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• W2039469985 abstract "Regulation of intra- and extracellular ion activities (e.g. H+, Cl−, Na+) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na+/HCO3− cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO3−transporters are known: a neutral Na+/ HCO3− cotransporter, a K+/ HCO3− cotransporter, and a Na+-dependent Cl−-HCO3− exchanger. We report the cloning and characterization of a Na+-coupled Cl−-HCO3− exchanger and a physiologically unique bicarbonate transporter superfamily member. ThisDrosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughoutDrosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl−, Na+, H+, and HCO3− but does not require HCO3−. Transport is blocked by the stilbene 4,4′-diisothiocyanodihydrostilbene- 2,2′-disulfonates and may not be strictly electroneutral. Our functional data suggest thisNa+driven anionexchanger (NDAE1) is responsible for the Na+-dependent Cl−-HCO3− exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva. Regulation of intra- and extracellular ion activities (e.g. H+, Cl−, Na+) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na+/HCO3− cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO3−transporters are known: a neutral Na+/ HCO3− cotransporter, a K+/ HCO3− cotransporter, and a Na+-dependent Cl−-HCO3− exchanger. We report the cloning and characterization of a Na+-coupled Cl−-HCO3− exchanger and a physiologically unique bicarbonate transporter superfamily member. ThisDrosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughoutDrosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl−, Na+, H+, and HCO3− but does not require HCO3−. Transport is blocked by the stilbene 4,4′-diisothiocyanodihydrostilbene- 2,2′-disulfonates and may not be strictly electroneutral. Our functional data suggest thisNa+driven anionexchanger (NDAE1) is responsible for the Na+-dependent Cl−-HCO3− exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva. central nervous system electrogenic Na+/HCO3−cotransporter (i.e. SLC4A4) bicarbonate transporter superfamily 4,4′-diisothiocyanodihydrostilbene- 2,2′-disulfonate Na+ driven anion exchanger anion exchanger base pair(s) reverse transcriptase polymerase chain reaction transmembrane span intracellular pH intracellular Cl− activity intracellular Na+ activity untranslated region Ionic homeostasis is the key to normal function of most biological systems. This regulation is especially important for tissues with highly specialized functions, such as the central nervous system (CNS),1 digestive tract, respiratory tract, and urinary system. Active transport of ions by ATPases (pumps) maintains ionic gradients and aid ion channels in “setting” the membrane potential. Secondary active transporters make use of one or more aspects of the membrane electrochemical gradient to specifically move ions and nutrients into and out of cellular compartments. With our cloning of an electrogenic Na+/HCO3− cotransporter (NBC; i.e.SLC4A4 2The electrogenic NBC is currently designated by several nomenclatures in the literature: NBC1, kNBC, pNBC, hhNBC, and SLC4A4 (see Ref. 3Romero M.F. Boron W.F. Annu. Rev. Physiol. 1999; 61: 699-723Crossref PubMed Scopus (183) Google Scholar for a detailed explanation). SLC4A4 is the human gene designation indicating “solute carrier family 4A, member 4” by the human genome nomenclature. The clones that are currently referred to as NBC2, mNBC3, and NBCn1 are likely splice variants of the same gene; however, this has not been explicitly demonstrated. Currently NBC2 is given a designation of SLC4A6 and mNBC3 as SLC4A7. Another apparently distinct human cDNA, SLC4A8, was deposited in GenBankTM (AF069512) but has not yet been functionally characterized.2The electrogenic NBC is currently designated by several nomenclatures in the literature: NBC1, kNBC, pNBC, hhNBC, and SLC4A4 (see Ref. 3Romero M.F. Boron W.F. Annu. Rev. Physiol. 1999; 61: 699-723Crossref PubMed Scopus (183) Google Scholar for a detailed explanation). SLC4A4 is the human gene designation indicating “solute carrier family 4A, member 4” by the human genome nomenclature. The clones that are currently referred to as NBC2, mNBC3, and NBCn1 are likely splice variants of the same gene; however, this has not been explicitly demonstrated. Currently NBC2 is given a designation of SLC4A6 and mNBC3 as SLC4A7. Another apparently distinct human cDNA, SLC4A8, was deposited in GenBankTM (AF069512) but has not yet been functionally characterized.), we found that NBC and the anion exchangers (AEs; i.e. SLC4A1-SLC4A3) form a bicarbonate transporter superfamily (BTS) (1Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (382) Google Scholar, 2Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar, 3Romero M.F. Boron W.F. Annu. Rev. Physiol. 1999; 61: 699-723Crossref PubMed Scopus (183) Google Scholar). More recently three groups have reported unique full-length cDNAs, which are additions to the BTS: NBC-2 from retina (13Ishibashi K. Sasaki S. Marumo F. Biochem. Biophys. Res. Commun. 1998; 246: 535-538Crossref PubMed Scopus (60) Google Scholar), an electroneutral NBC (NBCn1) (14Choi I. Aalkjaer C. Romero M.F. Boron W.F. FASEB J. 1999; 13 (abstr.): 400Google Scholar), and NBC-3 (SLC4A7) from muscle (15Pushkin 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 (173) Google Scholar). Functional data for NBC-2 have not been reported. NBCn1 is an electroneutral Na+/HCO3−cotransporter that is partially blocked by DIDS (14Choi I. Aalkjaer C. Romero M.F. Boron W.F. FASEB J. 1999; 13 (abstr.): 400Google Scholar). NBC-3 is currently characterized as a DIDS-insensitive, 5-(N-ethyl-N-isopropyl) amiloride-sensitive, Na+/HCO3− cotransporter (15Pushkin 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 (173) Google Scholar) whose electrical nature is not yet known. It is presently unclear whether these clones arise from separate genes or are splicing isoforms. Of the NBC clones reported, none are Cl−-dependent or transport Cl−. Physiologically two other HCO3− transporters are known, a K+/HCO3− cotransporter (5Hogan E.M. Cohen M.A. Boron W.F. J. Gen. Physiol. 1995; 106: 821-844Crossref PubMed Scopus (28) Google Scholar) and a Na+-dependent Cl−-HCO3− exchanger (6Russell J.M. Boron W.F. Nature. 1976; 264: 73-74Crossref PubMed Scopus (148) Google Scholar, 7Thomas R.C. J. Physiol. (Lond.). 1977; 273: 317-338Crossref Scopus (232) Google Scholar). Here we report the cloning and characterization of a cation-coupled Cl−- HCO3− exchanger and a physiologically unique BTS member from Drosophila. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl−, Na+, H+, and HCO3− but does not require HCO3−. Transport is blocked by the stilbene DIDS and may not be strictly electroneutral. Our expression data suggest this Na+driven anionexchanger (NDAE1) (GenBankTM accession numberAF047468) is responsible for the Na+-dependent Cl−- HCO3− exchange activity characterized in neurons (6Russell J.M. Boron W.F. Nature. 1976; 264: 73-74Crossref PubMed Scopus (148) Google Scholar, 7Thomas R.C. J. Physiol. (Lond.). 1977; 273: 317-338Crossref Scopus (232) Google Scholar, 8Schwiening C.J. Boron W.F. J. Physiol. (Lond.). 1994; 475: 59-67Crossref Scopus (160) Google Scholar), kidney (9Guggino W.B. London R. Boulpaep E.L. Giebisch G. J. Membr. Biol. 1983; 71: 227-240Crossref PubMed Scopus (91) Google Scholar, 10Ganz M.B. Boyarsky G. Sterzel R.B. Boron W.F. Nature. 1989; 337: 648-651Crossref PubMed Scopus (220) Google Scholar), and fibroblasts (11Kaplan D.L. Boron W.F. J. Biol. Chem. 1994; 269: 4116-4124Abstract Full Text PDF PubMed Google Scholar). We identified a Drosophilaexpressed sequence tag (AA567741, deposited by the BerkeleyDrosophila Genome Project) with similarity to both the AEs and NBCs. We obtained this Drosophila clone (Research Genetics, St. Louis, MO) and sequenced it (W. M. Keck Biotechnology Resource Laboratory, New Haven, CT). This 3225-base pair clone has an initial Met followed by a 3090-base pair open reading frame and a 3′-UTR (104 bp). We directionally subcloned the cDNA into a Xenopus expression plasmid as previously (2Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar). Linearized cDNA was used to make capped cRNA with the SP6 mMessage mMachine kit (Ambion, Austin, TX) as described previously (2Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar) for bothXenopus oocyte studies and in situ hybridization. The full cDNA sequence of Drosophila NDAE1 is GenBankTM accession number AF047468. We isolated poly(A)+ RNA from Drosophila developmental stages and body segments as described previously (1Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (382) Google Scholar). We used 2 μg of poly(A)+ RNA from these stages for denaturing electrophoresis and electroblotting. The NDAE1-cDNA was random primed and 32P-labeled. Hybridization overnight at 60 °C in ExpressHyb (CLONTECH) followed by low stringency washing (42 °C with 2× SSC) did not result in discrete hybridization. Reverse transcription was performed using SuperScript RT kit according to the manufacturer's directions (Life Technologies, Inc.) with Drosophila poly(A)+ RNA. UsingDrosophila NDAE1-specific primers, ExTaqpolymerase (Panvera, Madison, WI), and dNTPs, we performed PCR with 30 cycles of 94 °C (30 s), 55 °C (45 s), and 72 °C (45 s). Products were verified with a 0.65% agarose/Tris borate EDTA gel. The gel was Southern blotted onto Zeta-probe (Bio-Rad) and detected using random-primed, digoxigenin-labeled NDAE1 cDNA according to the manufacturer's instructions (Roche Molecular Biochemicals). Detection of digoxigenin-labeled DNA probe was performed using DIG Luminescent Detection (Roche Molecular Biochemicals), recorded on x-ray film, and digitized using Adobe PhotoShop (Fig.2 a). Multiple sequence alignments were performed using the Clustal method and the PAM250 residue weight table (DNA Star program, Lasergene, Madison, WI) with the percentage divergence and similarity calculated as previously reported (1Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (382) Google Scholar) and the alignment shaded and annotated using GeneDoc©. To determine NDAE1 mRNA cellular distribution in Drosophila, we made whole mounts of 0–24 hDrosophila embryos. To make antisense cRNA, the completeDrosophila NDAE1 was directionally cloned into pSport 2 (Life Technologies, Inc.) at EcoRI and SalI. Digoxigenin-labeled antisense NDAE1-cRNA was synthesized using the SP6 promoter and mMessage mMachine (Ambion) as described above and reduced to a mean size of ∼200 bp by alkaline hydrolysis (16Cox K.H. DeLeon D.V. Angerer L.M. Angerer R.C. Dev. Biol. 1984; 101: 485-502Crossref PubMed Scopus (1272) Google Scholar). Embryos were permeabilized using proteinase K treatment. Digoxigenin label was visualized using an anti-digoxigenin antibody coupled to alkaline phosphatase (17Tautz D. Pfeifle C. Chromosoma. 1989; 98: 81-85Crossref PubMed Scopus (2090) Google Scholar). Embryo staining was documented on slide film and subsequently digitized. Hybridization was determined specific if (i) NDAE1 staining was evident in discrete cells making DNA hybridization unlikely and (ii) staining with a sense RNA probe was negative. Chromosomes were prepared and hybridized by standard methods (18Pardue M.L. Methods Cell Biol. 1994; 44: 333-351Crossref PubMed Scopus (18) Google Scholar). Biotin-labeled probes were generated by random hexamer-priming with Biotin-HighPrime®(Roche Molecular Biochemicals) and the entire NDAE1-cDNA (i.e. the EcoRI/HindIII fragment of the pSport 2 construct), according to the manufacturer's instructions. Horseradish peroxidase-labeled anti-biotin antibodies were used for detection. The CO2/HCO3−-free ND96 contained 96 mm NaCl, 2 mm KCl, 1 mmMgCl2, 1.8 mm CaCl2, and 5 mm HEPES (pH 7.5 and 195–200 mosm). In CO2/HCO3−-equilibrated solutions, 10 mm NaHCO3 replaced 10 mm NaCl and was maintained by continuous bubbling with 1.5% CO2/98.5% O2. In O-Na+ solutions, choline replaced Na+. In O-Cl− solutions, gluconate replaced Cl−. Non- HCO3− solutions were bubbled with 100% O2 to remove trace CO2 and HCO3−. 50 nl of water (control) or RNA solution (35 ng of NDAE-cRNA) was injected into stage V/VIXenopus oocytes. Voltage electrodes, made from fiber-capillary borosilicate and filled with 3M KCl, had resistances of 1–10 MΩ (1Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (382) Google Scholar). Ion-selective electrodes (pH, Cl−, and Na+) were pulled similarly and silanized withbis-(dimethylamino)-dimethylsilane (Fluka Chemical Corp., Ronkonkoma, NY). pH electrodes tips were filled with hydrogen ionophore 1 mixture B (Fluka) and backfilled with phosphate buffer (pH 7.0). Cl− electrode tips were filled with a Cl− ionophore (Corning, Corning, NY) and backfilled with 0.5 m NaCl; Na+ electrode tips were filled with sodium ionophore 1 mixture B (Fluka) and backfilled with 0.15m NaCl. Electrodes were connected to a high impedance electrometer (WPI-FD223 for intracellular pH (pHi), intracellular Cl− activity (aCli), or intracellular Na+ activity (aNai) and Vm experiments), and digitized output data were acquired by computer. All ion-selective microelectrodes had slopes of −54 to −57 mV/decade ion concentration (or activity). pH electrodes were calibrated at pH 6.0 and 8.0; Cl− and Na+electrodes were calibrated with unbuffered 10 and 100 mmNaCl (ionic strength was not identical). Selectivity of Cl− was checked using unbuffered 100 mmNaHCO3 and for Na+ using 100 mmKCl. Na+ electrodes were greater than 50-fold selective for Na+ (19Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar) and Cl− electrodes were at least 10-fold selective versus HCO3−. For voltage-clamp experiments (Warner Inst. Co., Oocyte Clamp), electrodes were filled with 3 m KCl/agar and 3 m KCl and had resistances of 0.2–0.5 MΩ. Oocytes were clamped to −60 mV and stepped from −160 to +60 mV in 20 mV steps; the resulting data were filtered at 5 kHz (8 pole Bessel filter, Frequency Devices) and sampled at 1 kHz as previously reported (19Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar). Data were acquired and analyzed using Pulse and PulseFit (HEKA Instruments, Germany). Values quantitated are indicated as the mean ± S.E. Ion activities between control and NDAE1 oocytes were shown by a two-tailed t test to have a significance of p < 0.016 or less. DNA sequencing of our clone revealed a single, long open reading frame flanked by 5′- and 3′-UTRs (UTRs, 426 and 104 bp, respectively). This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to both the AEs and NBCs. The predicted protein is 43% similar to the cloned NBCs and 32% similar to the AEs (Fig.1, a and b). Although the NDAE1 hydropathy plot (Fig. 1 b) is similar to those of the BTS members, it is most similar to the AEs. A dendrogram of the published BTS sequences (Fig. 1 c) implies that NDAE1 forms a new branch of this superfamily. Our NDAE1 topology model (Fig.1 d) predicts (i) intracellular NH2 and COOH termini, (ii) 12 transmembrane spans (TMs), (iii) a central exofacial loop with putative N-glycosylation sites, and (iv) multiple putative phosphorylation sites. Recently the complete sequence of the Drosophila genome was reported (20Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4810) Google Scholar). Although a predicted gene product “CG4675” in two forms (AAF52496, alt 1 and 2 for proteins and AE003616 for the assembled genomic contig) encoding the ndae1 gene was identified, the sequence analysis is not completely accurate. The predicted protein sequences are missing thirteen NH2-terminal amino acids (MAEKNEYIELPWT) partly encoded from an additional 5′-intron. CG4675-alt 2 contains a 69-amino acid insertion (amino acids 32–100 of the NH2-truncated protein), which we have not found present in DrosophilamRNA. A second Drosophila gene and protein have homology to the BTS family (CG8177). Using a pileup analysis, gene product CG8177 (GenBankTM accession number AAF50207) is about 32% identical to NDAE1 and the NBCs, but about 34–40% identical to the AEs. Though CG8177 apparently encodes a HCO3− transporter protein, future transport experiments will be needed to determine the actual function. Next, we determined the location of NDAE1 mRNA in Drosophila. Using Northern blot analysis of poly(A)+ RNA, we were unable to detect NDAE1 mRNA in embryos, isolated adult heads, or body parts. However, by RT-PCR we could detect NDAE1 mRNA in heads as well as several embryonic stages (Fig.2 a). In situhybridization to NDAE1 mRNA in whole mount Drosophilaembryos (Fig. 2, b and c) illustrates that NDAE1 is present during embryogenesis. CNS staining is apparent throughout embryogenesis (Fig. 2, b and c). Staining of the gut primordium and mesoderm is evident in stage 6/7 (Fig.2 b). Staining of a specific subset of cells in the CNS is detectable by late embryogenesis (Fig. 2 c) as is staining of the anal plate (not shown), i.e. the larval absorptive apparatus. The NDAE1-sense strand controls did not stain (Fig. 2,d and e). The difference between RT-PCR andin situ hybridization verses Northern detection of NDAE1 mRNA likely reflects sensitivity by amplification or individual cell mRNA abundance of the two former techniques. To evaluate the physiologic function of NDAE1, we expressed it inXenopus oocytes. Fig. 3 is a model illustrating ion transport attributed to Na+-dependent Cl-HCO3 exchange activity. We tested this model with oocytes expressing NDAE1. Fig.4 a shows that removal and replacement of bath Na+, Cl−, or both, with and without HCO3− does not alter pHiof a water-injected control cell. However, expression of NDAE1 elevates resting pHi by ∼0.3 pH units (Fig. 4 b),i.e. control = 7.27 ± 0.03 (n = 9) and NDAE1 = 7.54 ± 0.03 (n = 18). The acidification elicited by CO2/HCO3−(Fig. 4 a) is markedly reduced in NDAE1 oocytes (Fig.4 b) and greatly increases intracellular [HCO3−] 3[HCO3−] is calculated using the pHi obtained just before CO2, steady-state pHi in the presence of CO2/HCO3−, and the Henderson-Hasselbalch equation (19Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar, 21Roos A. Boron W.F. Physiol. Rev. 1981; 61: 296-434Crossref PubMed Scopus (2288) Google Scholar).(control = 3.1 ± 0.2 mm, n = 9; NDAE1 = 7.4 ± 0.4 mm, n = 16). The higher resting pHi and elevated [HCO3−] are consistent with NDAE1's role as an acid extruder, “forward” transport in Fig. 3 a. Bath Na+ removal elicits a robust pHi decrease illustrating that NDAE1 is readily reversible (Fig. 3 b). Subsequent removal of Cl− stops and slightly reverses the acidification, whereas readdition of Na+ in the sustained absence of Cl− triggers a rapid pHi recovery (Fig. 4 b). A similar response is completely blocked by 200 μm DIDS (Fig. 4 g). Our results indicate that NDAE1 is indeed functionally unique in the BTS. These pHichanges are consistent with Na+ and HCO3− cotransport in exchange for Cl− and H+ as observed in snail neurons (7Thomas R.C. J. Physiol. (Lond.). 1977; 273: 317-338Crossref Scopus (232) Google Scholar) and squid axons (6Russell J.M. Boron W.F. Nature. 1976; 264: 73-74Crossref PubMed Scopus (148) Google Scholar).Figure 4Physiology of NDAE1 expressed inXenopus oocytes. Oocytes were injected with 50 nl of water or cRNA in water. a, c, and e are water-injected control oocytes. b, d,f–i are injected with 35 ng/oocyte of NDAE1 cRNA. All solutions are pH 7.5, and all HCO3−solutions are 1.5% CO2/10 mm HCO3−. Each panel shows the response of an oocyte to CO2/HCO3− addition, removal of Na+, removal of Na+ and Cl−, and removal of Cl−. a, pHi of water injected (control) oocyte. Both Na+ and Cl−are removed ± CO2/HCO3−.b, pHi of a NDAE1-injected oocyte. Similar experiment to a with a NDAE1-expressing oocyte. Starting pHi values for NDAE1-oocytes are ∼0.3 pH units higher than controls as expected for a HCO3− influx transporter, i.e. an acid extruder. c,aCli of a water-injected oocyte. Note thataCli is minimally altered by bath solution manipulations. d, aCli of a NDAE1-injected oocyte. Non-CO2/HCO3−solutions are bubbled with 100% O2, illustrating that NDAE1 does not require HCO3− to function. Starting aClis are ∼10 mm less than control oocyte indicating basal Cl− extrusion from the NDAE1-oocytes. e, aNai of a water-injected oocyte. The aNai is unaltered by any of the bath solution manipulations. f,aNai of a NDAE1-injected oocyte. The steady-state aNai is elevated in comparison to the control oocyte. g–i illustrate DIDS inhibition of ion transport via NDAE1. g, DIDS inhibition of NDAE1-mediated pHi changes. The oocyte was exposed twice to CO2/HCO3−, first without DIDS (not shown) and second with 200 μm DIDS. Exposure to DIDS appears to completely block NDAE1 activity, resulting in a response similar to control oocytes. h, DIDS inhibition of NDAE1-mediated aCli changes, second pulse shown.i, using a double CO2/HCO3− protocol as ing, DIDS also blocks the aNai changes. The hatched bar at the bottom right corner represents 10 min for that experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further tested our transport model (Fig. 3) by measuringaCli. Fig. 4 c shows that a control oocyte has ∼31 mm aCli (37.0 ± 1.6 mm, n = 9), which only slightly changes with ion replacement ± CO2/HCO3−. Fig. 4 dillustrates that an oocyte expressing the NDAE1 transporter has ∼22 mm aCli (29.5 ± 2.1 mm, n = 6). NDAE1 oocytes show both rapid and robust responses to ion replacement and addition of CO2/HCO3−, i.e. changes of 3–8 mm activity (Fig. 4 d). CO2/HCO3− supplied to the bath decreases aCli, and Na+ removal reverses this response. With both Na+ and Cl−removed aCli change stops, but readdition of Na+ elicits a large and rapid fall inaCli. The removal of bath CO2/HCO3− bringsaCli back to resting levels (Fig.4 d). These alterations of aCli are also blocked by 200 μm DIDS (Fig. 4 h). Moreover, the beginning of Fig. 4 d illustrates that HCO3− is not required for ionic movements through the transporter (solutions bubbled with 100% O2). This physiologic characteristic is reminiscent of the multiple transported anions (e.g. OH−, Br−, I−), and HCO3− stimulated activity of the AEs. To discriminate between Na+ dependence (“binding”)versus Na+ driven (transport), we measured the effect of NDAE1 function on aNai of oocytes. Fig. 4, e and f shows representative traces from control and NDAE1 oocyte experiments, respectively, using similar solution protocols as in Fig. 4, a–d. A control oocyte (Fig. 4 e) has ∼2.6 mm aNai (3.1 ± 0.5 mm,n = 10), which does not change with bath ion substitutions. Fig. 4 f shows that aNai is increased to ∼5 mm in NDAE1-expressing oocytes (4.6 ± 0.3 mm, n = 10). Na+ is transported by NDAE1 as evidenced by (i) increased aNai with the addition of CO2/HCO3−, (ii) reduced aNai with Na+ removal, and (iii) increased aNai with Cl−removal. Na+ transport via NDAE1 is blocked by 200 μm DIDS (Fig. 4 i). Changes ofaNai are always in the opposite direction asaCli changes indicating a Na+ for Cl− exchange. As shown for both the pHi andaCli responses, Na+ transport was also observed in the complete absence of HCO3−(not shown). Thus, our data indicate that this DrosophilaNa+-dependent Cl-HCO3 exchanger is more appropriately named a Na+-driven anion exchanger or NDAE1. We noted that Cl− removal or the addition of HCO3− resulted in significant depolarizations only in NDAE1 oocytes (Fig. 4 b). Therefore, we voltage-clamped and used anion transport inhibitors (DIDS, diphenylamine carboxylic acid, and niflumic acid) to evaluate the electrical nature of NDAE1 (Fig. 5). In a voltage-clamped oocyte, this depolarization is measured as an inward (negative) current. A comparison of water-injected control (Fig.5 a) and NDAE1 oocytes (Fig. 5 b) illustrates that both Cl− removal and HCO3− addition elicit current specific to NDAE1 expression. The reversal potential of both control (Fig. 5 c) and NDAE1 oocytes (Fig.5 d) is about −20 mV. In the absence of Cl−, there is also a HCO3−-stimulated current only in NDAE1 oocytes (Fig. 5 b). This current has a linear voltage dependence (Fig. 5 d). DIDS, diphenylamine carboxylic acid, and niflumic acid block the depolarization (unclamped cell) because of Cl− removal (Fig. 5 e). However, the measured currents in NDAE1 oocytes are small compared with the pHi,aCli, and aNai changes. The voltage deflections and associated currents are either endogenous to the oocyte uncovered by NDAE1 activity or more likely a “leak” current through the NDAE1 transporter. Present data imply that the NDAE1 current represents a leak current rather than NDAE1 being “electrogenic”: (i) the J(ion)/J(current) ratio 4The J(ion) was calculated from the initial rate of ionic change elicited by Cl− removal and the volume to surface area ratio of the oocyte ([diameter/2]/3). Similarly, J(current) was calculated from the current in 0 Cl− at −20 mV, i.e. the voltage obtained in unclamped oocytes with Cl− removal and SA/V. The resulting values was divided by the Faraday constant to yield a true flux, J(current).for Cl−, HCO3−, and Na+is > 1000; and (ii) the pHi changes are two to three times greater for NDAE1 than rkNBC, whereas the transport currents are at least 10-fold smaller (30 nA versus 300–500 nA, respectively) (19Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar). These transporter currents (or voltage changes) would not have been detectable in snail neurons (7Thomas R.C. J. Physiol. (Lond.). 1977; 273: 317-338Crossref Scopus (232) Google Scholar) or squid axons (6Russell J.M. Boron W.F. Nature. 1976; 264: 73-74Crossref PubMed Scopus (148) Google Scholar). To begin investigating the role of NDAE1 in vivo, we searched the Berkeley Drosophila genome Project (BDGP) data base with the NDAE1 sequence and identified a P element insertion mutation in the vicinity of ndae1. This insertion lies 408 bases 5′ of the predicted NDAE1 initiation codon (Fig.6 a). By RT-PCR we determined that the insertion site of this P element mutation lies within the NDAE1 5′-untranslated sequence, using wild type poly(A)+RNA and primers that flank the site of insertion (Fig. 6 b). These data suggest that this P element disrupts ndae1. Because this P element insertion was isolated in a screen for lethal P element-induced mutations (12Torok T. Tick G. Alvarado M. Kiss I. Genetics. 1993; 135: 71-80Crossref PubMed Google Scholar, 22Roch F. Serras F. Cifuentes F.J. Corominas M. Alsina B. Amoros M. Lopez-Varea A. Hernandez R. Guerra D. Cavicchi S. Baguna J. Garcia-Bellido A. Mol. Gen. Genet. 1998; 257: 103-112PubMed Google Scholar), our data further suggest thatndae1 may be essential for viability. Detailed phenotypic and physiological analysis of this mutant will be presented elsewhere. Expression of NDAE1 in Xenopus oocytes shows all the physiologic properties of the Na+ dependent Cl-HCO3 exchanger: Cl− transport, Na+ transport, Na+/HCO3−cotransport (or Na+-H+ exchange), and sensitivity to DIDS. NDAE1 does not require HCO3−and appears to be a more general anion exchanger. Our data indicate that NDAE1 exchanges Na+ and HCO3−(or an anion) for Cl− and H+ (Fig. 3). Thus based on our functional studies, it is likely that NDAE1 is theDrosophila form of the Na+-dependent Cl−-HCO3− exchanger functionally identified in neurons, fibroblasts, mesangial cells, and renal tubule cells. Physiologically, the activity of the Na+-dependent Cl−- HCO3− exchanger appears to be regulated. In mesangial cells, agents such as angiotensin II, serotonin, and vasopressin, which act locally as growth factors (23Ganz M.B. Boron W.F. Am. J. Physiol. 1994; 266: F576-F585Crossref PubMed Google Scholar), as well as epidermal growth factor and platelet-derived growth factor, stimulate ion transport activity including Na+-dependent Cl−-HCO3− exchange (10Ganz M.B. Boyarsky G. Sterzel R.B. Boron W.F. Nature. 1989; 337: 648-651Crossref PubMed Scopus (220) Google Scholar). Recently, Na+-dependent Cl−-HCO3− exchange activity was shown to increase during normal renal development (24Ganz M.B. Saksa B.A. Am. J. Physiol. 1998; 274: F550-F555PubMed Google Scholar). And, in NIH-3T3 fibroblasts, Kaplan and Boron (11Kaplan D.L. Boron W.F. J. Biol. Chem. 1994; 269: 4116-4124Abstract Full Text PDF PubMed Google Scholar) found that transformation with c-Ha-ras not only increased the activity of the Na+-dependent Cl−-HCO3− exchanger but also shifted activation to more alkaline pH values, effectively removing pHi as the transporter control mechanism. Moreover, some studies postulate that mis- or deregulation of stilbene-sensitive HCO3− transport (25Lee A.H. Tannock I.F. Cancer Res. 1998; 58: 1901-1908PubMed Google Scholar) or Na+-H+ exchange (26Rotin D. Steele-Norwood D. Grinstein S. Tannock I. Cancer Res. 1989; 49: 205-211PubMed Google Scholar) is involved in neoplasia. We postulate that future NDAE1 studies will elucidate the mechanisms for these regulatory observations. As the first cloned Na+ and Cl−-coupled HCO3− transporter, NDAE1 may assist the molecular identification of still other cation- and anion-coupled HCO3− transporters. The identification of NDAE1 in Drosophila presents the opportunity to use genetic analysis and manipulation to further understand NDAE1 function and importance in vivo. Because disruption of the NDAE1 gene and loss of the protein is apparently lethal (12Torok T. Tick G. Alvarado M. Kiss I. Genetics. 1993; 135: 71-80Crossref PubMed Google Scholar), NDAE1 is likely an important developmental protein. Cloning of mammalian NDAE1 homologs will provide novel insights to normal and pathologic roles they play in the CNS, circulatory system, digestive tract, respiratory tract (27Cassel D. Scharf O. Rotman M. Cragoe Jr., E.J. Katz M. J. Biol. Chem. 1988; 263: 6122-6127Abstract Full Text PDF PubMed Google Scholar), and urinary system. In the visual system, determining the localization of the NDAE1 may increase our understanding of fluid and ion transport by the ciliary body (28Butler G.A. Chen M. Stegman Z. Wolosin J.M. Exp. Eye Res. 1994; 59: 343-349Crossref PubMed Scopus (19) Google Scholar) and the lens (29Duncan G. Dart C. Croghan P.C. Gandolfi S.A. Exp. Eye Res. 1992; 54: 941-946Crossref PubMed Scopus (5) Google Scholar) (e.g. glaucoma) or neuronal transmission in the retina. In the kidney, understanding NDAE1 function may provide insights into disorders of excessive Na+ retention (e.g. hypertension) or impaired HCO3− reclamation (e.g. renal tubular acidosis). In the CNS elucidating NDAE1 function may lead to an understanding of how the Na+ driven Cl−-HCO3− exchanger modulates neuronal activity by altering pHi, pHo, andaCli (e.g. seizure disorders). NDAE1 is also likely an important acid-base homeostatic regulator for insects, particularly for the gut. Based on our in situhybridization, NDAE1 mRNA is present in the developing gut. The NDAE1 mRNA and the protein are likely to persist in the adult organism. The midguts of mosquito larvae (30Dadd R.H. J. Insect Physiol. 1975; 21: 1847-1853Crossref PubMed Scopus (129) Google Scholar) and lepidopteran larvae (31Dow J.A. J. Exp. Biol. 1992; 172: 355-375PubMed Google Scholar) are known to be extremely alkaline, i.e. pH 8–12. At least in mosquito larvae, this alkalinity is in part mediated by removal of H+ by a V-type ATPase (32Zhuang Z. Linser P.J. Harvey W.R. J. Exp. Biol. 1999; 202: 2449-2460PubMed Google Scholar) presumably working in concert with a yet unknown base secretory mechanism. Depending on the cellular location, an insect anion exchanger, e.g. NDAE1, could be involved in secretion of either acid or alkali, as in the α- and β-cells of the mammalian cortical collecting duct. In insect species with alkaline proximal guts, the distal gut is responsible for returning the food stream to a neutral or acidic pH (33Moffett D.F. Physiol. Zool. 1994; 67: 68-81Crossref Google Scholar). Thus, it is possible that NDAE1 could have a more widespread role in insect acid-base balance. From the current data, we hypothesize that NDAE1 may aid in maintaining the high gut pH of Drosophila and potentially mosquitoes. The gut and salivary gland pH of mosquitoes, in particular, is a factor contributing to transmission of disease such as encephalitis and malaria. That is, acidic environments (pH < 7) appear to promote cellular infection (34Hacker J.K. Hardy J.L. Virology. 1997; 235: 40-47Crossref PubMed Scopus (41) Google Scholar). The high pH of the gut likely protects the vector organism. Infectivity of Plasmodium berghei, present in mice, to mosquitoes is reduced with low pH and low blood HCO3− of the mice (35Butcher G.A. Sinden R.E. Billker O. Exp. Parasitol. 1996; 84: 371-379Crossref PubMed Scopus (16) Google Scholar). A corollary hypothesis is that the mosquitoes' ability to spread disease could be controlled by altering the transport activity of NDAE1. With the cloning of NDAE1 these questions may now be addressed at the genetic, molecular, and physiologic levels. We thank Marilyn McHugh and Dr. Ester P. Jane for technical assistance. We thank Dr. Marcello Jacobs-Lorena for helpful Drosophila discussions and Dr. Lamara D. Shrode for helpful discussions and manuscript comments. We also thank Dr. David F. Moffett for insights into insect gut pH regulation and relevance." @default.
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