From the Department of Cellular and Molecular
Physiology and the
Department of Genetics, Yale University
School of Medicine, New Haven, Connecticut 06520 and the ** Department
of Biology, Syracuse University, Syracuse, New York 13244
Received for publication, October 10, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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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) from
Drosophila. 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 in
Xenopus 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 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 (1-3), snail neurons (4-6), and barnacle muscle (7). 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 (8) in Fig. 1A. In
physiology experiments on mammalian cells, it is often extremely
difficult to distinguish this transporter from either an electroneutral
Na/HCO3 cotransporter (NBCn1, Fig. 1B) (9, 10)
or an electrogenic Na/HCO3 cotransporter (NBCe1, Fig.
1C) (11, 12) because of problems depleting cells of
Cl. Thus, NDCBE1 encodes a human, electroneutral
Na+-driven Cl-HCO3 exchanger.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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. 1D, which is a hybrid of those in Fig. 1,
A-C. The Na+-driven anion exchanger (NDAE1)
recently cloned from Drosophila (13) does not require
HCO
replacing HCO
View larger version (16K):
[in a new window]
Fig. 1.
Models of Na+-driven
HCO
In mammalian cells, increases in pHi that appear to depend on
Na+, Cl, and
HCO
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. 1A).
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EXPERIMENTAL PROCEDURES |
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Cloning of NDCBE1--
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 ZAPII 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
(27), sequenced the clone, and corrected PCR errors on the basis of the
consensus sequence. The full-length sequence (GenBankTM
accession number AF069512) was released in 1998.
FISH Mapping-- 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 (28).
Northern Analysis--
Northern blots from various human tissues
(catalogue numbers 7760-1 and 7759-1) were obtained from
CLONTECH. 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 ExpressHybTM 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.
Oocytes-- We transcribed NDCBE1 cDNA in vitro using an mMessage mMachineTM kit (Ambion, Austin, TX) with T7 RNA polymerase. Defolliculated Xenopus laevis oocytes (Stage V-VI) were prepared as described previously (29) 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.
Electrophysiology-- The voltage, pH- and sodium-sensitive microelectrodes, were prepared as described previously (10, 29, 30). 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 (31). 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 in Cl
-free solutions.
In some solutions we replaced 16 mM NaCl with 16 mM of n-butyric acid sodium salt (B-5887, Sigma).
36Cl Fluxes--
Ten to twenty oocytes were
incubated at room temperature for ~3 h in 250 µl of
36Cl "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 GoldTM 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 initial
36Cl content of the oocytes and the fractional rate of
36Cl loss during each sampling period in the experiment.
The CO2/HCO
, and the solution was equilibrated with 5%
CO2, 95% O2.
Statistics--
Data are expressed as mean ± S.E.
Statistical significance was judged from unpaired Student's
t tests.
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RESULTS |
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Molecular Characterization
Cloning-- Querying with the sequence of the cDNA encoding salamander NBCe1 (12), 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.
Sequence Analysis--
Fig.
2A compares the deduced amino
acid sequence of NDCBE1 to electroneutral NBCn1 from rat (NBCn1-D; 73%
identity) (10) and to electrogenic NBCe1 from rat kidney (rkNBC; 50%
identity) (32). NDCBE1 has two consensus sites for
N-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 (10), NDCBE1
has one potential DIDS motif (33, 34) (813-816/KLKK), corresponding to
the second of two similar motifs in electrogenic NBCs (12). Fig.
2B summarizes the relationships among the primary structures
of NDCBE1 and other members of the
HCO
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Chromosomal Mapping-- 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. 2C). The BAC clone hybridized 22% of the distance from the centromere to the telomere of arm 12q, corresponding to band 12q13 (Fig. 2D). In contrast, human NBCe1 (SLC4A5) maps (35) to chromosome 4q21, and human NBCn1 (SLC4A7) maps (36) to 3p22.
Tissue Distribution of mRNA-- A Northern blot analysis of multiple human tissues (Fig. 2E) 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 (35, 37). The very weak ~7.5-kb band (testis) may represent the human ortholog of NBCn1 (38). 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.
Physiological Characterization
Na+ Dependence of pHi Recovery--
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.
3A), extracellular 1.5%
CO2, 10 mM
HCO
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Electroneutrality--
In oocytes expressing NDCBE1 (Fig.
3A), 1.5% CO2, 10 mM
HCO
HCO
Inhibition of pHi Recovery by DIDS-- Applying 0.5 mM DIDS almost completely blocks the pHi recovery (Fig. 3C). In six experiments, the inhibition averaged 95% ± 10%. Thus, NDCBE1 is DIDS sensitive.
36Cl Efflux--
When we introduced 5%
CO2, 33 mM
HCO efflux with the properties expected of a
Na+-driven Cl-HCO3 exchanger.
Increase in [Na+]i--
To determine
whether NDCBE1 transports Na+, we used
Na+-sensitive microelectrodes to monitor
[Na+]i. In an oocyte-expressing NDCBE1,
extracellular 5% CO2, 33 mM
HCO1. In parallel experiments (not shown), we
determined dpHi/dt under identical conditions, and
computed the HCO
1.
Cl Dependence of Reversed NDCBE1--
We already
knew that the squid axon's Na+-driven Cl-HCO3
exchanger is very difficult to reverse (3), consistent with the slow
pHi decrease in 0-Na+ in Fig.
4A. 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
reversibly blocked
this decline (Fig. 4A) and caused a small hyperpolarization,
as in water-injected oocytes (not shown). In water-injected oocytes
(Fig. 4B), 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.
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DISCUSSION |
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Related Transporters--
After we submitted our NDCBE1 sequence
to GenBankTM, two other groups (41, 42) published partial
sequences of NDCBE1, one a 2-kb fragment referred to as "NBC-3"
(42). After we submitted our paper, a paper appeared by Wang et
al. (43), 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
(13) in that both exchange extracellular Na+ and "base"
for intracellular Cl. However, human NDCBE1 is strictly
HCO
in
the absence of HCO
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
]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. 1A).
Stoichiometry--
The ratio of net
HCO1)/(1.01 µM
s
1), or 2.17, consistent with the 2:1
stoichiometry expected of a Na+-driven Cl-HCO3
exchanger. Because NDCBE1 is electroneutral, we presume that the net
Cl
efflux is the same as the net Na+ influx
(Fig. 1A). 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
(13): 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
Conclusions--
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 (14). 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 (16, 58, 59), membrane excitability (60), and epileptiform activity (61). pHi
is important for CNS processes other than excitability. For example,
neurite formation (62) requires HCO
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ACKNOWLEDGEMENTS |
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We thank Dr. Nancy Lynn Johnston for
providing us with the human brain ZAPII cDNA library, Dr.
Cecilia M Canessa for providing us with the ENaC cRNA, and Drs. Anne
Marie Quinn, Cecilia M. Canessa, and David Ward for helpful
discussions. We thank Duncan Wong for computer support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS18400.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF069512 (for the electroneutral Na+-driven Cl-HCO3 exchanger gene).
The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number AAC82380.
§ Supported by the National Kidney Foundation. To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8026. Tel.: 203-785-5097; Fax: 203-785-4951; E-mail: ira_grich@hotmail.com.
¶ Supported by the American Heart Association.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.C000716200
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ABBREVIATIONS |
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The abbreviations used are: EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; nt, nucleotide(s); PCR, polymerase chain reaction; UTR, untranslated region; FISH, fluorescence in situ hybridization; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; bp, base pair(s); kb, kilobase(s); BAC, bacterial artificial chromosome.
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