Departments of 1 Physiology & Biophysics, 2 Genetics, and 3 Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970
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ABSTRACT |
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Na+-dependent Cl/HCO
/HCO
CWR57) recognizes NDAE1 electrophysiologically
characterized in Xenopus oocytes. Moreover, our results
begin to delineate the NDAE1 topology, i.e., both the NH2
and COOH termini are intracellular. NDAE1 is expressed throughout
Drosophila development in the central and peripheral nervous
systems, sensilla, and the alimentary tract (Malpighian tubules, gut,
and salivary glands). Coimmunolabeling of larval tissues with NDAE1
antibody and a monoclonal antibody to the
Na+-K+-ATPase
-subunit revealed that the
majority of NDAE1 is located at the basolateral membranes of Malpighian
tubule cells. These results suggest that NDAE1 may be a key
pHi regulatory protein and may contribute to basolateral
ion transport in epithelia and nervous system of Drosophila.
electrophysiology; fusion proteins; immunohistochemistry; development
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INTRODUCTION |
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SODIUM-DEPENDENT
Cl/HCO
/HCO
/HCO
We recently reported the cloning and characterization of a
Na+-driven anion exchanger, NDAE1, from Drosophila
melanogaster (47). NDAE1 is the first recombinant
protein functionally capable of Na+-dependent
Cl/HCO
-dependent HCO
Drosophila has been widely used as a model for studies of development, molecular, and population genetics. However, much less is known about Drosophila physiology. Insect Malpighian tubules (MTs) have been used as a model system of fluid secretion by epithelia (6, 12, 33, 34). The MT is a brush-border epithelium made up of principal cells and secondary stellate cells (57), which together regulate the osmolarity of hemolymph (36, 37, 64). The physiology of insect MT transport has been studied in a variety of species, and a general transport model has been proposed to which Drosophila (D. melanogaster and D. hydei) generally adhere (12, 14, 30). The MT and the gut of insects have many functional similarities to the mammalian kidney.
The rates of ionic and fluid transport across MTs are among the
highest known (33) and are driven primarily across
principal cells by an apical bafilomycin A1-sensitive,
V-type H+-ATPase (21, 24). Proton secretion,
in concert with K+/H+ exchange, drives apical
K+ secretion (24), whereas Cl
secretion seems to occur mainly via Cl
channels in
stellate cells (39). Less is known about the movement of
ions across the basolateral membrane of principal cells. The ouabain-sensitive Na+-K+-ATPase has been both
immunolocalized and functionally characterized at the basolateral
membrane of MTs (21, 30). Inhibitor studies using
bumetanide suggest that a
Na+-K+-2Cl
cotransporter may
mediate basolateral ion flux in some species (20, 38),
although not in D. melanogaster (14). A model proposed by Linton and O'Donnell (30) couples outward
movement of K+ via potassium channels with inwardly
directed (dihydroindenyl)oxy alkanoic acid-inhibitable KCl transport
and an unknown Na+/solute transporter (30). In
concert with the Na+-K+-ATPase, these
transporters drive basolateral flux of K+ and
Na+.
Control of systemic and intracellular pH is important for
nutrient absorption and insect viability (19) as well as
being a potential signaling mechanism for gametogenesis and
infiltration of malaria parasites into insect vector species (i.e.,
mosquitoes) (56). However, study of pHi
regulation in invertebrates has been mainly with squid giant axon
(49), snail neurons (59), and barnacle muscle
fiber (8), all of which contain Na+-dependent
Cl/HCO
To initiate the understanding of the role of NDAE1 in D. melanogaster physiology, we developed an NDAE1 antibody to
characterize the structural properties and subcellular localization of
the endogenous protein. Using this antibody and a monoclonal antibody to the Na+-K+-ATPase 5-subunit
(27), we have characterized the tissue and membrane
distribution of NDAE1 protein in developing D. melanogaster. Structurally, we have demonstrated that both the NH2 and
COOH termini of NDAE1 are located intracellularly as predicted by
hydropathy analysis.
Portions of this work have been presented in preliminary form (52).
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METHODS |
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Polyclonal Antibody Production and Affinity Purification
Recombinant glutathione S-transferase (GST)-NDAE1 fusion protein was used as an immunogen for the production of rabbit polyclonal antibodies by following standard production protocols (Cocalico Biologicals, Reamstown, PA). PCR primers designed to add 5' EcoRI (5'-GGGGAATTCATGG CCGAAAAGAATGAG-3') and 3' XhoI (5'-CCCCTCGAGCTCCACATCCTCCTCGAA-3') restriction enzyme sites onto the DNA sequence encoding to the first 100 amino acids of NDAE1 were used to amplify a 300-bp NH-terminal fragment (NTERM100) from NDAE1-pTLN cDNA. The amplified fragment was subcloned into the pGEX-4T.1 (GST) expression vector (Amersham Pharmacia, Piscataway, NJ). Sequence was verified by automated sequencing (Cleveland Genomics, Cleveland, OH). DH5To affinity purify antibodies specific to NDAE1 epitopes, GST-NTERM100
was run on a single-lane 10% SDS gel and Western blotted as described
(see Western Blot Analysis). A band of
nitrocellulose membrane containing the fusion protein was cut into
small pieces and incubated in PBS plus 5% BSA for 2 h at room
temperature (RT). To bind the NDAE1 antibody, we incubated the membrane
pieces in 5 ml of serum containing the target antibody overnight at
4°C. Subsequently, the membrane pieces were washed with PBS, and the antibody was eluted with 0.2 M glycine (pH 2.2) for 2 min. The pH was
then quickly titrated to 7.4 with 1 M Tris base. Affinity-purified antibodies were stored in 50% glycerol with 0.1% BSA at 20°C until use.
Tissue Membrane Preparations
Oregon-R strain D. melanogaster membrane preparations were prepared using a modified method of Wilcox (66). Briefly, ~150 mg of tissue were homogenized with a Polytron (Fisher, Pittsburgh, PA) in 5 ml of ice-cold modified balanced salt solution (BSS) containing (in mM) 10 Tris base, 55 NaCl, 40 KCl, 7 MgCl2, 5 CaCl2, 20 glucose, 50 sucrose, 0.2 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 benzamidine, and 1 EDTA as well as (in µg/ml) 5 aprotinin, 10 pepstatin, 10 leupeptin, and 150 phenylmethylsulfonyl fluoride. Homogenates were then centrifuged at 1,000 g for 5 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 1,500 g for 5 min at 4°C. The supernatant was then transferred to an ultracentrifuge tube and spun at 100,000 g for 90 min at 4°C. The membrane-enriched pellet was resuspended in 500 µl of BSS, and the protein concentration was quantified by Bradford colorimetric assay (Bio-Rad, Hercules, CA).Immunoprecipitation of NDAE1
COS-7 cells grown on 60-mm dishes were incubated for 10 min on ice in 1 ml of RIPA buffer containing (in mM) 50 NaCl and 20 Tris, pH 8.0, plus 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, and 10 µl/ml of mammalian protease cocktail (Sigma). Cells were scraped, and the lysate was centrifuged at 13,000 rpm to pellet-insoluble material. The supernatant was then incubated with primary antibody at 4°C overnight. Next, 50 µl of a 50% slurry of protein A-Sepharose beads were added, and the mixture was incubated at 4°C for 2 h. The beads were washed with RIPA buffer, and protein-antibody complexes were released from the beads and denatured by the addition of Laemmli sample buffer (2% SDS, 10% glycerol, 60 mM Tris, pH 6.8, and 0.02% bromphenol blue) and boiling for 5 min.Tagging NDAE1 With Enhanced Green Fluorescent Protein or Hemagglutinin A and Transient Transfection of COS-7 Cells
NDAE1-EGFP. A COOH terminal-tagged enhanced green fluorescent protein (EGFP) version of NDAE1 (NDAE1-EGFP) was created using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and appropriate primers to remove the stop codon of the NDAE1 cDNA and introduce an ApaI restriction site 3' of the original stop codon. The mutated NDAE1 cDNA was subsequently removed from the pTLN vector via KpnI and ApaI restriction sites and directionally subcloned into pEGFPN-1 (Clontech, Palo Alto, CA). The sequence was verified by automated DNA sequencing (Cleveland Genomics).
NDAE1-HA. To create a version of NDAE1 with a COOH-terminal hemagglutinin A (HA) tag (NDAE1-HA), site-directed mutagenesis primers were designed to remove the stop codon of NDAE1 and create a 3' EcoRI restriction site in NDAE1-pTLN. NDAE1 was nondirectionally subcloned via EcoRI restriction sites (second site in the 5' multiple cloning sites of pTLN) into the pMH mammalian expression vector (Boehringer Mannheim, Indianapolis, IN), and the sequence was verified by automated DNA sequencing (Cleveland Genomics).
Transient transfections. COS-7 cells plated on glass coverslips were grown to 50% confluence and transiently transfected using the Lipofectamine Plus kit (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's protocol and were used 48 h posttransfection.
Western Blot Analysis
Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using the indicated percentage of acrylamide and subsequently transferred to a nitrocellulose membrane. Membranes were blocked for 1-2 h with 10% skim milk in Tris-buffered saline containing (in mM) 20 Tris, 137 NaCl, and 0.1% Tween 20 titrated to pH 7.5 (TBST). Blots were then incubated overnight at 4°C in TBST plus 10% skim milk with the appropriate dilution of primary antibody. Next, blots were washed with TBST and incubated for 2 h at RT in TBST plus 10% skim milk with a 1:5,000 dilution of horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). Blots were washed with TBST, and signals were detected by enhanced chemiluminescence (ECL; Amersham Pharmacia).Cell and Tissue Preparation/Immunohistochemistry
COS-7 cell preparation. COS-7 cells attached to glass coverslips were fixed during a 30-min incubation at RT in PBS containing 4% paraformaldehyde (PFA). Cells were then incubated for 10 min in 100 mM glycine in PBS, washed with PBS, and permeabilized by 10 min of incubation in PBST.
Embryo preparation.
Oregon-R strain D. melanogaster embryos were collected, and
the chorion membranes (the outermost protective membrane) were removed
by 5 min of incubation in 2.6% sodium hypochlorite (50% bleach) and
then rinsed with a solution containing 0.9% NaCl and 0.03% Triton
X-100 (41). Embryos were placed into a 25-ml glass scintillation vial and suspended in 10 ml of heptane. Next, 10 ml of
fixing solution was added containing (in mM) 100 PIPES, 2 EGTA, and 1 MgSO4 and 4% PFA titrated to pH 7.0. Embryos were agitated
at RT for 30 min. The aqueous layer was then aspirated, and 100 mM
glycine in PBS was added for 60 min to clear remaining fixative. The
aqueous layer was then removed, and 10 ml of methanol were added. The
embryos were vigorously shaken for 1 min, and the vitelline
membranes at the heptane/methanol interface and the heptane were
removed. Embryos were washed with methanol and stored at 20°C until
rehydrated in PBS for use.
Larvae preparation. D. melanogaster third-instar larvae (43) were dissected in PBS using Teflon-coated no. 5 forceps and a fine dissecting probe. Tissues were washed in PBS, fixed for 20 min in 2% PFA in PBST, and treated with 100 mM glycine in PBS for 45 min.
Oocyte preparation.
Oocytes used for electrophysiological studies (see Oocyte
Preparation and Electrophysiology) were immediately
transferred to PBS containing 4% PFA for 1 h at RT. Subsequently,
oocytes were incubated in 100 mM glycine in PBS for 1 h and washed
in PBS. Oocytes were then frozen in OCT compound (EM Sciences, Ft. Washington, PA) using liquid nitrogen and sliced to 10 µm with a
cryotome. Slices were mounted on gelatin-coated microscope slides and
stored at 20°C until use.
Nuclei staining with 4',6-diamidino-2-phenylindole dihydrochloride. In some experiments 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) stain was used to demarcate cell nuclei. After staining was completed with the appropriate primary and secondary antibodies, samples were incubated in PBS with a 1:1,000 dilution of DAPI for 5 min at RT, followed by washes with PBS.
Immunohistochemistry.
Cells and tissues were incubated in PBST containing 1% BSA and 10%
donkey and/or 10% goat serum for 30-60 min at RT. Primary antibodies at the indicated dilution were then added for 2.5 h. Samples were then washed extensively in PBST and treated for 2 h
with the indicated secondary antibodies (Jackson ImmunoResearch) in
PBST containing 1% BSA, 10% donkey, and/or 10% goat serum, followed
by washes with PBST. Embryos were cleared by incubation in PBS plus
50% glycerol for 30 min. Samples were mounted on microscope slides
using DAKO fluorescence mounting medium (DAKO, Carpinteria, CA). Images
were acquired with an AxioCam digital camera and AxioVision software
(Carl Zeiss, Oberkochen, Germany). Confocal images were acquired using
a Zeiss LSM 410 confocal microscope. Samples treated with secondary
antibody alone or with affinity-purified CWR57 (
CWR57ap)
incubated for 1 h at RT with GST-NTERM fusion peptide were used as
negative controls.
Oocyte Preparation and Electrophysiology
Xenopus laevis oocytes were prepared for cRNA injection as previously described (48). cRNA (50 nl of 0.7 µg/µl) or water was injected into oocytes that were incubated at 18°C in OR3 medium (47). Oocytes were studied 3-11 days postinjection, and experimental procedures were repeated in two batches of oocytes from different frogs to take into account possible biological variation. Ion-selective microelectrode fabrication and experimentation were performed as previously described (54). ![]() |
RESULTS |
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Characterization of NH2-Terminal NDAE1 Antibody and Fusion Protein
We constructed an NH2-terminal NDAE1-GST fusion protein (GST-NTERM100) and used this recombinant protein for competition assays to determine the epitope specificity of the NH2-terminal NDAE1 antibody. Figure 1 shows a Western blot of the GST-NTERM100 peptide probed with the affinity-purified NH2-terminal antibody
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Detection of Full-Length NDAE1
We next tested the ability of
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Detection, Localization, and Physiology of Recombinant NDAE1 Expressed in Xenopus Oocytes
Initial characterization of NDAE1 as expressed in Xenopus oocytes revealed that it functions as a Na+-driven anion exchanger, with the capacity to mediate Na+-driven Cl
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We cryosectioned the oocytes tested in Fig. 3A and used
indirect immunofluorescent staining to determine the location of NDAE1 protein (n = 3). As shown in Fig. 3B,
staining with CWR57ap was observed in the NDAE1 oocyte sections as a
thin band that coincides with the margin of the plasma membrane. The
intracellular staining in Fig. 3B represents NDAE1 protein
not processed to the plasma membrane. No signal was observed in NDAE1
oocyte sections stained with secondary antibodies alone or in
water-injected control oocyte sections probed with
CWR57ap.
Together with previous physiological data showing that the
membrane-impermeable stilbene DIDS inhibits NDAE1 function
(47), these histological data indicate that NDAE1 can
traffic to the plasma membrane.
Plasma Membrane Sidedness of the NH2 and COOH Termini of NDAE1
To determine the location of the COOH terminus of NDAE1 with respect to the plasma membrane, we constructed an EGFP-tagged version of NDAE1 (NDAE1-EGFP). Figure 4A shows that when immunoprecipitated with
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To localize the NH2 terminus of NDAE1 relative to the
plasma membrane, COS-7 cells cotransfected with NDAE1 and EGFP were fixed either with or without Triton X-100 permeabilization and subjected to indirect immunofluorescent staining with CWR57ap primary antibody. As shown in Fig. 5,
although a punctate EGFP signal is apparent in both permeabilized
(top) and nonpermeabilized (bottom) cells, only
permeabilized cells show
CWR57ap staining. These data indicate that
the NH2 terminus of NDAE1 is intracellular, as predicted by
hydropathy analysis and structural analogy to AE1.
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The location of the COOH terminus of NDAE1 was determined using a
COOH-terminal, HA-tagged NDAE1 construct (NDAE1-HA). As shown in Fig.
6A, a band with an apparent
molecular mass of 105 kDa from COS-7 cells transfected with NDAE1-HA
(NDAE1 = 105 kDa, HA = 0.8 kDa) is recognized on a Western
blot by anti-HA monoclonal antibody (HA). Immunoprecipitation using
HA antibody followed by Western blot probed with
CWR57ap revealed
that this band was NDAE1-HA. COS-7 cells transiently cotransfected with
NDAE1-HA and EGFP (for visualization and control purposes) were fixed
either with or without Triton X-100 permeabilization and probed with
HA. As shown in Fig. 6B, only permeabilized cells had a
positive signal with
HA antibody. These results indicate that the
NDAE1 COOH terminus is also located on the cytosolic face of the plasma membrane.
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Localization of Endogenous NDAE1 in Drosophila melanogaster
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NDAE1 protein was next localized in various tissues of
third-instar larvae. Preliminary experiments with larvae and
adults revealed a high level of NDAE1 protein expression in four major tissues: MTs, gut, salivary gland, and brain (not shown). We dissected these tissues to characterize the tissue distribution of NDAE1. Like
many insects, D. melanogaster has anterior (AMT) and
posterior (PMT) MTs, each with three major segments: ureter/proximal,
medial, and distal. Figure 8A
shows the immunolocalization of NDAE1 and the
Na+-K+-ATPase in the AMT. Both proteins are
abundantly expressed in the ureter and proximal portions of the MT.
However, expression is attenuated in the medial tubule, and neither
protein is expressed in the distal (blind ending) AMT. In contrast,
Fig. 8B shows that both NDAE1 and
Na+-K+-ATPase are expressed along the entire
length of the PMT. Figure 8C shows that CWR57ap signal is
competed away by preincubation of this primary antibody for 1 h at
RT with GST-NTERM100 peptide.
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Confocal microscopy was used to more precisely visualize the expression
patterns of NDAE1 in MTs, gut, and salivary gland. Figure
9A shows a confocal micrograph
of the proximal segments of AMT (left) and PMT
(right). Both NDAE1 and the
Na+-K+-ATPase are more highly expressed in the
PMT. Additionally, NDAE1 expression is more discrete at the outer
perimeter of the AMT principal cells. Because the
Na+-K+-ATPase localizes at the basolateral
membrane of MTs in D. melanogaster, these results indicate
that the vast majority of NDAE1 protein is also located basolaterally,
although NDAE1 may be located apically. Figure 9B shows the
staining pattern in the mid/hindgut at the point of insertion of the
ureter of the AMT. The levels of NDAE1 and
Na+-K+-ATPase staining are similar in the
midgut and the AMTs. Figure 9C shows that NDAE1 is also
expressed in the salivary glands, apparently also basal in location.
The Na+-K+-ATPase protein is much less abundant
in the salivary gland than in MTs or gut. The data in Fig. 9 show that
NDAE1 expression persists throughout gut development and may play an
important role in the function and/or pH regulation of all parts of the
alimentary tract in the mature fly.
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DISCUSSION |
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Na+-dependent Cl/HCO
transport across secretory
epithelia, we developed a polyclonal antibody to the NDAE1
NH2 terminus. We have characterized NDAE1 protein
distribution in embryos and larvae of D. melanogaster.
Antibody Specificity and Subcellular Localization of NDAE1
TheAlthough the predicted molecular mass of NDAE1 is 114 kDa,
CWR57ap recognizes a band with an apparent molecular mass of ~105 kDa from D. melanogaster tissue membrane preparations and a
slightly larger band in COS-7 cells transiently transfected with NDAE1 cDNA. The band from native tissue is much more discrete than that of
the COS-7 transfections, perhaps indicating a difference in posttranslational modification of the protein. The observation of a
28-kDa degradation band in Fig. 1 indicates that the epitope recognized
by
CWR57ap is likely located within the first 20-30 amino acids
of the NH2 terminus of NDAE1. Accordingly, it is unlikely that this ~105-kDa band on Western blots is due to an
NH2-terminal truncation. Our cell transfection studies with
HA- and EGFP-tagged versions of NDAE1 imply that the COOH terminus
remains intact when expressed in COS-7 cells. Initial characterization
of an antibody raised against the COOH terminus of NDAE1 also
recognizes a band on Western blots with an apparent molecular mass of
~105 kDa from D. melanogaster tissue, indicating that the
COOH terminus remains intact in vivo (unpublished observations) and
that the NDAE1 protein runs at a lower molecular mass on an SDS-PAGE
gel than predicted.
The primary sequence of NDAE1 contains one putative protein
kinase A and seven putative protein kinase C regulatory sites on the
NH2- and COOH-terminal tails. cAMP
(14), cGMP (13), and intracellular
Ca2+ as well as nitric oxide have been shown to regulate
fluid secretion in the D. melanogaster MTs. In mammalian
cells, Na+-dependent
Cl/HCO
Developmental Expression of NDAE1 Protein in the Alimentary Tract of D. melanogaster
The alimentary tract of insects is responsible for the absorption of nutrients and maintenance of solute concentration and water balance of the hemolymph. Luminal gut pH in many insect species, including those in the order Diptera, i.e., Drosophila and mosquitoes, is quite alkaline (pH 8-11) (19). This alkaline pH facilitates proper nutrient absorption and may be a key signal for the movement of malarial parasites (Plasmodium) from lumen to cell in mosquitoes (56). An apical V-type H+-ATPase, in part, facilitates this alkaline pH across the gut epithelia (19). Yet, the epithelia maintains pHi homeostasis, indicating that another acid-base transporter at the basolateral membrane facilitates this process.On the basis of determinations of ion content across the basolateral membrane (5, 64, 65), as well as the pH gradient and membrane potential, a basolaterally located NDAE1 likely functions as an acid extruder in MTs, gut, and salivary glands. The current model of basolateral ion transport includes a Na+-K+-ATPase, KCl cotransporter, and an unidentified Na+/solute transport mechanism (30). Basolateral localization of NDAE1 in MTs intimate the role of NDAE1 basolateral solute transport. This hypothesis is further supported by the colocalization of NDAE1 and the Na+-K+- ATPase. Dow and coworkers (14) showed that PMTs are capable of fluid secretion along their entire length, whereas AMT only secrete fluid at their proximal end. Our findings that both NDAE1 and the Na+-K+- ATPase are homogenously expressed along the length of the PMTs but only proximally in AMTs indicate that protein expression of these proteins parallels the secretory function of MT epithelia.
We also observed that NDAE1 is abundant in the cuboidal cells of the
salivary gland, whereas the Na+-K+-ATPase is
much less abundant (Fig. 9). This low level of
Na+-K+-ATPase expression correlates with the
low mRNA signal detected by Northern blot analysis, presented in the
original characterization of the 5 antibody in D. melanogaster (27). NDAE1 is apparently located on the
basolateral membrane (Fig. 9) because fluorescence signal was observed
on the outer edge, but not the interior (not shown), of the salivary
gland cells. Thus the role of NDAE1 may be to mediate basal
HCO
Future studies should explore the in vivo role of NDAE1 in
Drosophila as well as NDAE1 expression in
Diptera. Perhaps NDAE1, functioning as an acid-extruding
Na+-dependent Cl/HCO
NDAE1 Expression in D. melanogaster Nervous System
Work in our laboratory has shown that NDAE1, when expressed in Xenopus oocytes, functions as a Na+-driven ClThis study demonstrates that the Na+-driven
Cl/HCO
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NOTE ADDED IN PROOF |
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Since this manuscript was accepted, other investigators
(Grichtchenko II, Choi I, Zhong X, Bray-Ward P, Russell JM, and Boron WF. Cloning, characterization, and chromosomal mapping of a human electroneutral Na+-driven Cl-HCO3 exchanger.
J Biol Chem 276: 8358-8363, 2001) have reported
another cDNA encoding a mammalian Na+-driven
Cl/HCO
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ACKNOWLEDGEMENTS |
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We thank Montelle C. Sanders for technical assistance and Maryanne Pendergast for assistance in obtaining confocal images.
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FOOTNOTES |
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This work was supported by an American Heart Association grant, a Howard Hughes Medical Institute Grant to Case Western Reserve University (M. F. Romero), and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-56218 (M. F. Romero). C. M. Sciortino was supported by an NIDDK Predoctoral Fellowship (DK-07678). L. D. Shrode was supported by a National Heart, Lung, and Blood Institute Postdoctoral Fellowship (HL-07415).
Address for reprint requests and other correspondence: M. F. Romero, Dept. of Physiology & Biophysics, Case Western Reserve Univ. School of Medicine, 2119 Abington Rd., Cleveland, OH 44106-4970 (E-mail: mfr2{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 January 2001; accepted in final form 5 March 2001.
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