes denatured DNA. After the prehybridization, the filters were incubated with the 32P probe using 25 ml of hybridization buffer. The probes were denatured and added to the hybridization solution at 107 dpm/ml. The filters were probed at 42 °C for 18 h and washed in 1× SSC, 0.1% SDS at 45 °C for 60 min (3 changes, 350 ml/wash); after exposure for 9.5 h, the filters were rewashed in 1× SSC, 0.1%SDS at 65 °C for 30 min and 0.1× SSC, 0.1%SDS at 65 °C for 1 h. The glyceraldehyde-3-phosphate dehydrogenase DNA was T4 polynucleotide kinase (New England Biolabs, Beverly, MA) labeled with [32P]γATP to a specific activity of 2.5 μCi/pmol. The filters were prehybridized at 42 °C for 2 h using 50% formamide, 6x SSPE, 0.5% SDS, Denhardt's solution, and 0.1 mg/ml sheared herring testes denatured DNA. After the prehybridization, the filters were probed with the 32P probe using 25 ml of hybridization buffer. The probe was denatured and added to the hybridization solution at 0.5 pmol/ml. The filters were probed at 42 °C for 18 h and then washed 3 times in 1x SSC, 0.1%SDS at 45 °C for 30 min. The following probes were used in the Northern blot studies: 1) a 159-bp PCR product with a sequence common to both pNBC and kNBC (nucleotides 2795–2954 pNBC sequence and 2695 to 2854 in the kNBC sequence); 2) synthetic oligonucleotide specific for pNBC (nucleotides 118 to 212 in the pNBC sequence); 3) synthetic oligonucleotide specific for kNBC (nucleotides 175 to 268 in the kNBC sequence). To prepare the riboprobes, the insert (9.2.1) was subcloned into pPCR-script SK(+) (Stratagene) Riboprobes were synthesized by in vitro transcription and labeled with 35S-CTP. For generation of the antisense riboprobe, the plasmid was linearized withSstI and transcribed by T7 RNA polymerase. For generation of the sense riboprobe, the plasmid was linearized with KpnI and transcribed with T3 RNA polymerase. The RNA transcripts were purified by phenol-chloroform extractions and Sephadex G-50 spin columns (Sigma). The final products were suspended in Tris-EDTA buffer with 0.1 m dithiothreitol. The RNA transcripts were then sheared by alkaline hydrolysis at 68 °C for 5 min. After the shearing, the reaction was neutralized by adding 3 m sodium acetate, pH 5, to make a final acetate concentration of 0.3m. Slices of mouse pancreas (1 mm) were fixed in 4% formalin, and 5-μm sections were attached to glass slides (Fisher). The slides were prewashed and digested for 15 min at 37 °C with proteinase K. To reduce nonspecific background staining, the slides were succinylated with succinic anhydride and acetylated with acetic anhydride. The riboprobes were hybridized for 18 h at 45 °C. The slides were then washed for 15 min in 2× SSC at room temperature, followed by a wash (15 min) in 1 × SSC/50% formamide at 45 °C, then three washes in 2 × SSC/0.1% Triton X-100 at 60 °C for 15 min each, followed by two washes in 0.1 SSC at 60 °C for 15 min each. The slides were then digested by RNase A (25 μg/ml; Sigma) and RNase T1(25 units/ml; Sigma) for 40 min at 37 °C. The slides were washed twice in 2× SSC at 60 °C for 15 min each and then dehydrated in 0.3 m ammonium acetate, 70% ethanol for 5 min followed by a further 5 min of dehydration in 0.3 mammonium acetate, 95% ethanol. The slides were dipped into NTB2 emulsion solution (Eastman Kodak Co.) and exposed for 3 days at 4 °C followed by hematoxylin/eosin staining. The sections were imaged using a Zeiss Axiophot microscope (Max Erb, Los Angeles, CA) and digitized using a Sony 3CCD color video camera (model DXC-960MD, Compix Imaging Systems, Tuscon, AZ) with C Imaging software (Compix Imaging Systems, Tuscon, AZ). The PCR probe used to screen the pancreatic cDNA library was also used to screen an arrayed PAC human genomic library (Genome Systems, St. Louis, MO). DNA from clone F335 was identified by sequencing and was then labeled with digoxigenin dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2× SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with 4′,6-diamidino-2-phenylindole, dihydrochloride for one color experiments. Probe detection for two color experiments was accomplished by incubating the slides in fluoresceinated antidigoxigenin antibodies and Texas red avidin followed by counterstaining with 4′,6-diamidino-2-phenylindole, dihydrochloride. The plasmid containing the complete coding sequence of pNBC was linearized by digestion withKpnI, and capped cRNA was prepared with T3 RNA polymerase using a T3 mMessage mMachine kit RNA capping kit (Ambion, Austin, TX) as recommended by the manufacturer. This cRNA was used for theXenopus oocyte expression studies. An aliquot of the synthesized cRNA was run on a denaturing gel to verify the expected size before oocyte injection. Defolliculated oocytes were injected with 50 nl of sterile water or a solution containing 1 ng/nl capped pNBC cRNA (prepared as described above). They were then bathed in Barth's medium at 18 °C. 3–6 days post-injection, optical recordings were made at 22–24 °C. Intracellular pH was monitored using the fluorescent probe 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a microfluorometer coupled to the microscope (41Kurtz I. J. Clin. Invest. 1987; 80: 928-935Crossref PubMed Scopus (97) Google Scholar). Individual defolliculated oocytes were held in place with pipettes attached to low suction with vegetal pole surface closest to the 40× objective. Before loading with BCECF, the background intensity from each oocyte was digitized at 500 nm and 440 nm (530-nm emission). The oocytes were loaded with 32 μm BCECF-acetoxymethyl ester for 1 h before experimentation in the following solution: NaCl (108 mm), KCl (2 mm), CaCl2 (1 mm), MgCl2 (1 mm), and Hepes (8 mm), pH 7.4. Calibration of intracellular BCECF in the oocytes was performed at the end of each experiment by monitoring the 500/440-nm fluorescence excitation ratio at various values of pHi in the presence of high K+ nigericin standards as described previously (42Sasaki S. Ishibashi K. Nagai T. Marumo F. Biochim. Biophys. Acta. 1992; 1137: 45-51Crossref PubMed Scopus (62) Google Scholar). Three experimental protocols were performed: 1) Na+ removal/addition. The oocytes were bathed in the following Na+-containing solution for 30 min: NaCl (100 mm); KCl (2 mm); CaCl2 (1 mm); MgCl2 (1 mm); NaHCO3 (8 mm) and bubbled with 1.5% CO2, pH 7.4. After a steady state was reached, Na+ was removed by bathing the oocytes in the following Na+-free solution: TMA-Cl (100 mm), KCl (2 mm), CaCl2 (1 mm), MgCl2 (1 mm), TMA-HCO3 (8 mm) bubbled with 1.5% CO2, pH 7.4; 2) Na+ removal/addition with DIDS (0.3 mm); and 3) Na+ removal/addition in Cl−-free solutions with EIPA (10 μm). For the latter experiments, the oocytes were loaded with BCECF in the following Cl−-free solution: sodium gluconate (108 mm), potassium gluconate (2 mm), calcium gluconate (7 mm), magnesium gluconate (2 mm), Hepes (8 mm), pH 7.4. The oocytes were bathed in the following Na+-containing Cl−-free solution for ∼30 min with EIPA (10 μm): sodium gluconate (100 mm), potassium gluconate (2 mm), calcium gluconate (7 mm), magnesium gluconate (2 mm), NaHCO3 (8 mm), pH 7.4. bubbled with 1.5% CO2, pH 7.4. After a steady state was reached, Na+ was removed by bathing the oocytes in the following Na+-free, Cl−-free solution with EIPA (10 μm): TMA-OH (100 mm), d-gluconic acid lactone (100 mm), potassium gluconate (2 mm), calcium gluconate (7 mm), magnesium gluconate (2 mm), TMA-HCO3 (8 mm) bubbled with 1.5% CO2, pH 7.4. Defolliculated oocytes were injected with pNBC cRNA (50 nl, 1 μg/ul) or water and incubated in Barth's medium for 3–6 days at 18 °C before study. The oocytes were preincubated for 1 h in 1 ml of a Na+-free solution containing: TMA-Cl (108 mm), KCl (2 mm), CaCl2 (1 mm), MgCl2 (1 mm), and Hepes (8 mm), pH 7.4. The oocytes were then transferred into 1.4 ml of the following Na+-containing solution: NaCl (100 mm), KCl (2 mm), CaCl2 (1 mm), MgCl2 (1 mm), NaHCO3 (8 mm) bubbled with 1.5% CO2, pH 7.4 with 2 μCi of 22Na+. A 10-μl aliquot was removed from the influx solution for later determination of 22Na+-specific activity.22Na+ influx was measured after 15 min and terminated with three washes of ice-cold Na+-free stop solution. The influx experiments were repeated in the presence of DIDS (0.3 mm). In the DIDS-containing experiments, the oocytes were exposed to 0.3 mm DIDS for 30 min before and throughout the influx period. The oocytes were preincubated in 1 ml for 1 h in a Cl−-free solution containing sodium gluconate (108 mm), potassium gluconate (2 mm), calcium gluconate (7 mm), magnesium gluconate (2 mm), and Hepes (8 mm), pH 7.4. The oocytes were then transferred into 1.4 ml of the following Cl−-containing solution: NaCl (100 mm), KCl (2 mm), CaCl2 (1 mm), MgCl2 (1 mm), NaHCO3 (8 mm) bubbled with 1.5% CO2, pH 7.4. with 3.3 μCi of36Cl−. A 10-μl aliquot was removed from the influx solution for later determination of36Cl−-specific activity.36Cl− influx was measured after 15 min and terminated with three washes of ice-cold stop solution. The sequences submitted to GenBankTM have been scanned against the data base, and the following related human sequences have been identified (AF007216, P02730, P48751, U62531). The cloning of pNBC was based on the identification of human pancreatic cDNA clone in the GenBankTM data base. To obtain full-length pNBC, we screened a human pancreas λgt10 cDNA library using a probe generated by PCR amplification of the human EST sequence. Two overlapping clones were obtained that were fused at a shared SpeI restriction site to generate a full-length clone containing the entire open reading frame. To confirm that the pNBC sequence was derived from a bona fide transcript, reverse transcription-PCR was used to generate a full-length PCR product containing the complete open reading frame. Analysis of the full-length clone revealed a 1079-amino acid open reading frame beginning with the initial methionine as well as 117 bp of 5′-untranslated region. Additional overlapping 3′-untranslated rapid amplification of cDNA ends sequences were almost identical to the sequence recently published by Burnham et al. (30Burnham C.E. Amlal H. Wang Z. Shull G.E. Soleimani M. J. Biol. Chem. 1997; 272: 19111-19114Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) except for minor changes likely due to polymorphisms. The nucleotide sequence of human pNBC has been submitted to the GenBankTM (accession number AF011390). The overall structure of pNBC is similar to kNBC (30Burnham C.E. Amlal H. Wang Z. Shull G.E. Soleimani M. J. Biol. Chem. 1997; 272: 19111-19114Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) and other members of the anion exchange gene family (43Alper S. Annu. Rev. Physiol. 1991; 53: 549-564Crossref PubMed Scopus (205) Google Scholar). Specifically, pNBC has 12 predicted transmembrane regions and hydrophilic intracellular N- and C-terminal regions. As shown in Fig. 1, the sequences of pNBC and kNBC are identical after the Ser residue at position 42 of kNBC and position 86 of the pancreatic sequence. Unlike the kidney sequence, the N terminus of pNBC before the region common to both polypeptides contains blocks of charged amino acids. Further distinctive features of the N terminus of pNBC are 1) the consensus phosphorylation site for protein kinase A beginning at Lys46, 2) the consensus phosphorylation sites for protein kinase C beginning at Ser38 and Ser65, and 3) the casein kinase II phosphorylation si

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