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• W2095758052 abstract "The electroneutral Na+-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pHi) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBankTM accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); ∼50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; ∼73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na+-driven anion exchanger (NDAE1) fromDrosophila. Northern blot analysis of NDCBE1 shows a robust ∼12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed inXenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pHi and [Na+]i (monitored with microelectrodes) that require HCO 3− and are blocked by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The pHi increase also requires extracellular Na+. The Na+:HCO 3−stoichiometry is 1:2. Forward-running NDCBE1 mediates a36Cl efflux that requires extracellular Na+ and HCO 3− and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl−. Thus, NDCBE1 encodes a human, electroneutral Na+-driven Cl-HCO3 exchangerAF069512AAC82380. The electroneutral Na+-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pHi) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBankTM accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); ∼50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; ∼73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na+-driven anion exchanger (NDAE1) fromDrosophila. Northern blot analysis of NDCBE1 shows a robust ∼12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed inXenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pHi and [Na+]i (monitored with microelectrodes) that require HCO 3− and are blocked by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The pHi increase also requires extracellular Na+. The Na+:HCO 3−stoichiometry is 1:2. Forward-running NDCBE1 mediates a36Cl efflux that requires extracellular Na+ and HCO 3− and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl−. Thus, NDCBE1 encodes a human, electroneutral Na+-driven Cl-HCO3 exchangerAF069512AAC82380. expressed sequence tag rapid amplification of cDNA ends nucleotide(s) polymerase chain reaction untranslated region fluorescence in situ hybridization 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid base pair(s) kilobase(s) bacterial artificial chromosome The first transporter shown to be involved in the regulation of intracellular pH (pHi) was the Na+-driven Cl-HCO3 exchanger, initially described in squid axons (1Boron W.F. De Weer P. Nature. 1976; 259: 240-241Crossref PubMed Scopus (92) Google Scholar, 2Russell J.M. Boron W.F. Nature. 1976; 264: 73-74Crossref PubMed Scopus (145) Google Scholar, 3Boron W.F. Russell J.M. J. Gen. Physiol. 1983; 81: 373-399Crossref PubMed Scopus (103) Google Scholar), snail neurons (4Thomas R.C. J. Physiol. 1976; 255: 715-735Crossref PubMed Scopus (141) Google Scholar, 5Thomas R.C. Nature. 1976; 262: 54-55Crossref PubMed Scopus (74) Google Scholar, 6Thomas R.C. J. Physiol. 1977; 273: 317-338Crossref PubMed Scopus (231) Google Scholar), and barnacle muscle (7Boron W.F. Am. J. Physiol. 1977; 233: C61-C73Crossref PubMed Google Scholar). This acid extruder (i.e. a transporter that behaves as if it mediates net H+ efflux) could function according to any of the four schemes (8Boron W.F. J. Gen. Physiol. 1985; 85: 325-345Crossref PubMed Scopus (57) Google Scholar) in Fig. 1 A. In physiology experiments on mammalian cells, it is often extremely difficult to distinguish this transporter from either an electroneutral Na/HCO3 cotransporter (NBCn1, Fig. 1 B) (9Aalkjaer C. Hughes A. J. Physiol. ( Lond. ). 1991; 436: 57-73Crossref PubMed Scopus (48) Google Scholar, 10Choi I. Aalkjaer C. Boulpaep E.L. Boron W.F. Nature. 2000; 405: 571-575Crossref PubMed Scopus (213) Google Scholar) or an electrogenic Na/HCO3 cotransporter (NBCe1, Fig.1 C) (11Boron W.F. Boulpaep E.L. J. Gen. Physiol. 1983; 81: 53-94Crossref PubMed Scopus (385) Google Scholar, 12Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (378) Google Scholar) because of problems depleting cells of Cl− or measuring very small electrical changes. In the absence of electrical data, one could not distinguish an electroneutral Na+-driven Cl-HCO3 exchanger from the scheme in Fig. 1 D, which is a hybrid of those in Fig. 1,A–C. The Na+-driven anion exchanger (NDAE1) recently cloned from Drosophila (13Romero M.F. Henry D. Nelson S. Harte P.J. Dillon A.K. Sciortino C.M. J. Biol. Chem. 2000; 275: 24552-24559Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) does not require HCO 3− and could function according to either of the top two schemes in Fig. 1 A, but with OH− replacing HCO 3−. In mammalian cells, increases in pHi that appear to depend on Na+, Cl−, and HCO 3−have been described in neurons (14Schwiening C.J. Boron W.F. J. Physiol. ( Lond. ). 1994; 475: 59-67Crossref PubMed Scopus (159) Google Scholar, 15Smith G.A. Brett C. Church J. J. Physiol. ( Lond. ). 1998; 512: 487-505Crossref PubMed Scopus (38) Google Scholar, 16Bonnet U. Leniger T. Wiemann M. Brain Res. 2000; 872: 116-124Crossref PubMed Scopus (39) Google Scholar), astrocytes (17Ko Y.P. Lang H.J. Loh S.H. Chu K.C. Wu M.L. Chin. J. Physiol. 1999; 42: 237-248PubMed Google Scholar, 18Shrode L.D. Putnam R.W. Glia. 1994; 12: 196-210Crossref PubMed Scopus (75) Google Scholar), renal mesangial cells (19Boyarsky G. Ganz M.B. Sterzel B. Boron W.F. Am. J. Physiol. 1988; 255: C857-C869Crossref PubMed Google Scholar), corneal endothelium (20Lane J. Wigham C.G. Hodson S.A. Biophys. J. 2000; 78: 2493-2498Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), bile duct (21Strazzabosco M. Joplin R. Zsembery A. Wallace L. Spirli C. Fabris L. Granato A. Rossanese A. Poci C. Neuberger J.M. Okolicsanyi L. Crepaldi G. Hepatology. 1997; 25: 976-985Crossref PubMed Scopus (50) Google Scholar), aortic endothelium (22Faber S. Lang H.J. Hock F.J. Scholkens B.A. Mutschler E. Cell Physiol. Biochem. 1998; 8: 202-211Crossref PubMed Scopus (18) Google Scholar), spermatozoa (23Zeng Y. Oberdorf J.A. Florman H.M. Dev. Biol. 1996; 173: 510-520Crossref PubMed Scopus (175) Google Scholar), and various transformed cells (24Kaplan D. Boron W.F. J. Biol. Chem. 1994; 269: 4116-4124Abstract Full Text PDF PubMed Google Scholar, 25Ladoux A. Krawice I. Cragoe Jr., E.J. Abita J.P. Frelin C. Eur. J. Biochem. 1987; 170: 43-49Crossref PubMed Scopus (24) Google Scholar, 26Kottgen M. Leipziger J. Fischer K.G. Nitschke R. Greger R. Pflügers Arch. 1994; 428: 179-185Crossref PubMed Scopus (31) Google Scholar). However, given the difficulties noted above, definitively ascribing a cell phenotype to a transporter will require molecular tools. It is therefore extremely important not only to clone the genes encoding various Na+-driven HCO 3−transporters, but also to assign their function unambiguously to one of the schemes in Fig. 1. Here we report the tissue distribution, chromosomal location, and functional characterization of a cDNA that we cloned from human brain (GenBankTM accession number AF069512 and NCBI accession number AAC82380). Our physiological analysis indicates that this cDNA encodes an electroneutral Na+-driven Cl-HCO3 exchanger (NDCBE1, Fig. 1 A). We cloned NDCBE1 in three parts. After performing a BLAST search, using the salamander NBCe1 cDNA sequence (GenBankTM accession number AF001958) as the query, of the GenBankTM data base, we obtained the central part as a cDNA expressed sequence tag (EST)1 clone AA775966(catalogue number CDNA-1401, Genome System Inc., St. Louis, MO). We obtained the 5′-end by performing rapid amplification of cDNA ends (RACE). Using human brain poly(A)+ RNA (CLONTECH, Palo Alto, CA) as the template, we generated cDNA using an NDCBE1-specific primer corresponding to nucleotide sequence 598–627 (numbered from first nucleotide of open reading frame). The downstream, NDCBE1-specific primers for RACE corresponded to nt 547–579 and nt 328–358. We used the two upstream primers provided in the RACE kit (Life Technologies, Inc.). We obtained the 3′-end by performing a nested polymerase chain reaction (PCR), using a human brain λZAPII cDNA library (gift of Dr. Nancy Lynn Johnston, John Hopkins University) as the template. The upstream, NDCBE1-specific primers corresponded to nt 1876–1905 and nt 2014–2043, and the downstream primer corresponded to a sequence near the polycloning site in the pBluescript vector. We verified that the three cDNA fragments represent a single transcript by performing PCR using an upstream primer corresponding to a region (nt −44 to −18) in the 5′-untranslated region (UTR) and a downstream primer corresponding to a region in the 3′-UTR (nt 3136–3165). We obtained the consensus sequence by directly sequencing the full-length PCR product (Keck Sequencing Center, Yale University). We also subcloned the full-length PCR product into the oocyte expression vector pGH19 (27Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1075) Google Scholar), sequenced the clone, and corrected PCR errors on the basis of the consensus sequence. The full-length sequence (GenBankTMaccession number AF069512) was released in 1998. Using a NDCBE1 cDNA as template, we generated a 304-bp cDNA probe, corresponding to a unique region (nt 54–358). DNA clone 477 L 11 from the RPCI-11 human BAC library was identified by Research Genetics, Inc. (Huntsville, AL). The purified BAC DNA was labeled with biotin-dUTP by nick translation. DNA of a chromosome-12 painting library was labeled with Cy3-dUTP by PCR. A biotin-labeled BAC probe, alone or together with Cy3-labeled chromosome-12 painting probe, was hybridized to metaphase chromosome spreads in the presence of human Cot-1 DNA and salmon sperm DNA. The biotin-labeled probe was detected by avidin-fluorescein isothiocyanate. Fifty metaphase spreads were taken for analysis and measurements. Gray scale images were obtained using an Olympus epifluorescence microscope coupled to a cooled CCD camera (Photometrics Ltd., Tucson, AZ). Fractional length measurement and band assignment were established by analysis of ten chromosomes (28Francke U. Cytogenet. Cell Genet. 1994; 65: 206-218Crossref PubMed Google Scholar). Northern blots from various human tissues (catalogue numbers 7760-1 and 7759-1) were obtained fromCLONTECH. The [32P]dCTP-labeled, randomly primed 671-bp cDNA probe was generated to the unique 5′-region of NDCBE1 (nt −44 to 627). Membranes were incubated overnight at 68 °C in ExpressHyb™ hybridization buffer (CLONTECH) containing the 32P-labeled probe. Subsequently, membranes were washed at room temperature in 2 × SSC, 0.05% SDS for 40 min and then at 50 °C in 0.1 × SSC, 0.1% SDS for 1.5 h, before being exposed to Kodak X-Omat film at −80 °C for 24 h for detection of high-intensity signals. We transcribed NDCBE1 cDNA in vitrousing an mMessage mMachine™ kit (Ambion, Austin, TX) with T7 RNA polymerase. Defolliculated Xenopus laevis oocytes (Stage V–VI) were prepared as described previously (29Grichtchenko I.I. Romero M.F. Boron W.F. J. Gen. Physiol. 2000; 115: 533-545Crossref PubMed Scopus (32) Google Scholar) and injected with 50 nl of NDCBE1 cRNA (1 μg/μl) or water and incubated in OR3 media. Injected oocytes were maintained for 3–7 days at 18 °C before use. For experiments in which we reversed NDCBE1, the 50-nl injectate contained not only NDCBE1 cRNA (1 μg/μl), but also cRNA encoding the amiloride-sensitive epithelial Na+channel (ENaC; 0.2 μg/μl, gift of Dr. Cecilia Canessa, Yale University). Immediately after this coinjection, we added 20 μm amiloride to the oocyte culture media. One hour prior to the experiment, we transferred coinjected oocytes into amiloride-free HEPES solution. The voltage, pH- and sodium-sensitive microelectrodes, were prepared as described previously (10Choi I. Aalkjaer C. Boulpaep E.L. Boron W.F. Nature. 2000; 405: 571-575Crossref PubMed Scopus (213) Google Scholar, 29Grichtchenko I.I. Romero M.F. Boron W.F. J. Gen. Physiol. 2000; 115: 533-545Crossref PubMed Scopus (32) Google Scholar, 30Siebens A.W. Boron W.F. J. Gen. Physiol. 1987; 90: 799-831Crossref PubMed Scopus (66) Google Scholar). The pH electrode tip was filled with proton ionophore 1 mixture B (Fluka Chemical Corp., Ronkonkoma, NY) and back-filled with a pH 7 phosphate buffer (31Chao P. Ammann D. Oesch U. Simon W. Lang F. Pflügers Arch. 1988; 411: 216-219Crossref PubMed Scopus (59) Google Scholar). The Na+ electrode tip was filled with sodium ionophore 1 mixture A (Fluka Chemical Corp.) and back-filled with 10 mm NaCl. Electrodes were connected to high-impedance electrometers (model FD-223; World Precision Instruments, Inc., Sarasota, FL), which in turn were connected to the A-D converter of a computer. In electrophysiological experiments, the CO2/HCO 3−-free ND96 solution contained (in mm) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5); osmolality was 188–200 mOsm/kg, 22 °C. In solutions equilibrated with 1.5% CO2 (pH 7.50), 5% CO2 (pH 7.50), and 20% CO2 (pH 6.9), we replaced 10, 33, and 33 mm NaCl, respectively, with an equivalent molarity of NaHCO3. EtherN-methyl-d-glucammonium (NMDG+) replaced Na+ in Na+-free solutions, and gluconate replaced Cl− in Cl−-free solutions. In some solutions we replaced 16 mm NaCl with 16 mm of n-butyric acid sodium salt (B-5887, Sigma). Ten to twenty oocytes were incubated at room temperature for ∼3 h in 250 μl of36Cl “loading solution”, which consisted of (in mm): 70 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, and 32 HEPES titrated with NaOH to pH 7.5;36Cl was present as 190 μCi/mmol of total Cl−. We then rapidly washed the oocytes five times with 0.5 ml of ice-cold HEPES flux solution (same as “loading solution,” but without 36Cl). The washed oocytes were transferred to one-half of a 1-ml equilibrium-dialysis chamber (BelArts Products, Pequannock, NJ) containing ∼0.5 ml of ice-cold HEPES flux solution. The other half of the dialysis chamber, modified to permit continuous inflow and outflow of solution, was placed open-side up. We added a small magnetic stirring flea, covered the opening with a nylon mesh membrane (which permits free exchange of solution between the two chamber halves), and lightly coated the open edges of the chamber half with silicon stopcock grease (High Vacuum Grease, Dow-Corning, Midland, MI), which acted as a gasket when the two chamber halves were joined and placed oocyte-side up on a magnetic stirring plate. We flowed ice-cold HEPES flux solution at 8 ml/min for 2 min to wash out extracellular 36Cl and then flowed room temperature solution at 3 ml/min for 4 min before beginning to collect samples of the chamber effluent at 3 ml/min every 3 min. Experiments with dye indicated that the time to exchange 95% of the fluid in the upper (i.e. oocyte) chamber half was ∼2 min. All samples were collected directly into plastic scintillation vials, to which we later added 9 ml of Ultima Gold™ liquid scintillation counting mixture (Packard Instrument Co., Meriden, CT). At the end of the experiment, the chamber was rapidly taken apart, the oocytes were transferred to 150 μl of a 10% SDS solution in 1 n NaOH for digestion, and a 50-μl aliquot of the digest was prepared for liquid scintillation counting. We calculated the initial36Cl content of the oocytes and the fractional rate of36Cl loss during each sampling period in the experiment. The CO2/HCO 3− flux solution used in the 36Cl flux experiments was the same as the HEPES flux solution, except that 33 mmHCO 3− replaced 33 mmCl−, and the solution was equilibrated with 5% CO2, 95% O2. Data are expressed as mean ± S.E. Statistical significance was judged from unpaired Student'st tests. Querying with the sequence of the cDNA encoding salamander NBCe1 (12Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (378) Google Scholar), we searched the GenBankTM data base and found a human brain EST clone (accession number AA775966) that, at one end, was 53% identical to query. Sequencing this EST clone revealed a 2-kb open reading frame, representing the center of the full-length clone. We obtained the 5′-end by RACE on human brain RNA and the 3′-end by PCR on a human frontal-lobe cDNA library. We obtained the full-length clone, which encodes 1044 amino acids, by performing PCR on human brain cDNA, using primers designed to amplify the entire open reading frame as well as portions of the 5′- and 3′-UTRs. Fig.2 A compares the deduced amino acid sequence of NDCBE1 to electroneutral NBCn1 from rat (NBCn1-D; 73% identity) (10Choi I. Aalkjaer C. Boulpaep E.L. Boron W.F. Nature. 2000; 405: 571-575Crossref PubMed Scopus (213) Google Scholar) and to electrogenic NBCe1 from rat kidney (rkNBC; 50% identity) (32Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar). NDCBE1 has two consensus sites forN-glycosylation on the presumed 5,6 extracellular loop (residues 646–649/NHTL and 666–669/NLTV), 12 for protein kinase C, and one for protein kinase A (243–246/KKQS). Like NBCn1 (10Choi I. Aalkjaer C. Boulpaep E.L. Boron W.F. Nature. 2000; 405: 571-575Crossref PubMed Scopus (213) Google Scholar), NDCBE1 has one potential DIDS motif (33Kopito R.R. Lee B.S. Simmons D.M. Lindsey A.E. Morgans C.W. Schneider K. Cell. 1989; 59: 927-937Abstract Full Text PDF PubMed Scopus (202) Google Scholar, 34Okubo K. Kang D. Hamasaki N. Jennings M. J. Biol. Chem. 1994; 269: 1918-1926Abstract Full Text PDF PubMed Google Scholar) (813–816/KLKK), corresponding to the second of two similar motifs in electrogenic NBCs (12Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (378) Google Scholar). Fig.2 B summarizes the relationships among the primary structures of NDCBE1 and other members of the HCO 3− transporter superfamily. An NDCBE1 BAC clone produced clear FISH signals on a pair of chromosomes (not shown), which, on the basis of their size, morphology, and DAPI stain-banding pattern, we identified as chromosome 12. Cohybridization of this BAC clone with a chromosome-12 painting probe confirmed the identification (Fig.2 C). The BAC clone hybridized 22% of the distance from the centromere to the telomere of arm 12q, corresponding to band 12q13 (Fig. 2 D). In contrast, human NBCe1 (SLC4A5) maps (35Abuladze N. Lee I. Newman D. Hwang J. Boorer K. Pushkin A. Kurtz I. J. Biol. Chem. 1998; 273: 17689-17695Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) to chromosome 4q21, and human NBCn1 (SLC4A7) maps (36Pushkin A. Abuladze N. Lee I. Newman D. Hwang J. Kurtz I. Genomics. 1999; 57: 321-322Crossref PubMed Scopus (23) Google Scholar) to 3p22. A Northern blot analysis of multiple human tissues (Fig. 2 E) revealed a ∼12-kb transcript, with strong signals in brain and testis and a weaker signals in kidney > ovary. The weak ∼9.5-kb bands (pancreas > kidney) may represent NBCe1 (35Abuladze N. Lee I. Newman D. Hwang J. Boorer K. Pushkin A. Kurtz I. J. Biol. Chem. 1998; 273: 17689-17695Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 37Choi I. Romero M.F. Khandoudi N. Bril A. Boron W.F. Am. J. Physiol. 1999; 276: C576-C584Crossref PubMed Google Scholar). The very weak ∼7.5-kb band (testis) may represent the human ortholog of NBCn1 (38Pushkin A. Abuladze N. Lee I. Newman D. Hwang J. Kurtz I. J. Biol. Chem. 1999; 274: 16569-16575Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The bands at ∼6.3 kb (brain > testis > kidney), ∼4.2 kb (testis), and ∼3.3 kb (brain > testis) may represent alternative splicing of the NDCBE1 primary transcript or products of different but related genes. We found that the three bands that appear in the Northern blot of whole brain also are present in multiple brain regions (not shown), including cerebral cortex, cerebellum, medulla, thalamus, and hippocampus. However, NDCBE1 was notably absent from spinal cord. To determine the function of NDCBE1, we injected cRNA into oocytes and used microelectrodes to monitor pHi and membrane potential (Vm). In oocytes expressing NDCBE1 (Fig.3 A), extracellular 1.5% CO2, 10 mmHCO 3−elicited a rapid fall in pHi due to CO2 influx (39Boron W.F. De Weer P. J. Gen. Physiol. 1976; 67: 91-112Crossref PubMed Scopus (669) Google Scholar), followed by an increase due to HCO 3−uptake. Removing extracellular Na+ converted the pHi recovery to a very slow acidification, reflecting reversal of NDCBE1. In water-injected oocytes, 1.5% CO2, 10 mmHCO 3−caused the usual pHifall, followed by a long period of stability, with or without Na+ (not shown). Thus, NDCBE1 requires Na+. In oocytes expressing NDCBE1 (Fig.3 A), 1.5% CO2, 10 mmHCO 3−caused a small, slow depolarization (arrow), and removing Na+ caused a slight hyperpolarization (arrowhead), as observed in H2O-injected oocytes (not shown). In contrast, with oocytes expressing electrogenic NBCs, applying CO2/HCO 3−elicits a large and rapid hyperpolarization, whereas removing Na+ elicits a large and rapid depolarization (12Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (378) Google Scholar, 29Grichtchenko I.I. Romero M.F. Boron W.F. J. Gen. Physiol. 2000; 115: 533-545Crossref PubMed Scopus (32) Google Scholar, 40Bevensee M.O. Schmitt B.M. Choi I. Romero M.F. Boron W.F. Am. J. Physiol. Cell Physiol. 2000; 278: C1200-C1211Crossref PubMed Google Scholar). Thus, NDCBE1 is electroneutral. When we acidified NDCBE1-expressing oocytes with butyric acid, rather than CO2, pHifailed to recover in the presence of Na+ (Fig.3 B). However, after we removed the butyric acid and applied CO2/HCO 3−, pHiinitially recovered rapidly, even though pHi was substantially higher than in the presence of butyric acid. Thus, NDCBE1 requires HCO 3−. Applying 0.5 mm DIDS almost completely blocks the pHi recovery (Fig. 3 C). In six experiments, the inhibition averaged 95% ± 10%. Thus, NDCBE1 is DIDS sensitive. When we introduced 5% CO2, 33 mmHCO 3−(pH 7.50) to NDCBE1-expressing oocytes, the 36Cl efflux increased more than 3-fold (Fig.3 D). The slow decline in 36Cl efflux probably reflects a NDCBE1-mediated increase in pHi, [HCO 3−]i and [Na+]i. These changes were absent in oocytes injected with water, rather than NDCBE1 cRNA. Moreover, in NDCBE1-expressing oocytes, the transition from HEPES to CO2/HCO 3−did not affect36Cl efflux in either the absence of Na+ or presence of 0.5 mm DIDS. Thus, while NDCBE1 is mediating Na+-dependent HCO 3−uptake, it also mediates a Cl− efflux with the properties expected of a Na+-driven Cl-HCO3 exchanger. To determine whether NDCBE1 transports Na+, we used Na+-sensitive microelectrodes to monitor [Na+]i. In an oocyte-expressing NDCBE1, extracellular 5% CO2, 33 mmHCO 3−caused [Na+]i to increase (Fig. 3 E). The mean rate of this [Na+]i increase was substantially higher than in the presence of DIDS or in water-injected oocytes (Fig.3 F). The average, DIDS-sensitive d[Na+]i/dt was 1.01 μms−1. In parallel experiments (not shown), we determined dpHi/dt under identical conditions, and computed the HCO 3−pseudo-flux (Fig.3 F), the DIDS-sensitive component of which averaged 2.19 μm s−1. We already knew that the squid axon's Na+-driven Cl-HCO3exchanger is very difficult to reverse (3Boron W.F. Russell J.M. J. Gen. Physiol. 1983; 81: 373-399Crossref PubMed Scopus (103) Google Scholar), consistent with the slow pHi decrease in 0-Na+ in Fig.4 A. We took two steps in an attempt to speed the reversed NDCBE1. First, we coexpressed ENaC Na+ channels to increase [Na+]i. Second, we exposed the oocyte to 20% CO2 to increase [HCO 3−]i. Removing Na+ reversed NDCBE1, causing pHi to decline. However, removing extracellular Cl− reversibly blocked this decline (Fig. 4 A) and caused a small hyperpolarization, as in water-injected oocytes (not shown). In water-injected oocytes (Fig. 4 B), Na+ removal blocked a very slow pHi recovery (probably due to endogenous Na-H exchange at very low pHi), but Cl− removal had no effect on the pHi trajectory. Thus, the reversed NDCBE1 requires external Cl−, as expected of a Na+-driven Cl-HCO3 exchanger. After we submitted our NDCBE1 sequence to GenBankTM, two other groups (41Nagase T. Ishikawa K. Suyama M. Kikuno R. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 355-364Crossref PubMed Scopus (204) Google Scholar, 42Amlal H. Burnham C.E. Soleimani M. Am. J. Physiol. 1999; 276: F903-F913PubMed Google Scholar) published partial sequences of NDCBE1, one a 2-kb fragment referred to as “NBC-3” (42Amlal H. Burnham C.E. Soleimani M. Am. J. Physiol. 1999; 276: F903-F913PubMed Google Scholar). After we submitted our paper, a paper appeared by Wang et al. (43Wang C.Z. Yano H. Nagashima K. Seino S. J. Biol. Chem. 2000; 275: 35486-35490Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), who cloned from mouse insulinoma cells a cDNA named NCBE, 71% identical on the amino acid level to NDCBE1. Mouse NCBE's 5.5-kb transcript, like the transcripts of human NDCBE1, is robustly expressed in cerebrum and cerebellum. However, mouse NCBE mRNA is only weakly present in testis. The function of mouse NCBE is unclear. It mediates a 22Na influx that largely depends on extracellular [Cl−]. In addition, oocytes expressing this mouse clone mediate a 36Cl efflux that is only partially external Na+-dependent or DIDS-sensitive. Moreover, because no 36Cl-efflux data are available from water-injected oocytes, it is impossible to know whether the 36Cl-efflux data represent NCBE activity. Finally, no electrical data are available. Human NDCBE1 is functionally similar to Drosophila NDAE1 (13Romero M.F. Henry D. Nelson S. Harte P.J. Dillon A.K. Sciortino C.M. J. Biol. Chem. 2000; 275: 24552-24559Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) in that both exchange extracellular Na+ and “base” for intracellular Cl−. However, human NDCBE1 is strictly HCO 3−-dependent, whereasDrosophila NDAE1 apparently transports OH− in the absence of HCO 3−. Although CO2/HCO 3−enhances pHi changes, it is not clear that Drosophila NDAE1 can transport HCO 3−. The CO2/HCO 3−could act strictly as a buffer, dissipating OH− unstirred layers. Another difference is that expression of Drosophila NDAE1 in oocytes is associated with a Cl− current, as well as an inward current caused by applying CO2/HCO 3−. On the other hand, in oocytes expressing human NDCBE1, the V mchanges caused by altering [HCO 3−]o or [Cl−]o are no different than in water-injected oocytes. Because Drosophila NDAE1 and human NDCBE1 come from distantly related phyla and not closely related in terms of deduced amino acid sequence (47% identity), one must keep open the possibility that, although they appear superficially similar in some respects,Drosophila NDAE1 and human NDCBE1 may function by different molecular mechanisms (Fig. 1 A). The ratio of net HCO 3−and Na+ fluxes mediated by NDCBE1 was (2.19 μms−1)/(1.01 μms−1), or 2.17, consistent with the 2:1 stoichiometry expected of a Na+-driven Cl-HCO3exchanger. Because NDCBE1 is electroneutral, we presume that the net Cl− efflux is the same as the net Na+ influx (Fig. 1 A). However, it was impractical to measure the net Cl− efflux directly with ion-sensitive microelectrodes, because [Cl−]i is too high relative to NDBCE1 expression. However, we can calculate the unidirectional Cl− efflux from the DIDS-sensitive component of the rate constant for 36Cl efflux in NDCBE1-expressing oocytes and the resting [Cl−]i of NDAE-expressing oocytes (13Romero M.F. Henry D. Nelson S. Harte P.J. Dillon A.K. Sciortino C.M. J. Biol. Chem. 2000; 275: 24552-24559Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar): 0.00025 s−1 × 29.5 mm = 7.4 μm s−1. This unidirectional flux is ∼7.3-fold higher than the expected net flux, suggesting that NDCBE1 mediates substantial Cl-Cl exchange in parallel with Na+-driven Cl-HCO3 exchange. Indeed, the unidirectional Cl− efflux from barnacle muscle fibers is also much higher than the net HCO 3−efflux mediated by the endogenous Na+-driven Cl-HCO3 exchanger (44Boron W.F. Russell J.M. Brodwick M.S. Keifer D.W. Roos A. Nature. 1978; 276: 511-513Crossref PubMed Scopus (44) Google Scholar). We have now cloned the electroneutral Na+-driven Cl-HCO3 exchanger, the first transporter shown to regulate pHi in any cell. The heavy expression of the NDCBE1 transcript in multiple brain regions, including hippocampus, suggests that NDCBE1 plays a major role in pHi regulation in human neurons. In the rat, functional data show that the Na+-driven Cl-HCO3 exchanger is a key pHi regulator in pyramidal neurons from the hippocampal CA1 region (14Schwiening C.J. Boron W.F. J. Physiol. ( Lond. ). 1994; 475: 59-67Crossref PubMed Scopus (159) Google Scholar). pHi is critically important for neuronal function because pHi changes substantially modulate the activity of a variety of CNS channels (45–57). Low pHi inhibits (and/or high pHi stimulates) spontaneous firing in neurons (16Bonnet U. Leniger T. Wiemann M. Brain Res. 2000; 872: 116-124Crossref PubMed Scopus (39) Google Scholar, 58Bonnet U. Wiemann M. Brain Res. 1999; 840: 16-22Crossref PubMed Scopus (34) Google Scholar, 59Meyer T.M. Munsch T. Pape H.C. Neuroreport. 2000; 11: 33-37Crossref PubMed Scopus (22) Google Scholar), membrane excitability (60Church J. J. Physiol. ( Lond. ). 1992; 455: 51-71Crossref PubMed Scopus (44) Google Scholar), and epileptiform activity (61Xiong Z.Q. Saggau P. Stringer J.L. J. Neurosci. 2000; 20: 1290-1296Crossref PubMed Google Scholar). pHiis important for CNS processes other than excitability. For example, neurite formation (62Kostenko M.A. Musienko V.S. Smolikhina T.I. Brain Res. 1983; 276: 43-50Crossref PubMed Scopus (25) Google Scholar) requires HCO 3−. Thus, NDCBE1 is in a position to influence a wide range of neuronal behaviors." @default.
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• W2095758052 title "Cloning, Characterization, and Chromosomal Mapping of a Human Electroneutral Na+-driven Cl-HCO3Exchanger" @default.
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