Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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We screened rat brain cDNA libraries and used 5'
rapid amplification of cDNA ends to clone two electrogenic
Na+-HCO3 cotransporter
(NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid
level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The
other clone (rb2NBC) is identical to rb1NBC except for 61 unique
COOH-terminal amino acids, the result of a 97-bp deletion near the
3' end of the open-reading frame. Using RT-PCR, we confirmed that
mRNA from rat brain contains this 97-bp deletion. Furthermore, we
generated rabbit polyclonal antibodies that distinguish between the
unique COOH-termini of rb1NBC (
rb1NBC) and rb2NBC (
rb2NBC).
rb1NBC labels an ~130-kDa protein predominantly from kidney, and
rb2NBC labels an ~130-kDa protein predominantly from brain.
rb2NBC labels a protein that is more highly expressed in cortical
neurons than astrocytes cultured from rat brain;
rb1NBC exhibits the
opposite pattern. In expression studies, applying 1.5%
CO2/10 mM HCO
3 to
Xenopus oocytes injected with rb2NBC cRNA causes 1)
pHi to recover from the initial CO2-induced
acidification and 2) the cell to hyperpolarize. Subsequently,
removing external Na+ reverses the pHi increase
and elicits a rapid depolarization. In the presence of 450 µM DIDS,
removing external Na+ has no effect on pHi and
elicits a small hyperpolarization. The rate of the pHi
decrease elicited by removing Na+ is insensitive to
removing external Cl
. Thus rb2NBC is a
DIDS-sensitive, electrogenic NBC that is predominantly expressed in
brain of at least rat.
intracellular pH; pH regulation; alternative splice variant; bicarbonate; sodium
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INTRODUCTION |
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THE REGULATION OF pH is important in the central
nervous system (CNS) because changes in either the pH of the
intracellular fluid (pHi) or the pH of the extracellular
fluid (pHECF) can alter the level of neuronal activity (see
Refs. 15 and 31). In general, decreases in pHECF inhibit,
whereas increases in pHECF stimulate neuronal firing,
probably because many cellular functions are sensitive to changes in
pHECF and/or accompanying changes in pHi. For
example, decreases in pHECF inhibit many voltage- and
ligand-activated ion channels (see Refs. 43 and 49). The relationship
between changes in brain pH and neuronal activity is complicated,
however, because neuronal firing itself elicits changes in both
pHi and pHECF. Neuronal firing typically causes
a decrease in the pHi of the neurons involved and a
corresponding increase in pHECF due to acid-base transport
across neuronal plasma membranes. The activity-evoked pHi
decrease of neurons can occur via HCO3 efflux through GABAA-stimulated Cl
channels that are permeable to HCO
3
(13, 14, 25, 26) and also via H+ influx mediated by the
Ca2+-H+ pump (27, 28, 39, 42, 48). Because
neurons and astrocytes are in such close apposition to one another in
the CNS, the increase in pHECF will also increase the
pHi of the inactive adjacent cells by altering the activity
of acid-base transporters that are sensitive to extracellular pH. For
instance, increases in pHECF typically stimulate acid
extruders (e.g., Na+/H+ exchanger and
Na+-HCO
3 cotransporter
with a 1:2 or 1:1 Na+:HCO
3
stoichiometry) that move H+ out of or base equivalents such
as HCO
3 or OH
into
cells. In addition, increases in pHECF can inhibit acid loaders (e.g.,
Cl
/HCO
3 exchanger)
that move H+ into or base equivalents out of cells.
Therefore, acid-base transport mechanisms in brain cells not only
contribute to the long-term pHi/pHECF
homeostasis of the CNS, but they also contribute to transient changes
in pHi/pHECF during neuronal firing.
Some of the most powerful pHi-regulating transporters in
neurons and glia are HCO3 dependent
(see Refs. 5, 18, 19, and 35). For example, in freshly dissociated CA1 neurons with a relatively low resting pHi
from immature rats, a Na+-driven
Cl
/HCO
3 exchanger
is the predominant acid extruder responsible for the pHi
recovery from an acid load in the presence of
CO2/HCO
3 (38). Similarly,
in cultured hippocampal neurons, the activity of a
HCO
3-dependent acid extruder appears
to be about twofold greater than that of the Na+/H+ exchanger during the pHi
recovery from an acid load in the presence of
CO2/HCO
3 (30).
HCO
3-dependent transporters also play
an important role in regulating the pHi of glial cells. For
instance, an electrogenic
Na+-HCO
3 cotransporter
with a 1:2 stoichiometry was first documented in leech glial cells (20,
21). Subsequently, similar
Na+-HCO
3 cotransporters
have been identified in several other nonmammalian glial cells, and
evidence has been presented for the existence of
Na+-HCO
3 cotransporters in
mammalian glial cells (see Refs. 18, 19, and 35). More recently, we
have shown that the Na+-driven
HCO
3 transporter in cultured
hippocampal astrocytes elicits membrane voltage changes of a magnitude
that is consistent with the transporter being an electrogenic
Na+-HCO
3 cotransporter
with a 1:2 stoichiometry (4, 7). As proposed by Chesler (15) and Ransom
(31), an electrogenic
Na+-HCO
3 cotransporter in
glial cells may be stimulated by cell depolarization elicited by
increases in the extracellular K+ concentration
([K+]o) in response to neuronal
activity. Such a depolarization-induced alkalinization indeed occurs in
cultured astrocytes exposed to high-[K+]o solutions (10). The
accompanying decrease in pHECF could slow further neuronal
firing. Clearly, HCO
3 transport
mechanisms are abundant in the nervous system, and they are likely to
regulate the pH of the neurons, the glial cells, and the surrounding
extracellular fluid, particularly during neuronal activity.
Except for the anion exchangers (AEs), little was known about
HCO3 transporters at the molecular
level until Romero et al. (34) expression cloned the electrogenic
Na+-HCO
3 cotransporter
(NBC) from Ambystoma kidney (akNBC). Subsequently, others have
identified NBCs from human kidney (11), rat kidney (12,
33), human pancreas (1) and heart (17), human skeletal muscle (29),
and rat aorta and pulmonary artery (16). Rat kidney NBC (rkNBC) is 33%
identical to rat AE1, and both proteins are predicted to possess at
least 10 membrane-spanning segments. Several investigators have
detected NBC-related mRNA in brain from rat and human, based on
Northern blotting (2, 11, 12, 17, 33, 36) and in situ hybridization (24, 36). Investigators have also identified NBC-related proteins in
rat brain, based on immunoblotting (22, 36) and immunolocalization (36).
In the present study, we cloned two NBC-related cDNAs from rat brain
(rb1NBC and rb2NBC), based on their homology to rkNBC. At the protein
level, rb1NBC is 96% identical to the NBC encoded by the cDNA
previously cloned from human pancreas and heart (hpNBC). rb2NBC is
identical to rb1NBC at the cDNA level, except for a 97-bp deletion near
the 3' end of its open-reading frame (ORF). The resultant frame
shift yields a protein with 61 unique COOH-terminal amino acids (AA);
these replace the 46 COOH-terminal AA of rb1NBC. At the protein level,
rb2NBC is 92% identical to hpNBC. We have generated rabbit polyclonal
antibodies that distinguish between the COOH-termini of rb1NBC and
rb2NBC. In rat, a protein with the COOH-terminus of rb2NBC is
predominantly expressed in brain and the protein is expressed heavily
in cultured cortical neurons compared with astrocytes.
When expressed in Xenopus oocytes, rb2NBC displays all of the
hallmarks of a DIDS-sensitive, electrogenic Na+-HCO3 cotransporter.
Portions of this work have been published in abstract form (6).
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METHODS |
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Cloning
Library screening.
We screened the following two size-selected, ZAP II cDNA libraries:
1) an oligo(dT)-primed rat brain cDNA library (
RB-L; kindly
provided by Dr. Terry Snutch, Univ. of British Colombia) and 2)
a random-primed rat forebrain cDNA library (
ZAPRFB; kindly provided
by the Molecular Neurobiology Laboratory, Salk Institute, La Jolla,
CA). Both libraries were first titered and then plated at ~50,000
plaque-forming units per plate. DNA from the plaques was transferred
and ultravioletly cross-linked to Hybond-N nylon membranes (Amersham
Pharmacia Biotech, Piscataway, NJ). Subsequently, the membranes were
probed at 68°C for 15-18 h with a
[32P]cDNA probe (see below) in Express-Hyb
(Clontech Laboratories, Palo Alto, CA). Membranes were then washed with
1) 2× saline sodium citrate (SSC)/0.05% SDS [1-,
5-, and 15-min washes at room temperature (RT), then 2 × 20-min
washes at 44°C] and 2) 0.1× SSC/0.1% SDS (3 × 20-min washes at 44°C). Washed filters were wrapped in
Saran wrap and exposed to Kodak X-Omat film (Kodak, Rochester, NY)
overnight. Positive plaques were isolated and replated, and a secondary
screening was performed as described above. Positive single plaques
from the secondary screen were isolated, and the corresponding
pBluescript SK(
) phagemids were excised ("rescued") from
the
ZAP II vector using the F1 helper phage (Stratagene, La Jolla,
CA). The cloned cDNA inserts within the phagemids were sequenced by the
Keck Sequencing Center (New Haven, CT) using fluorescent dideoxy
sequencing. All nucleic acid sequences were analyzed with DNAsis
(Hitachi Software, San Bruno, CA).
Probes for cDNA library screening.
For the RB-L cDNA library screen, the cDNA probe was obtained by
PCR, using as a template cDNA from rat hippocampal astrocytes; the
primers were degenerate primers to a portion of the rkNBC ORF.
Hippocampal astrocytes were grown as previously described (7), and
total RNA was harvested using the TRIzol Reagent (GIBCO-BRL Life
Technologies, Gaithersburg, MD). mRNA was then isolated using the
Oligotex mRNA kit (Qiagen, Valencia, CA). The sense
degenerate primer
[5'-gCTAT(A/T/C)CCggCTTTgCT(T/C)GTIACC-3']
was to nucleotides 2221-2244 of the rkNBC ORF; the antisense
degenerate primer
[5'-gAg(g/A)TCgTgCTgggA(g/A)AAIAg-3'] was to nucleotides 2825-2845 of the rkNBC ORF. The PCR product was
then labeled with [32P]CTP by random-hexamer
priming (GIBCO-BRL Life Technologies).
5' Rapid amplification of cDNA ends. The above library screening yielded two types of clones with different 3' ends, but lacking the 5' ends of the ORF. The 5' ends of the two types of clones were obtained by using two rounds of 5' rapid amplification of cDNA ends (5'-RACE; GIBCO-BRL Life Technologies) using mRNA from an adult rat brain. The brain was excised from a decapitated rat and was immediately homogenized in TRIzol Reagent. Total RNA and mRNA were then isolated as described above for rat hippocampal astrocytes.
In the first round of 5'-RACE, cDNA from rat brain mRNA was generated using RT and the gene-specific primer 5'-gTCAgACATCAAggTggCgATggCTCTTCC-3'. After tailing the cDNA with a poly(C) homopolymer, we used PCR to amplify the cDNA using a primer complementary to the poly(C) tail and a primer to an internal gene-specific sequence (5'-gggCACAggCACCTCAgTCAgggC-3'). The former primer is engineered to contain an additional "abridged" sequence. The cDNA was further amplified using PCR and primers to the abridged sequence and a second internal gene-specific sequence (5'-AgTgTCCAAgAAgTCAACCTCCCC-3'). The same methodology was used to perform the second round of 5'-RACE, using 5'-TgTCTTCCCAATgTCAGCCAgggA-3' to make the cDNA and primers to internal gene-specific sequences (5'-ggATTTCTTggTTTgATgTCggTg-3' and 5'-CCgCAgCAgCgTATAggTgAC-3') to amplify the cDNA. Full-length clones were amplified using RT-PCR and primers to the beginning and to the end of the ORFs of the two clones. The PCR products were subcloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced.Generating Polyclonal Antibodies
Immunogens were prepared using the maltose-binding protein (MBP) fusion-protein system, as described by Schmitt et al. (37). Briefly, a purified PCR product encoding either the COOH-terminal 46 AA of rb1NBC or the COOH-terminal 61 AA of rb2NBC was subcloned into the expression vector pMAL-c2 (NEB, Beverly, MA). DH5Immunoblotting
Our immunoblotting procedure is described in greater detail by Schmitt et al. (37).Culturing astrocytes and neurons. We cultured astrocytes from rat cortex as previously described for astrocytes from rat hippocampus (7). We obtained cultured neurons from rat cortex from Ying Xia and Gabriel G. Haddad (Dept. of Pediatrics, Yale University), who cultured the neurons as described by Xia et al. (50).
Preparing microsomes. Tissues isolated from Sprague-Dawley rats, or cultured astrocytes or neurons scraped from tissue-culture flasks, were placed in ice-cold HB and were homogenized using a Polytron (model PT-MR 3100; Kinematica, Littau, Switzerland). The homogenate was then centrifuged for 15 min at ~600 g (4°C) to remove cell debris and nuclei. The supernatant was centrifuged again for 45 min at ~28,000 g (4°C) to pellet microsomes containing plasma and organellar membranes. The pellet was resuspended in HB, and the protein concentration was determined with the BCA kit.
Preparing protein from Xenopus oocytes.
Oocytes injected with either H2O or cRNA were pooled in an
Eppendorf tube and were suspended in 20 µl/oocyte of fresh Triton HB
(100 mM NaCl, 20 mM Tris · HCl, and 1% Triton X-100,
pH 7.6). The oocytes were homogenized with a pellet pestle and were
centrifuged for 10 min at ~12,000 g (4°C) to pellet cell
debris. The protein-containing supernatant was then removed and stored
at 70°C.
Gel electrophoresis and protein transfer. Microsomal proteins were separated by SDS-PAGE. Proteins in the gel were transferred to polyvinylidene difluoride membranes (Immobilin-P; Millipore, Bedford, MA) using a semi-dry blotting apparatus (Bio-Rad Laboratories, Richmond, CA) and a discontinuous buffer system. Proteins on membranes were visualized by Coomassie blue staining.
Antibody detection.
Membranes were incubated for 30 min at RT in a "blocking"
solution (Blotto) that contained PBS (137 mM NaCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4,
and 2.7 mM KCl, pH 7.4), 5% Carnation dry milk powder (Nestle Food,
Glendale, CA), and 0.05% Tween 20. Membranes were then incubated first
for 1 h at RT in Blotto containing the primary antibody, and then for 1 h at RT in Blotto containing a 1:3,000 dilution of the secondary
antibody [goat -rabbit-whole IgG-horseradish peroxidase (HRP)
(Sigma)]. Membranes were then washed with copious amounts of
antibody-free Blotto. In a preabsorption experiment, Blotto containing
the primary antibody was mixed for ~30 min with 10 µg/ml fusion
protein before being applied to the membranes. Bound HRP was detected
by the SuperSignal chemiluminescence detection kit (Pierce), and
membranes were exposed to Kodak X-Omat film (Kodak).
Functional Characterization of rb2NBC
Harvesting and injecting oocytes. Stage V/VI oocytes were harvested from female Xenopus laevis frogs as described by Romero et al. (33). cDNA encoding rb2NBC was subcloned into the Xenopus expression vector pTLN2 (33), and cRNA encoding rb2NBC was transcribed in vitro using the T7 promoter and the mMessage mMachine kit (Ambion, Austin, TX). The cRNA was then capped and purified by phenol/chloroform extraction. The oocytes in OR3 medium (50% Leibovitz L-15 media containing 1 mM L-glutamine and 5 mM HEPES, pH 7.5, supplemented with 5 U/ml penicillin-streptomycin) were visualized with a dissecting microscope and were injected with 50 nl of RNase-free H2O or solution containing cRNA. The sterile pipettes had tip diameters of 20-50 µm and were backfilled with paraffin oil before being connected to a Drummond "Nanoject" microinjector (Drummond Scientific, Broomall, PA). Injected oocytes were maintained at 18°C and were studied 3-8 days postinjection.
pHi and membrane potential experiments.
Our technique for measuring pHi and membrane potential
(Vm) of oocytes has been described previously (17,
33). pH- and voltage-sensitive microelectrodes were pulled from
borosilicate fiber-capillary glass (Warner Instruments, West Haven, CT)
using a microelectrode puller (model P-97; Sutter Instrument, Novato, CA). The pH microelectrodes were then dried for at least 2 h at 200°C before being silanized with bis(dimethylamino)dimethyl
silane vapor. The electrode tips were filled with hydrogen ionophore I-cocktail B (Fluka Chemical), backfilled with a phosphate buffer (150 mM NaCl, 40 mM KH2PO4, and 23 mM NaOH, pH 7.0),
and connected to a high-impedance electrometer. The
Vm microelectrodes were filled with 3 M KCl and
were connected to a high-impedance electrometer. Vm
microelectrodes had resistances of 0.2-2 M. All experiments were performed at RT.
Solutions
The standard ND-96 solution contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, and was titrated to pH 7.5 with NaOH. CO2 (1.5%)/HCOStatistics
Data are reported as means ± SE. Levels of significance were assessed using the paired and unpaired Student's t-test. A P value < 0.05 was considered significant. Rates of change of pHi (dpHi/dt values) were fitted by a line using a least-squares method. ![]() |
RESULTS |
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Cloning of Two NBC-Related cDNAs From Rat Brain
Cloning strategy.
We screened a rat brain cDNA library (RB-L) with a
[32P]cDNA fragment of rkNBC and obtained
several partial-length clones that fell into two groups. The clones in
one group had ORFs that were identical to the corresponding ORF of
rkNBC. The clones in the other group had ORFs that were nearly
identical to the corresponding ORF of rkNBC, except for a 97-bp
deletion near the 3' end. None of the clones in either group
contained the 5' end of the ORF.
Sequence analysis of rb1NBC and rb2NBC.
At the AA level, the clone without the 97-bp deletion (rb1NBC) is 96%
identical to hpNBC. This clone has an ORF of 3237 bp, which encodes a
protein of 1079 AA. As is the case for the comparable human NBCs,
rb1NBC and rkNBC are identical, except that the
NH2-terminal 85 AA in rb1NBC replace the
NH2-terminal 41 AA in rkNBC. As shown in Fig.
1A, the NH2-terminal 85 AA of rb1NBC and hpNBC are identical except for two AA.
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rb2NBC mRNA Expressed in Rat Brain
We used RT-PCR techniques to confirm that rb2NBC (i.e., with the 97-bp deletion) is present in mRNA isolated from rat whole brain. After synthesizing rat brain cDNA by reverse transcribing mRNA, we amplified the cDNA using PCR techniques and primers to sequences flanking the 97-bp deletion. Analyzing a portion of the reaction on a 1.5% agarose gel, we observed two bands (Fig. 2). The ~640-bp band was the expected product from rb1NBC (without the deletion), and the ~540-bp band was the expected product from rb2NBC (with the 97-bp deletion). We subcloned and sequenced similar PCR products to confirm their identity. We observed no bands with H2O as the template in the PCR.
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In another series of RT-PCR experiments with cDNA made from isolated regions of rat brain, we used similar primers to detect bands representative of rb1NBC and rb2NBC. In whole brain, cerebral cortex, brainstem diencephalon, and cerebellum, we detected each of the two PCR products (B. M. Schmitt, U. V. Berger, R. Douglas, M. O. Bevensee, M. A. Hediger, G. G. Haddad, and W. F. Boron, unpublished observations).
rb2NBC Protein Expressed in Rat Brain
Polyclonal antibodies to rb1NBC and rb2NBC.
To determine if rb1NBC and rb2NBC proteins are expressed in rat brain,
we generated rabbit polyclonal antibodies that would discriminate
between the COOH-termini of the two proteins. We generated two
polyclonal antibodies, one against the COOH-terminal 46 AA of rb1NBC
(rb1NBC) and the other against the COOH-terminal 61 AA of rb2NBC
(
rb2NBC). We separated microsomal proteins from rat brain by
SDS-7.5% PAGE and then immunoblotted the protein with preimmune rabbit
serum as well as
rb1NBC (Fig.
3A) and preimmune serum as well as
rb2NBC (Fig. 3B). Neither of the preimmune sera labeled
brain protein (Fig. 3, A and B, lane 1). In
contrast, both
rb1NBC and
rb2NBC labeled ~130-kDa proteins in
brain (Fig. 3, A and B, lane 2). Therefore, rat
brain expresses protein containing the COOH-termini of both rb1NBC and
rb2NBC.
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Recognition of distinct epitopes by rb1NBC and
rb2NBC.
As also shown in Fig. 3, we examined the specificity of labeling by
attempting to preabsorb the antibodies with various fusion proteins
(Fig. 3, A and B, lanes 3-5). In Fig.
3A,
rb1NBC labeled an ~130-kDa protein even after the
antiserum was exposed to the MBP-gal and MBP-rb2NBC fusion proteins
(lanes 3 and 4, respectively). In contrast,
rb1NBC
failed to label a protein after the serum was exposed to the immunogen,
the MBP-rb1NBC fusion protein (Fig. 3A, lane 5). Note
that the labeling of the ~70-kDa protein by
rb1NBC (Fig.
3A, lanes 2-5) is nonspecific because it occurs even in the presence of MBP-rb1NBC (Fig. 3A, lane
5).1 Thus
rb1NBC labels
an ~130-kDa brain protein by recognizing the unique 46 AA of rb1NBC.
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Localization of rb1NBC and rb2NBC in neurons and astrocytes.
We also studied the cellular distribution of the two NBC clones in rat
brain by performing immunoblots of protein isolated from neurons and
astrocytes cultured from rat cortex. We separated microsomal proteins
from cultured neurons and astrocytes by SDS-7.5% PAGE and then
immunoblotted the protein with rb1NBC or
rb2NBC. As a positive
control, we also immunoblotted protein from whole brain of rat. As
shown in Fig. 5A,
rb1NBC labeled
an ~130-kDa protein from astrocytes more intensely than from neurons.
In contrast,
rb2NBC labeled an ~130-kDa protein from neurons more
intensely than from astrocytes (Fig. 5B). Thus cultured neurons
and astrocytes from rat cortex differentially express NBCs, with the
COOH-terminus of rb1NBC being more prevalent in astrocytes and the
COOH-terminus of rb2NBC being more prevalent in neurons.
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Functional Characterization of rb2NBC Expressed in Xenopus Oocytes
Choi et al. (17) have shown that hpNBC expressed in Xenopus oocytes has all the basic properties expected of an electrogenic Na+-HCOImmunolabeling of rb2NBC expressed in Xenopus oocytes.
We used rb2NBC to confirm that oocytes injected with rb2NBC cRNA did
in fact express rb2NBC protein. We used SDS-5% PAGE to separate total
protein from oocytes injected with either H2O or rb2NBC
cRNA, or total protein from whole brain. As shown in Fig.
6, we then immunoblotted the protein using
rb2NBC. Our first approach for evaluating the specificity of
labeling was to compare immunoblots for oocytes injected with
H2O with those for oocytes injected with rb2NBC cRNA. As
expected, we found that
rb2NBC detected an ~130-kDa protein in
oocytes injected with rb2NBC (lane 2) but not in
H2O-injected oocytes (lane 1). Our second approach for evaluating the specificity of
rb2NBC labeling was to attempt to
preabsorb the antibody with various fusion proteins. Preabsorption with
the MBP-rb2NBC fusion protein eliminated the detection of the
~130-kDa band from rb2NBC-expressing oocytes (lane 3). In contrast, preabsorption with MBP-rb1NBC did not reduce the intensity of
the ~130-kDa band (lane 4). For a positive control, we probed whole rat brain with
rb2NBC that we had pretreated with MBP-rb1NBC (lane 5). Thus, based on immunoblotting, rb2NBC protein is
expressed in oocytes injected with rb2NBC cRNA.
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Effect of applying
CO2/HCO3 and
removing external Na+.
Figure 7A is a record of an
experiment performed on an oocyte injected with cRNA encoding rb2NBC.
In the standard ND-96 solution, the oocyte had a pHi of
~7.3 and a Vm of approximately
55 mV (before point a/a'). Exposing the oocyte to a
solution buffered with 1.5% CO2/10 mM
HCO
3 elicited an initial pHi decrease (ab), due to CO2 influx,
followed by a rapid pHi recovery (bc), consistent
with rb2NBC-mediated HCO
3 transport
into the oocyte. Applying
CO2/HCO
3 also elicited an
abrupt hyperpolarization of ~40 mV
(a'b'), consistent with transport of net
negative charge into the cell. In the continued presence of
CO2/HCO
3, replacing
external Na+ with Li+ had two effects. First,
the replacement blocked the pHi recovery and caused
pHi to decrease (cd). Second, the
replacement elicited an abrupt depolarization (at point
c'). Both findings are consistent with reversal of an
electrogenic NBC (4, 8, 17, 33, 34). We observed similar effects when
we used either NMDG+ or choline instead of Li+
as the Na+ substitute. In voltage-clamp experiments,
Sciortino and Romero (40) have shown that Li+ is only
poorly transported by rkNBC expressed in Xenopus oocytes. Returning external Na+ caused the pHi to
increase once again (de) and caused the oocyte to hyperpolarize
(at point d').
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Effect of removing external Cl.
Because some HCO
3-dependent acid-base
transporters move Cl
(e.g.,
Na+-dependent and -independent
Cl
/HCO
3 exchange),
we performed the experiment shown in Fig.
8A to evaluate the
Cl
dependence of rb2NBC. The first part of the
protocol was the same as that shown in Fig. 7A. After we had
exposed the rb2NBC-injected oocyte to
CO2/HCO
3, removing
external Na+ elicited the expected pHi decrease
(cd) and depolarization (at point c'). If the
pHi decrease elicited by removing external Na+
(and thereby reversing rb2NBC) were due to a
Cl
-dependent process such as a
Na+-driven
Cl
/HCO
3 exchanger,
then removing external Cl
should block this
pHi decrease. However, removing and returning external
Cl
had little effect on the pHi decrease
observed in the absence of external Na+ (cdef).
Returning Na+ again elicited a pHi recovery
(fg) and also hyperpolarized the oocyte (at point
f'). Switching back to the nominally
CO2/HCO
3-free ND-96
solution reversed the pHi (gh) and
Vm (g'h') effects
produced by applying
CO2/HCO
3.
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Effect of applying DIDS.
Because most HCO3-dependent
transporters are sensitive to stilbene derivatives such as DIDS, we
examined the effect of DIDS on rb2NBC. The experiment shown in Fig.
9A is similar to that shown in Fig.
7A and Fig. 8A in that we exposed an rb2NBC-expressing
oocyte to CO2/HCO
3 and then removed and returned external Na+. However, in Fig.
9A, we assessed the effect of 450 µM DIDS on the
Na+-dependent changes of pHi and
Vm. As noted previously, returning the
Na+ elicited a pHi recovery (de) and a
hyperpolarization (at point d'). Exposing the oocyte to
450 µM DIDS not only blocked the pHi recovery, but also
unmasked a sizeable acidification (ef). The DIDS also
elicited a depolarization (at point e'). Subsequently, removing external Na+ in the presence of DIDS did not
affect pHi (fg) and generated only a small
hyperpolarization (point f') followed by a slow
nonspecific depolarization to point g'.
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DISCUSSION |
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97-bp Deletion of rb2NBC
In screening rat brain cDNA libraries and using 5'-RACE, we cloned two NBC-related cDNAs from rat brain. One clone, rb1NBC, is very similar to the NBC cloned from human pancreas and heart. The other clone, rb2NBC, is identical to rb1NBC except for a 97-bp deletion near the 3' end of the ORF, resulting in a protein with 61 novel COOH-terminal AA.As to the origins of rb2NBC vs. rb1NBC, the most likely explanation is that rb2NBC is an alternative splice variant of rb1NBC in which an exon lacking the 97-bp region replaces an exon containing this region. Alternatively, the 97-bp segment is an intron that the splicing machinery fails to excise in the production of rb1NBC. However, the 5' and 3' termini of this 97-bp segment do not contain the well-conserved 5'-GT and 3'-AG consensus splice sequences found in the majority of premessenger RNA introns. The segment also lacks the 5'-AT and 3'-AC consensus splice sequences found in a small percentage of premessenger RNA introns (see Ref. 46). If this region is indeed an intron, then the 5' and 3' sequences may represent a new class of consensus splice sequences. Finally, rb1NBC and rb2NBC may represent different genes. Presumably, electrogenic NBCs have been localized to human chromosomes 4 (1, 32) and 17 (32).
Regardless of its origin, the 97-bp deletion causes a shift in the translational reading frame because 97 is not divisible by three. Consequently, the stop codon in rb2NBC is further 3' than that in rb1NBC, and the COOH-terminus of rb2NBC is unique. Although rare in eukaryotes, there are examples of such alternative splicing leading to a shift in the ORF. For example, an alternative splice variant of gastrin-releasing peptide contains an additional 19 nucleotides near the 3'-ORF, resulting in a translational frame shift and a unique COOH-terminus (44).
Novel COOH-Terminus of rb2NBC
According to our data, rb2NBC expressed in oocytes has the same general functional properties as akNBC (34), rkNBC (33), and hpNBC (17). Although we did not express and study the function of rb1NBC in oocytes, this clone is likely to be very similar in function to hpNBC, inasmuch as the two clones are 96% identical, and the 4% divergence is more-or-less evenly scattered throughout the clones. rkNBC, hpNBC, and rb2NBC are all electrogenic, Na+ dependent, ClAlthough rb2NBC expressed in oocytes is electrogenic, we do not know if
the transporter has a Na+:
HCO3 stoichiometry of 1:2 or 1:3.
Based on the electrochemical gradients, an NBC with either
stoichiometry would transport Na+ and
HCO
3 into the oocyte. Determination of the Na+:HCO
3 stoichiometry
of rb2NBC (or rb1NBC) expressed in oocytes will require a more detailed
study in which one compares at least two of the three following
parameters in voltage-clamped oocytes: NBC-mediated Na+
fluxes, HCO
3 fluxes, and membrane currents.
The unique COOH-terminal 61 AA of rb2NBC are not homologous to any
other sequence in GenBank. However, one interesting feature of this
COOH-terminus is that the last three AA (TTL) resemble the
COOH-terminal consensus sequence (S/TXV) that is recognized by PDZ
domains of proteins that are involved in protein-protein interactions
(see Ref. 23). PDZ is an acronym that stands for the first three
proteins identified that contain conserved, 80-90 AA repeats that
are involved in protein binding. These three proteins are a
brain-specific synaptic protein (SD-95),
the Drosophilia septate junction protein disks-large
(
lg), and the epithelial tight-junction
protein zonula occludens-1 (
O1). PDZ
domains are found in proteins involved in signal transduction, and they
may play a role in clustering other proteins in the plasma membrane (see Ref. 23). Although the COOH-terminal AA in rb2NBC is L rather than
the V in the consensus sequence, there are examples in which the
PDZ-binding domain contains an L instead of V. For example, LIN-7 of
Caenorhabditis elegans, which is a component of the
ras-signaling pathway, appears to interact with the
COOH-terminal ETCL of the receptor tyrosine kinase LET-23
(41). Also, using a yeast two-hybrid screen, Strehler et
al. (45) identified PDZ proteins that interact with the COOH-terminal
ETSL of the 2b isoform of the plasma membrane
Ca2+-H+ pump. Therefore, it is intriguing to
speculate that the ETTL sequence in rb2NBC may be a PDZ-binding domain
and may be important for either membrane targeting or functional
regulation of rb2NBC.
Based on our functional studies, rb2NBC is expressed at the plasma
membrane of oocytes. However, it is possible that NBCs such as rb1NBC
and rb2NBC may also be expressed in intracellular compartments in vivo.
Indeed, based on preliminary electron microscopy studies on rat brain
slices, rb1NBC appears to label intracellular proteins in neurons
from rat cerebellum (A. Maunsbach, personal communication). We also
cannot exclude the possibility that the subcellular localization of
NBCs may vary among different brain regions.
Distribution of rb2NBC
As mentioned in the INTRODUCTION, considerable functional data demonstrate the existence of Na+-HCOThe question arises as to the function of an electrogenic NBC in
neurons. An electrogenic NBC with a
Na+:HCO3 stoichiometry of
1:2 would probably function as an acid extruder and raise
pHi at all physiological Vm (40) and
would probably be accelerated at the positive Vm
accompanying an action potential. Even an NBC with a 1:3 stoichiometry
would transport Na+ and
HCO
3 into the cell during action
potentials. However, most experimenters have observed that neuronal
firing causes a decrease in pHi rather than an increase
(see Ref. 3). The major causes of these pHi decreases are
the influx of H+ mediated by the
Ca2+-H+ pump and the efflux of
HCO
3 mediated by the GABAA
channel. If electrogenic
Na+-HCO
3 cotransport is
indeed stimulated during neuronal firing, then this cotransporter may
minimize the extent of the activity-induced decrease in pHi
and concomitant increase in pHECF. Thus electrogenic NBCs
in both neurons and glial cells may influence the extent to which
pHi and pHECF change in response to neuronal
firing. In addition, these NBCs may have different functional
properties and sensitivities to various signaling pathways and may thus
respond differently to a range of electrical and chemical stimuli.
![]() |
NOTE ADDED IN PROOF |
---|
Giffard et al. recently reported the cloning of an NBC (GenBank accession number AF210250) from a rat brain hippocampal cDNA library (Giffard RG, Papadopoulos MC, van Hooft JA, Xu L, Giuffrida R, and Monyer H. The electrogenic sodium bicarbonate cotransporter: developmental expression in rat brain and possible role in acid vulnerability. J Neurosci 20: 1001-1008, 2000). The translated protein is identical to rb1NBC, except for four amino acids.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Terry P. Snutch (Biotechnology Laboratory, Univ. of
British Colombia) for kindly allowing us to use the RB-L cDNA
library, William J. Joiner (Kaczmarek Laboratory, Dept. of Pharmacology, Yale University) for providing us with the
RB-L cDNA
library and for assisting in the library screening, Drs. Ying Xia and
Gabriel G. Haddad (Dept. of Pediatrics, Yale University) for generously
providing us with cultured cortical neurons, Dr. Christopher B. Burge
(Center for Cancer Research, Massachusetts Institute of Technology) for
helping us analyze the clones, and Duncan Wong for providing computer support.
![]() |
FOOTNOTES |
---|
M. O. Bevensee was supported by National Institutes of Health (NIH) Training Grant TG HL-07778. B. M. Schmitt was supported by a Forschungsstipendium from the Deutsche Forschungsgemeinschaft. I. Choi was supported by a postdoctoral fellowship from the American Heart Association. M. F. Romero was supported by NIH Service Award DK-09342 and by a grant from the American Heart Association. This work was supported by NIH Grants P01 HD-32573 and DK-30344.
GenBank accession numbers are AF254802 for rb1NBC and AF124441 for rb2NBC.
Present addresses: B. M. Schmitt, Dept. of Anatomy, University of Würzburg, 97070 Würzburg, Germany; M. F. Romero, Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970.
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. §1734 solely to indicate this fact.
1
In other immunoblot experiments on rat brain
protein, we often observed that rb1NBC and
rb2NBC at higher
concentrations (e.g., <1:1,000 dilution) labeled one or two other
proteins in the 70- to 100-kDa range. The labeling of these other bands
(i.e., not the ~70-kDa band in Fig. 3A) is specific
because it can be competed away with the appropriate fusion proteins.
These lower-molecular-weight proteins could be either breakdown
products of rb1NBC and rb2NBC, or they could be smaller-sized NBCs.
Multiple proteins are also labeled by an antibody to the COOH-terminal
108 AA of rkNBC (37).
Address for reprint requests and other correspondence: M. O. Bevensee, Dept. of Cellular and Molecular Physiology (12-17-99), Yale Univ. School of Medicine, 333 Cedar St., Rm. B-127 SHM, New Haven, CT 06520.
Received 3 September 1999; accepted in final form 30 December 1999.
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