Cloning, Tissue Distribution, Genomic Organization, and
Functional Characterization of NBC3, a New Member of the Sodium
Bicarbonate Cotransporter Family*
Alexander
Pushkin,
Natalia
Abuladze
,
Ivan
Lee,
Debra
Newman,
James
Hwang, and
Ira
Kurtz§
From the Division of Nephrology, Center for Health Sciences, UCLA
School of Medicine, Los Angeles, California 90095-1698
 |
ABSTRACT |
Previous functional studies have demonstrated
that muscle intracellular pH regulation is mediated by sodium-coupled
bicarbonate transport, Na+/H+ exchange,
and Cl
/bicarbonate exchange. We report the cloning,
sequence analysis, tissue distribution, genomic organization, and
functional analysis of a new member of the sodium bicarbonate
cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3
encodes a 1214-residue polypeptide with 12 putative membrane-spanning
domains. The ~ 7.8-kilobase transcript is expressed uniquely in
skeletal muscle and heart. The NBC3 gene (SLC4A7) spans ~80 kb and is
composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA in
Xenopus laevis oocytes demonstrated that the protein
encodes a novel stilbene-insensitive
5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.
 |
INTRODUCTION |
Intracellular pH regulatory mechanisms are critically important
for the maintenance of many cellular processes in skeletal muscle,
smooth muscle, and myocardial cells (1-8). In muscle cells,
contractile processes, metabolic reactions, and membrane transport
processes are influenced by pH. Importantly, during periods of
increased energy demands and ischemia, muscle cells produce large
amounts of lactic acid (3). In these circumstances, intracellular
pH (pHi)1 regulatory
processes prevent the acidification of the sarcoplasm due to
lactic acid accumulation.
Several different transport mechanisms have been described in muscle
cells, which maintain a relatively constant intracellular pH during
changes in metabolic proton production or an elevation in ambient
CO2. The relative contribution of each process varies with
cell type, the metabolic requirements of the cell, and local environmental conditions. Intracellular pH regulatory processes that
have been characterized functionally in skeletal, smooth muscle, and
cardiac cells include: Na+/H+ exchange (1-3,
9-22), Na(HCO3)n cotransport (12, 13, 16, 17, 21,
23-29), Na+-dependent (12, 18, 19, 26, 30, 31)
and -independent (19, 24, 26, 29, 32, 33) Cl
/base
exchange, and lactate/proton cotransport (3).
With the exception of guinea pig femoral artery, when present,
Na+-dependent
HCO3
transport plays a significant
role in regulating pHi in the tissues where it has been
investigated. However, the characteristics of
Na+-dependent
HCO3
transport vary between different
muscle cell preparations. Specifically, the
Na(HCO3)n cotransporter in cardiac Purkinje fibers and ventricular myocytes (17, 24) and in certain smooth muscle cells
(11, 26-29) appears to be electroneutral in contrast to the
electrogenicity reported in other cell types (13, 25, 34). Furthermore,
stilbene-insensitive Na+-dependent
HCO3
transport has been reported in
various smooth muscle cells (27, 28). Finally, EIPA-inhibitable
stilbene-insensitive Na+-dependent
HCO3
transport has been described in
vascular smooth muscle cells (12, 15, 21). The functional differences
that have been reported in various skeletal muscle, smooth muscle, and
cardiac myocytes preparations suggest that several transporters may
exist that differ in their functional properties and sensitivity to inhibitors. However, the molecular mechanisms mediating
Na+-dependent
HCO3
transport in muscle cells have
not previously been investigated. An electrogenic sodium bicarbonate
cotransporter (NBC1) has recently been cloned from salamander and rat
kidney (35-37). Furthermore, a sodium bicarbonate cotransporter has
also been cloned from human kidney (kNBC1) and human pancreas (pNBC1)
(38, 39). The kidney uniquely expresses the kNBC1 isoform,
predominantly in the proximal tubule, whereas expression of pNBC1 is
more widespread (39, 40). pNBC1 was only weakly expressed in human
skeletal muscle on Northern blots. Ishibashi et al. (41)
have recently cloned an additional member of the NBC family (NBC2) from
human retina whose function is unknown.
To gain additional insight into the reason for the functional
variability in Na+-dependent
HCO3
transport among various muscle
cell preparations, we screened a human skeletal muscle cell library and
obtained a unique clone that is expressed specifically in skeletal
muscle and heart. In this paper, we report the cloning, sequence
analysis, tissue distribution, genomic organization, and functional
analysis of this new member of the NBC family, NBC3.
 |
MATERIALS AND METHODS |
Cloning and Sequencing of mNBC3--
Human expressed sequence
tag clones were identified by a homology search of the expressed
sequence tag data base for pNBC1-related sequences. The clones were
used as a template for sequencing and 5' and 3' RACE products were
generated. 5' and 3' oligonucleotide probes based on the RACE products
were synthesized, random primer labeled with 32P, and used
to screen a human muscle
gt11 cDNA library
(CLONTECH, Palo Alto, CA). Positive clones were
verified by sequencing. Two overlapping clones were obtained that
contained the entire coding region. To obtain a full-length clone
containing the complete open reading frame, these two clones were
spliced together using a common BamHI restriction site and
subcloned into pPCR-Script SK+ (Stratagene, La Jolla, CA).
The 5' end of the coding sequence for mNBC3 was confirmed by 5' RACE
PCR amplification. Furthermore, to confirm that the mNBC3 amino acid
sequence was derived from a bona fide transcript, we
amplified the entire open reading frame of human mNBC3 by reverse
transcription-PCR using Marathon Ready cDNA prepared from
human muscle (CLONTECH) as a template.
Nucleotide sequences were determined bidirectionally by automated
sequencing (ABI 310, Perkin-Elmer, Foster City, CA) using
Taq Polymerase (Ampli-Taq FS, Perkin-Elmer).
Sequence assembly and analysis was carried out using
Geneworks software (Oxford, UK). Secondary structure and
hydropathy analyses were performed using the Tmpred program (www.isrec.isb-sib.ch/software/TMPRED_form.html).
Northern Analysis and Tissue Distribution--
Northern blots
with various human tissues were obtained from
CLONTECH. The probe was primed with
[32P]dCTP and [32P]dATP to a specific
activity of about 1.5 × 109 dpm/µg. The filters
were prehybridized at 42 °C for 2 h using 50% formamide, 6×
SSPE, 0.5% SDS, Denhardt's solution, and 0.1 mg/ml of sheared herring
testes-denatured DNA. Following the prehybridization, the filters were
incubated with the 32P probe using 25 ml of hybridization
buffer. The probes were denatured and added to the hybridization
solution at 107 dpm/ml. The filters were probed at 42 °C
for 18 h and washed in 1× SSC,0.1% SDS at 45 °C for 60 min
(three changes, 350 ml/wash); after exposure for 9.5 h, the
filters were rewashed in 1× SSC, 0.1% SDS at 65 °C for 30 min and
0.1× SSC, 0.1% SDS at 65 °C for 1 h. A labeled synthetic
95-bp oligonucleotide probe (nucleotides 1002-1096) specific for the
unique N-terminal region of mNBC3 was randomly primed and labeled with
[32P]dCTP and [32P]dATP.
Genomic Organization--
To isolate the NBC3 gene, we screened
a BAC human genomic DNA library (Genome Systems, St.Louis, MO) using a
PCR approach. The PCR primers designed for screening the BAC human
library were: 5'-CTGCAACAGTGTCAATAAGTCATG-3' (2995) and
5'-ACTGAAGTCATGAACACAGAGAGGC-3' (3130). The PCR product was 160 bp. BAC
clone 15 was isolated, and sequence analysis revealed that this clone
encompassed the entire human NBC3 gene and was therefore used for all
subsequent analysis. A series of primers were designed based on the
known mNBC3 cDNA sequence (AF047033) to determine the structure of the NBC3 gene. Sequences at the intron-exon boundaries of the NBC3 gene
were determined by aligning the cDNA sequence of mNBC3 with the
genomic sequence.
Xenopus Oocyte Expression--
The mNBC3 insert in pPCR-Script
SK+ was subcloned into the Xenopus expression
vector ppT7TS (obtained from Dr. Paul Krieg), which contains the 5'-
and 3'-untranslated regions of Xenopus
-globin mRNA
inserted into pGEM4Z. The plasmid containing the complete coding
sequence of mNBC3 was linearized by digestion with SalI, and
capped cRNA was prepared with T7 RNA polymerase using a T7 mMessage
mMachine kit RNA capping kit (Ambion, Austin, TX) as recommended by the
manufacturer. This cRNA was used for the Xenopus oocyte
expression studies. An aliquot of the synthesized cRNA was run on a
denaturing gel to verify the expected size prior to oocyte injection.
Defolliculated oocytes were injected with 50 nl of sterile water or a
solution containing 0.5-1 ng/nl of capped mNBC3 cRNA (prepared as
described above). They were then bathed in Barth's medium at 18 °C
and studied 3-14 days post-injection.
22Na+ Influx
Measurements--
Defolliculated oocytes were injected with mNBC3 cRNA
(50 nl, 0.5-1 µg/ul) or water and incubated in Barth's medium at
18 °C prior to study. In uptake studies performed in the presence of chloride, the oocytes were preincubated for 1 h in 10 ml of a Na+-free solution containing 108 mM
tetramethylammonium Cl, 2 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, and 8 mM Hepes, pH 7.4. The oocytes were then transferred into
1.4 ml of the following Na+-containing solution: 22 mM NaCl, 78 mM tetramethylammonium Cl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 8 mM
NaHCO3 bubbled with 1.5% CO2, pH 7.4 with 2 µCi of 22Na+. A 10-µl aliquot was removed
from the influx solution for later determination of
22Na+ specific activity.
22Na+ influx was measured after 15 min and
terminated with three washes of ice-cold Na+-free stop
solution. The influx experiments were repeated in the presence of DIDS
(1 mM). In the DIDS-containing experiments, the oocytes
were exposed to 1 mM DIDS for 1 h prior to and
throughout the influx period. The effect of EIPA (50 µM)
on 22Na+ uptake was also studied. In uptake
studies performed in the absence of chloride, the oocytes were
preincubated for 1 h in 10 ml of a Na+-free
Cl
-free solution containing 108 mM
tetramethylammonium hydroxide, 108 mM gluconic acid
lactone, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM
Hepes, pH 7.4. The oocytes were then transferred into 1.4 ml of the
following Na+-containing solution: 22 mM sodium
gluconate, 78 mM tetramethylammonium hydroxide, 78 mM gluconic acid lactone, 2 mM potassium
gluconate, 7 mM calcium gluconate, 2 mM
magnesium gluconate, and 8 mM NaHCO3 bubbled
with 1.5% CO2, pH 7.4, with 2 µCi of
22Na+.
86Rb Influx Measurements--
The oocytes were
preincubated in 10 ml for 1 h in a K+-free solution
containing 98 mM NaCl, 10 mM RbCl, 1 mM CaCl2, 1 mM MgCl2, and 8 mM Hepes, pH 7.4, containing 0.1 mM
ouabain. The oocytes were then transferred into 1.4 ml of the following
solution: 90 mM NaCl, 10 mM RbCl, 1 mM CaCl2, 1 mM MgCl2,
and 8 mM NaHCO3 bubbled with 1.5%
CO2, pH 7.4, containing 0.1 mM ouabain with 2 µCi of 86Rb. A 10-µl aliquot was removed from the
influx solution for later determination of 86Rb specific
activity. 86Rb influx was measured after 15 min and
terminated with three washes of ice-cold stop solution.
Measurement of Oocyte pHi--
Optical recordings were
made at 21 °C. Intracellular pH was monitored using the fluorescent
probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a
microflourometer coupled to the microscope (42). Individual
defolliculated oocytes were held in place with pipettes attached to low
suction with vegetal pole surface closest to the 40× objective. Prior
to loading with BCECF, the background intensity from each oocyte was
digitized at 500 and 440 nm (530 nm emission). The oocytes were loaded
with 32 µM BCECF-AM for 30-60 min prior to
experimentation in the following solution: 108 mM sodium
gluconate, 2 mM potassium gluconate, 7 mM
calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4. Calibration of intracellular BCECF in the
oocytes was performed at the end of each experiment by monitoring the
500/440 nm fluorescence excitation ratio at various values of
pHi in the presence of high K+ nigericin standards
as described previously (43). The following experimental protocols were
performed: 1) For Na+ removal/addition in
Cl
-free bicarbonate-buffered solutions, the oocytes were
bathed in the following Na+-containing
Cl
-free solution for 1-8 h prior to loading with BCECF:
108 mM sodium gluconate, 2 mM potassium
gluconate, 7 mM calcium gluconate, 2 mM
magnesium gluconate, and 8 mM Hepes, pH 7.4. After a steady state was reached, the oocytes were exposed to a bicarbonate-buffered Cl
-free solution containing 100 mM sodium
gluconate, 2 mM potassium gluconate, 7 mM
calcium gluconate, 2 mM magnesium gluconate, 8 mM NaHCO3
bubbled with
1.5% CO2, pH 7.4. After a new steady state was achieved, Na+ was removed by exposing the oocytes to a solution
containing 100 mM tetramethylammonium hydroxide, 100 mM D-gluconic acid lactone, 2 mM
potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, 8 mM
tetramethylammonium HCO3 bubbled with 1.5%
CO2, pH 7.4. Additional experiments were performed in
oocytes bathed in 1 mM DIDS or 50 µM EIPA. 2)
For Na+ removal/addition in Cl
-free
Hepes-buffered solutions bubbled continuously with 100% O2, the oocytes were bathed in the following
Na+-containing Cl
-free solution for 1-8 h
prior to loading with BCECF: 108 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate,
2 mM magnesium gluconate, and 8 mM Hepes, pH
7.4. After a steady state was reached in the same solution,
Na+ was removed by bathing the oocytes in the following
Hepes-buffered Na+-free solution: 108 mM
tetramethylammonium hydroxide, 108 mM
D-gluconic acid lactone, 2 mM potassium
gluconate, 7 mM calcium gluconate, 2 mM
magnesium gluconate, and 8 mM Hepes, pH 7.4, bubbled
continuously with 100% O2. 3) For K+
addition/removal in Cl
-free bicarbonate-buffered
solutions, the oocytes were bathed in the following
Na+-containing Cl
-free solution for 1-8 h
prior to loading with BCECF: 108 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate,
2 mM magnesium gluconate, and 8 mM Hepes, pH
7.4. After a steady state was reached, the oocytes were bathed in a
solution containing 50 mM sodium gluconate, 2 mM potassium gluconate, 48 mM
tetramethylammonium hydroxide, 48 mM gluconic acid lactone,
7 mM calcium gluconate, 2 mM magnesium
gluconate, and 8 mM NaHCO3
bubbled with 1.5% CO2, pH 7.4. The K+
concentration was acutely increased by exposing the oocytes to a
solution containing the identical Na+ concentration and a
K+ concentration of 50 mM: 50 mM
sodium gluconate, 50 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate,
and 8 mM NaHCO3
bubbled
with 1.5% CO2, pH 7.4.
 |
RESULTS AND DISCUSSION |
We report the cloning, sequence analysis, tissue distribution,
genomic organization, and functional characterization of a new member
of the NBC family, NBC3, which is uniquely expressed in skeletal muscle
and heart muscle. Partial sequence information obtained from human
expressed sequence tag clones and 5' and 3' RACE products were used for
generating probes to screen a human muscle
gt11 cDNA library to
obtain full-length mNBC3. The nucleotide sequence of mNBC3 is 7785 nucleotides in length, with a predicted open reading frame of 3642 nucleotides, as 5'-untranslated region of 71 nucleotides and a
3'-untranslated region of 4072 nucleotides (GenBankTM/EMBL
Data Bank with the accession number AF047033). mNBC3 is homologous to
several expressed sequence tag sequences (human: AA205898, AA535856
AA569234, AA188932, AA852668, AA243166, AA476540, AA227419, AA216661,
AA243228, AA218565, AA101282, Z19373, R57438, AA494043, AA630595, H66521, AF00103, and Z20394; mouse: AA166155, AA689098, and AA592390).
Beginning with methionine at position 1, an open reading frame codes
for a protein of 1214 amino acids. Fig. 1
demonstrates the protein alignment of mNBC3 with other members of the
NBC family and the anion exchanger bAE3. mNBC3 is 78% homologous to
NBC2; 39 and 46% homologous to pNBC1 and kNBC1, respectively, and 29%
homologous to bAE3.
The secondary structure analyses and hydropathy profile of the protein
predict an intrinsic membrane protein with 12 putative transmembrane
domains (Fig. 2); N-linked
glycosylation sites in exofacial loops 1-2 and 5-6; protein kinases A
and C, casein kinase II, and ATP/GTP-binding consensus phosphorylation
sites; potential sites for myristylation and amidation; and cytoplasmic
N and C termini. A large exofacial loop is present between predicted
membrane domains 1 and 2. The first of two putative stilbene-binding
motifs (K(M/L)XK) shared by other members of the anion
exchanger superfamily (AE1, AE2, AE3, pNBC1, and kNBC1) is lacking in
mNBC3, which has the sequence KLFD (amino acids 742-745). The
replacement of the highly conserved lysine at position 745 by aspartic
acid may block negatively charged stilbene disulfonates from binding
and alter the Ki of these compounds for mNBC3 (44,
45). It is of interest that stilbene-insensitive
Na+-dependent bicarbonate transport has been
reported in cultured smooth muscle cells from human internal mammary
artery (15, 21), rat aorta (12), guinea pig femoral artery (27), and guinea pig ureter (28). The second putative stilbene-binding motif KLKK
(amino acids 944-947) in mNBC3 is identical in sequence to the
homologous stilbene-binding site in human kNBC1, pNBC1, and NBC2
(38, 39, 41).

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Fig. 2.
Putative topology of human mNBC3 in the
membrane. Charged amino acids are depicted by plus and
minus signs. Potential N-linked glycosylation
sites are depicted by . mNBC3 also has numerous potential
intracellular consensus phosphorylation sites: 1) protein kinase A:
Lys222 and Lys239; 2) protein kinase C:
Ser39, Ser55, Ser76,
Ser91, Thr132, Ser203,
The225, Ser260, Ser271,
Ser288, Thr320, Ser324,
Ser327, Thr847,
Thr1127, Ser1141, and
Ser1194; 3) casein kinase II: Thr30,
Ser76, Thr100, Thr114,
Ser161, Ser181, Thr182,
Ser203, Ser233, Ser407,
Ser412, Thr424, Ser639,
Thr858, Thr1127, Ser1132, and
Ser1198; and 4) ATP-GTP-binding site motif A:
Ala17.
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The expression of mNBC3 was examined in various human tissues by
Northern blot analysis (Fig. 3). A
synthetic 95-bp oligonucleotide probe (nucleotides 1002-1096) specific
for the unique region of mNBC3 was randomly primed and labeled with
[32P]dCTP and [32P]dATP. This probe
hybridized to a ~7.8-kb transcript in mRNA from skeletal muscle
and heart. These results indicate the mNBC3 transcript is
muscle-specific. We have recently determined the chromosomal
localization of the mNBC3 gene by fluorescent in situ hybridization (46). mNBC3 maps to a position that is 19% of the
distance from the centromere to the telomere of chromosome arm 3p, an
area that corresponds to band 3p22. Recently, Olsen and Keating (47)
have studied a family with dilated cardiomyopathy associated with sinus
node dysfunction, supraventricular tachyarrythmias, conduction delay,
and stroke. The disease gene has been mapped to a 30 cM region of
chromosome 3p22-3p25 excluding genes encoding a G-protein (GNA12),
calcium channel (CACNL1A2), sodium channel (SCN5A), and inositol
triphosphate receptor. The finding that mNBC3 maps to this region
suggests that it is a new candidate gene potentially causing familial
dilated cardiomyopathy.

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Fig. 3.
Northern blot analysis of expression of mNBC3
in human tissues. Lane 1, heart; lane 2,
brain; lane 3, placenta; lane 4, lung; lane
5, liver; lane 6, skeletal muscle; lane 7,
kidney; lane 8, pancreas; lane 9, spleen;
lane 10, thymus; lane 11, prostate; lane
12, testis; lane 13, ovary; lane 14, small
intestine; lane 15, colon; lane 16, peripheral
blood leukocytes; lane 17, stomach; lane 18,
thyroid; lane 19, spinal chord; lane 20, lymph
node; lane 21, trachea; lane 22, adrenal gland;
lane 23, bone marrow. Each lane was loaded with ~2 µg of
poly(A)+ human RNA. The multiple tissue Northern blots were
from CLONTECH. The following
32P-labeled mNBC3 probe was used: a synthetic 95-bp
oligonucleotide specific for mNBC3 (nucleotides 1002-1096).
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NBC3 (approved symbol SLC4A7) is the first member of the NBC family in
which the genomic structure has been analyzed (Fig. 4). The gene coding for mNBC3 is composed
of 25 exons and 24 introns and spans ~80 kb. The exon sizes ranged
from 82 to 4123 bp. The large 3'-untranslated region is contained in
exon 25. Intron sizes were not determined. All intron sequences match
known consensus donor and acceptor splice sites. The genomic
organization of the related anion exchange gene family differs in
several respects. There are 20 exons in the AE1 gene and 23 exons in
the AE2 and AE3 genes if alternate exons transcribed from internal
promoters are excluded. For all AE family members the first exon
consists of the 5'-untranslated region, and the second exon contains
translation start site. In the NBC3 gene there are 25 exons, and the
translation start site is contained in the first exon. In NBC3
(SLC4A7), the large 3'-end exon (exon 25) codes for the 3'-terminus of
the polypeptide and the 3'-untranslated region.

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Fig. 4.
Intron/exon boundaries of the human NBC3
(approved symbol SLC4A7) gene. Nucleotide sequences at the
intron/exon boundaries are shown, with exon sequences in
uppercase letters and intron sequences in lowercase
letters. Exons are numbered from left to
right. The length of each exon determined by sequence
analysis is shown.
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We next examined the functional properties of mNBC3 using measurements
of pHi, 22Na, and 86Rb uptake after
injecting the corresponding polyadenylated cRNA into Xenopus
oocytes. Polyadenylated cRNA prepared as described above was injected
into oocytes and allowed to express for at least 72 h. As shown in
Fig. 5, augmented 22Na uptake
was observed in oocytes injected with mNBC3 cRNA. The uptake was
increased by approximately 12 nmol/h/oocyte in cRNA-injected oocytes
(3.44 ± 0.34 nmol/h/oocyte in control oocytes (n = 24) versus 15.3 ± 1.44 nmol/h/oocyte
(n = 32), p < 0.001). 22Na
uptake was not significantly different in oocytes exposed to Cl
-free solutions (15.3 ± 1.44 nmol/h/oocyte
(n = 32) versus 13.8 ± 1.30 nmol/h/oocyte (n = 16) in oocytes exposed to
Cl
-free solutions). Uptake was not significantly affected
by DIDS (1 mM; 16.5 ± 1.56 nmol/h/oocyte
(n = 8)) but was completely inhibited by EIPA (50 µM; 3.28 ± 0.38 nmol/h/oocyte (n = 16), p < 0.02). 86Rb uptake was similar in
control oocytes and cRNA-injected oocytes (controls 1.04 ± 0.04 nmol/h/oocyte (n = 16) versus 2.55 ± 0.04 nmol/h/oocyte (n = 16) in cRNA-injected
oocytes).

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Fig. 5.
Functional expression of mNBC3 in
Xenopus laevis oocytes. 22Na uptake
(expressed in nmol/h/oocyte) in control oocytes or with mNBC3
cRNA-injected oocytes 3-6 days after injection was measured in
chloride-free solutions and in the presence of DIDS (1 mM)
or EIPA (50 µM). Each bar represents the
mean ± S.E. of 8-32 oocytes. 22Na uptake was
significantly increased by mNBC3 cRNA versus control oocytes
(p < 0.001) and inhibited in the presence of 50 µM EIPA (p < 0.02). Furthermore, in
cRNA-injected oocytes 22Na uptake was not affected by
chloride removal or 1 mM DIDS. 86Rb uptake
(expressed as nmol/h/oocyte) was also assessed in control oocytes and
in mNBC3 cRNA-injected oocytes. 86Rb uptake was similar in
both groups.
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Further studies were done measuring pHi transients in control
oocytes and those injected with mNBC3 cRNA (Fig.
6). All studies were performed in the
absence of chloride given the lack of dependence of the
22Na uptake results on the presence of chloride. In
bicarbonate-buffered solutions, resting pHi was 7.14 ± 0.02 (n = 4) in control oocytes and 7.29 ± 0.04 (n = 5) in cRNA-injected oocytes (p < 0.05). After extracellular Na+ was removed in
bicarbonate-buffered solutions, in control oocytes, pHi failed
to decrease (Fig. 6a). In contrast, in the cRNA-injected
oocytes, at a resting pHi of 7.29 ± 0.04 (n = 5), pHi decreased at a rate of
0.14 ± 0.02 pH/min (n = 5), p < 0.001 (Fig. 6b). Further experiments were done to determine
whether K+-induced pHi transients would be present.
As shown in a typical experiment in Fig. 6c, in
bicarbonate-buffered solutions, increasing the external K+
from 2 to 50 mM in cRNA-injected oocytes had no effect on
pHi. These results suggest that 1) mNBC3 is
K+-independent as was also shown by the 86Rb
flux measurements and 2) depolarizing the oocyte membrane potential using high K+ solutions (48) has no measurable effect on
pHi in cRNA-injected oocytes, suggesting that the transport
process is electroneutral. In Hepes buffered-solutions, following
Na+ removal at a resting pHi of ~7.5, pHi
decreased at a rate of
0.17 ± 0.02 pH/min (n = 4) (Fig. 6d). The rate of change of pHi in
Hepes-buffered solutions when pHi had decreased to ~7.3
(resting pHi in bicarbonate-buffered solutions), was
0.15 ± 0.01 pH/min (n = 4). Furthermore, at a minimum pHi of ~6.5, following the readdition of
Na+ in bicarbonate-buffered solutions pHi increased
at a rate of 0.13 ± 0.01 pH/min versus 0.12 ± 0.01 pH/min in Hepes-buffered solutions. Given the difference in
intracellular buffer capacities in Hepes and bicarbonate-containing
solutions, the results suggest that the rate of equivalent base flux is
greater in the presence of bicarbonate in cRNA-injected oocytes. In
separate experiments the effect of DIDS and EIPA inhibition was
studied. In cRNA-injected oocytes, DIDS (1 mM) did not
significantly modify the decrease in pHi induced by
Na+ removal (
0.12 ± 0.01 pH/min (n = 3), p = NS; Fig. 6e). In contrast, EIPA
(50 µM) completely blocked the Na+-induced
pHi transients in cRNA-injected oocytes (0.02 pH/min ± 0.01 pH/min (n = 3), p < 0.001; Fig.
6f). The dependence of mNBC3 transport on external
Na+ is shown in Fig.
7a. In these studies,
Na+ was repeatedly removed to acidify pHi to a
value of ~7.0. Various concentrations of Na+ were added
to determine the external Na+ dependence of the
transporter. The data indicate that the external Km
for external Na+ under these conditions is ~24
mM (Fig. 7b).

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Fig. 6.
Intracellular pH measurements in X. laevis oocytes 5-14 days after injection. Oocytes were
bathed initially in a Hepes-buffered chloride-free solution.
a and b, following the addition of
bicarbonate-buffered chloride-free solutions a steady state was
achieved, and Na+ removal/readdition experiments were
performed in control oocytes (a) and cRNA-injected oocytes
(b). c, increasing the solution K+
from 2 mM to 50 mM had no effect on pHi
in cRNA-injected oocytes. d, Na+
removal/readdition in Hepes-buffered chloride-free solutions in
cRNA-injected oocytes. e, Na+ removal/readdition
in bicarbonate-buffered chloride free solutions in cRNA-injected
oocytes in the presence of 1 mM DIDS. f,
Na+ removal/readdition in bicarbonate-buffered
chloride-free solutions in cRNA-injected oocytes in the presence of 50 µM EIPA.
|
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Fig. 7.
a, dependence of mNBC3 transport on
extracellular Na+. Na+ was repeatedly removed
to acidify pHi to a value of ~7.0. Various concentrations of
Na+ were added when pHi decreased to ~7.0. The
Na+-dependent rate (at each Na+
concentration) was calculated by subtracting the rate of change in
pHi immediately prior to the addition of Na+ (the
acidification rate in zero Na+ at pH ~ 7.0) from the
initial rate of change in pHi following the addition of a
particular concentration of Na+. Once the initial rate of
change in pHi was measured for a given Na+
concentration, pHi was returned more rapidly to ~7.5 by the
addition of 108 mM Na+ followed by another
phase of Na+ removal. b, a Hanes-Woolf analysis
of the data revealed that the external Km for
Na+ is ~24 mM.
|
|
The properties of mNBC3 closely resemble those of
Na+-dependent
HCO3
transport activity previously
described in certain smooth muscle cells. A novel Na+ and
HCO3
-dependent mechanism,
sensitive to EIPA but insensitive to disulfonic stilbenes, has
been described in cultured human internal mammary smooth muscle cells
(15, 21) and cultured rat aortic smooth muscle cells (12).
DIDS-insensitive electroneutral NaHCO3 cotransport has also
been described in guinea pig ureter and guinea pig femoral artery (27,
28).) Stilbene-insensitive NaHCO3 cotransport has also been
demonstrated in cultured oligodendrocytes from mouse spinal cord (49),
rat hippocampal slices (50), and cultured rat cerebellar
oligodendrocytes (51). Previous studies have shown that
H2DIDS reacts covalently with Lys539 and
Lys851 in human AE1 (45). Replacement of the highly
conserved lysine at position 745 by aspartic acid in the highly
conserved putative stilbene-binding motif (K(M/L)XK) in
mNBC3, corresponding to Lys542 in human AE1, may block
negatively charged stilbene disulfonates from binding. The negatively
charged aspartic acid residue at position 745 in mNBC3 may alter the
Ki of these compounds for mNBC3 and account for the
lack of DIDS inhibition of 22Na influx and
Na+-induced pHi changes that we observed.
Electroneutral Na(HCO3)n cotransport has also been
identified in sheep cardiac Purkinje fibers (17), guinea pig
ventricular myocytes (24), the smooth muscle-like cell line BC3H-1
(26), and rat mesenteric vessels (11, 29). However, in these
preparations, the transporter is stilbene-sensitive, suggesting that a
different protein is likely involved.
The novel finding that EIPA inhibits the transport of mNBC3 is of
interest. Further studies are required to determine which residues
react with the EIPA molecule. EIPA has been shown to provide protection
against cardiac ischemia and reperfusion and to significantly reduce
infarction size in rat and rabbit hearts (52-55). The protective
effect of EIPA is not altered by K+ channel blockers or PKC
inhibitors and has been attributed to inhibition of
Na+/H+ exchange (54). The results of the
present study suggest the interesting possibility that inhibition of
mNBC3 may also contribute to the protective effect of EIPA.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DK46976, the Iris and B. Gerald Cantor Foundation, the Max Factor
Family Foundation, the Verna Harrah Foundation, the Richard and Hinda
Rosenthal Foundation, and the Fredericka Taubitz Foundation.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) AF047033.
Supported by Training Grant J891002 from the National Kidney
Foundation of Southern California.
§
To whom correspondence should be addressed: UCLA Div. of
Nephrology, 10833 Le Conte Ave., Rm. 7-155 Factor Bldg., Los Angeles, CA 90095-1689. Tel.: 310-206-6741; Fax: 310-825-6309; E-mail: IKurtz{at}med1.medsch.ucla.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
pHi, intracellular pH;
NBC, sodium bicarbonate cotransporter;
EIPA, 5-(N-ethyl-N-isopropyl)-amiloride;
RACE, rapid
amplification of cDNA ends;
PCR, polymerase chain reaction;
bp, base pair(s);
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid;
BCECF, 2',7'- biscarboxyethyl-5,6-carboxyfluorescein;
mNBC3, muscle
sodium bicarbonate cotransporter, 3;
AE, anion exchanger.
 |
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