1 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Department of Cardiovascular Pharmacology, SmithKline Beecham, Laboratory Pharmaceutiques, 35760 Saint Grégoire, France
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ABSTRACT |
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Our group recently
cloned the electrogenic
Na+-HCO3
cotransporter (NBC) from salamander kidney and later from mammalian
kidney. Here we report cloning an NBC isoform (hhNBC) from a human
heart cDNA library. hhNBC is identical to human renal NBC (hkNBC),
except for the amino terminus, where the first 85 amino acids in hhNBC
replace the first 41 amino acids of hkNBC. About 50% of the amino acid
residues in this unique amino terminus are charged, compared with
~22% for the corresponding 41 residues in hkNBC. Northern blot
analysis, with the use of the unique 5' fragment of hhNBC as a
probe, shows strong expression in pancreas and expression in heart and
brain, although at much lower levels. In
Xenopus oocytes expressing hhNBC,
adding 1.5% CO2/10 mM
HCO
3 hyperpolarizes the membrane and
causes a rapid fall in intracellular pH
(pHi), followed by a
pHi recovery. Subsequent removal
of Na+ causes a depolarization and
a reduced rate of pHi recovery.
Removal of Cl
from the bath
does not affect the pHi recovery.
The stilbene derivative DIDS (200 µM) greatly reduces the
hyperpolarization caused by adding
CO2/HCO
3.
In oocytes expressing hkNBC, the effects of adding
CO2/HCO
3
and then removing Na+ were similar
to those observed in oocytes expressing hhNBC. We conclude that hhNBC
is an electrogenic
Na+-HCO
3
cotransporter and that hkNBC is also electrogenic.
intracellular pH; acid extruder; oocytes; bicarbonate
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INTRODUCTION |
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BECAUSE ALMOST EVERY cellular process is sensitive to
changes in intracellular pH
(pHi), the regulation of
pHi is important for normal cell
function (9). Steady-state pHi is
determined by the balance between acid extruders (i.e., transporters
that move acid out of cells or alkali into cells) and acid loaders. Acid extruders include
Na+/H+
exchangers (the NHEs) (6), various
H+ pumps, and the
Na+-driven
Cl/HCO
3
exchanger, which is present in invertebrate cells (34, 39) as well as
cultured fibroblasts (28), cultured mesangial cells (13), and central
nervous system neurons (37). Acid loaders include
Cl
/HCO
3
exchangers (the AEs) (3). In addition, a
K+-HCO
3
cotransporter, which would function as an acid loader, has been
reported in squid giant axons (21, 22).
Electrogenic
Na+-HCO3
cotransporters (10) can function either as acid extruders or acid
loaders. In renal proximal tubules, the electrogenic
Na+-HCO
3
cotransporter has a Na+ to
HCO
3 stoichiometry of 1:3 and moves
HCO
3 out of the cell (4, 12); i.e.,
the cotransporter functions as an acid loader. In glial cells (8, 19),
however, the electrogenic Na+-HCO
3
cotransporter appears to have a stoichiometry of 1:2 and moves
HCO
3 into the cell (i.e., it is an
acid extruder). Finally, electroneutral
Na+-HCO
3
cotransporters have been reported in vascular smooth muscle cells (1)
and in the heart (18, 27). These transporters would have a
stoichiometry of 1:1 and mediate HCO
3 uptake. On the other hand, there is also a report that the cardiac cotransporter is electrogenic (15), presumably with a stoichiometry of
1:2.
Romero and co-workers (32) expression cloned the cDNA encoding an
electrogenic
Na+-HCO3
cotransporter (NBC) in the kidney of the tiger salamander
Ambystoma tigrinum. Others
subsequently cloned similar cDNAs from mammalian kidneys (14, 33). The
open reading frame of the NBC cDNA is ~3.2 kb in length,
corresponding to 1,035 amino acids. The amino acid sequences of the NBC
are ~30% identical to those of the AEs, and these two families of
HCO
3 transporters have very similar
hydropathy profiles. Expression in
Xenopus oocytes of
Ambystoma NBC (aNBC) and rat kidney
NBC (rkNBC) confirms that both clones encode electrogenic
Na+-HCO
3
cotransporters that are blocked by the stilbene DIDS (32, 33).
These two NBCs have similar apparent Km values for
extracellular HCO
3, ~10 mM (20). Antibodies specific for rkNBC localize the cotransporter to the basolateral membrane of the renal proximal tubules of rat and rabbit
(36).
As noted above, unlike the
Na+-HCO3
cotransporters identified in kidney and glial cells, the cardiac
Na+-HCO
3
cotransporter described by one group is electroneutral (18, 27).
Therefore, we decided to attempt to clone the cardiac NBC by screening
a human heart cDNA library with rkNBC. We identified a cDNA from human
heart (hhNBC) that is identical to the one from human kidney (hkNBC),
except that the 85 amino-terminal amino acids in hkNBC are replaced by
41 amino-terminal amino acids in hhNBC. While our paper was being submitted, a paper appeared by Abuladze et al. (2), reporting the
cloning of a cDNA with an identical sequence from pancreas. We found
that both hhNBC and hkNBC, when expressed in
Xenopus oocytes, are electrogenic.
This work is the first demonstration that a human NBC is electrogenic.
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METHODS |
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Cloning hhNBC. The open reading frame
of rkNBC (GenBank Accession no. AF004017) was cut into three pieces
with BstX I, radiolabeled with
[-32P]dCTP by
random priming, pooled, and used to screen a human heart
-ZAPII cDNA
library (Stratagene, La Jolla, CA). The titrated plaques (0.64 × 106) were plated and
blotted on nitrocellulose filters. Hybridization was performed in 0.5 M
sodium phosphate, 7% SDS, 1% BSA at 65°C overnight. The membranes
were washed at 50°C in 1× standard sodium citrate (SSC; 150 mM NaCl/15 mM sodium citrate)/0.1% SDS and then autoradiographed. We
then performed a PCR with pBluescript vector primers adjacent to the
cloning sites to amplify the cDNA inserts from primary positive
plaques.1
PCR products were separated on 1.0% agarose gel, transferred via
capillary to a Hybond membrane (Clontech, Palo Alto, CA), and
separately hybridized with each probe used for the screening library.
Positive plaques were plated for a second screening with the same
probes. We isolated four positive phage clones and excised the inserts
by rescuing the plasmids. Sequencing was performed by the Keck
Sequencing Center at Yale University. We analyzed the sequence using
DNAsis (Hitachi Software, San Bruno, CA).
The above approach yielded nucleotide sequence data from both the 5' end and the 3' end of the putative clone. However, we were missing the middle of the putative clone. We performed RT-PCR with human heart poly(A)+ RNA Clontech (catalog no. 6533-1; a pool of whole heart from Caucasians ages 20-78 who had died of trauma) to obtain the missing middle portion. The upstream primer sequence was 5'-CCG GAG AAG GAC CAG CTG AAG-3', corresponding to a region near the 3' end of clone 15.1, which contains the 5' fragment of hhNBC. The downstream primer sequence was 5'-ATC AGA GTA GGG AGG AAA GAG-3', corresponding to a sequence at the TAG stop codon of clone 4.1, which contains the 3' fragment of hhNBC. The result of the PCR2 was a DNA fragment that represented the middle and 3' end of the putative clone, which we sequenced. We ligated this PCR product to clone 15.1 (the 5' end) at the Pml I site, to obtain the putative full-length clone of hhNBC (GenBank Accession no. AF069510). We confirmed the sequence of this full-length clone near the ligation site.
To confirm that clones 15.1 and 4.1 represent, respectively, the 5' and 3' ends of the same clone, we ran a PCR between the ATG start codon of clone 15.1 and the TAG stop codon of clone 4.1. The result was a 3.2-kb PCR product, the sequence of which corresponded to the ligation product discussed above.
Cloning hkNBC. The 5' fragment
of hkNBC cDNA was isolated from human pancreas
poly(A)+ RNA by RT-PCR with
primers designed on the basis of the published hkNBC sequence (14). The
upstream primer was 5'-TTG GGA GGC TTA GCA GGA AAG ATG
TCC-3' (21 to +6), and the downstream primer was
5'-TTT CTT GGT TTG ATG CCG GTG CTT CCG-3'(+496 to +522).
PCR was performed as described above,2 and the final single
PCR product was cloned into the pCRII vector (Invitrogen, Carlsbad,
CA). To obtain a full-length hkNBC, we ligated the above PCR product to
hhNBC at the Afl II site. The inserted
DNA fragment, as well as the ligation site, were sequenced to confirm
that the ligation product is identical to the published hkNBC sequence
(GenBank accession no. AF007216).
Northern blots. A human
multiple-tissue Northern blot (catalog no. 7760-1) was purchased
from Clontech. A 32P-labeled,
random-hexamer-primed cDNA probe (GIBCO BRL, Gaithersburg, MD) was made
from the unique 5' region of the hhNBC (7-271 bp of the
coding region of hhNBC). Hybridization was performed in the ExpressHyb
hybridization buffer (Clontech) at 68°C for 2 h, at a probe
concentration of 0.83 × 106
counts · min1 · ml
1.
The membrane was washed at 37°C in 2× SSC/0.1% SDS for 40 min and then at 50°C in 0.1× SSC/0.1% SDS for 1.5 h. The
membrane was exposed to Kodak X-Omat film (Kodak, Rochester, NY), which was developed 1 day later for the detection of high-intensity signals.
For the detection of low-intensity signals, the film was
autoradiographed for 7 days.
Membrane isolation and Western blot analysis. Oocyte plasma membranes were isolated as described by Preston et al. (30), with some modification. Groups of four oocytes were washed with the PBS and homogenized in 0.5 ml of fresh hypotonic lysis buffer (7.5 mM potassium phosphate, pH 7.4, 1 mM EDTA, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml leupeptin). The cellular debris was removed by centrifuging at 810 g for 5 min. For the collection of membranes, the supernatant was then centrifuged at 15,000 g for 30 min at 4°C. The pellets were gently washed with the lysis buffer and were dissolved in 40 µl of a sample-loading buffer containing 2% SDS. The samples were separated on a 7.5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Blots were preincubated for 1 h in blocking buffer containing 0.2% I-block (Tropix, Bedford, MA) and 0.05% Tween 20 in Tris-buffered saline (TBS; 50 mM Tris, pH 7.4; 150 mM NaCl) and then were incubated with an antibody specific to the carboxy terminus of rkNBC (1:500 dilution) (36). After several washes with TBS containing 0.05% Tween 20, blots were incubated with anti-rabbit IgG conjugated to horseradish peroxidase (HRP; Sigma, St. Louis, MO) for 1 h (1:2,000 dilution in blocking buffer). Blots were washed and developed by HRP/hydrogen peroxide-catalyzed oxidation of luminol under alkaline conditions (Pierce, Rockford, IL).
pHi and membrane potential measurements in oocytes. The hhNBC insert in the plasmid described above contains 44 bp of the 5'-untranslated region (UTR), as well as the entire coding region of hhNBC. The insert was digested with EcoR I and ligated into pGH19, an oocyte expression vector (40). The resulting construct and the hkNBC construct in pGH19 were linearized with Not I before transcription and then in vitro transcribed with the mMessage mMachine kit (Ambion, Austin, TX) using T7 RNA polymerase. The ratio of cap analog to GTP was decreased by increasing the final GTP concentration to 3 mM, thereby maximizing the synthesis of full-length transcripts. Oocytes of X. laevis were obtained as described by Romero et al. (32). Defolliculated oocytes (stages V and VI) were injected with 50 nl of RNA (0.5 mg/ml) or water and incubated in OR3 buffer (50% Leibovitz L-15 media with L-glutamine, 5 mM HEPES, pH 7.5) supplemented with 5 U/ml penicillin-streptomycin (33). Injected oocytes were maintained for 3-7 days at 18°C before use.
pH-sensitive microelectrodes were made from borosilicate glass capillaries (2.0-mm outer diameter; Warner Instrument, Hamden, CT) that were pulled on a microelectrode puller (model P-97; Sutter, Novato, CA), dried in an oven at 200°C for at least 2 h, and silanized in the vapor of bis(dimethylamino)-dimethyl silane (Fluka, Ronkonkoma, NY) in a closed vessel. The electrode tip was then filled with hydrogen ionophore 1 cocktail B (Fluka) and backfilled with a pH 7.0 phosphate buffer (38). The electrode was connected to a high-impedance electrometer (model FD-223; WPI, Sarasota, FL) and calibrated in standard solutions of pH 6 and 8. The slope was generally 55-57 mV/pH unit. Physiological solutions. Table 1 summarizes the composition of the solutions used in the electrophysiological experiments. Choline, gluconate, and DIDS were all obtained from Sigma.
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RESULTS |
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Sequence analysis of hhNBC cDNA. By
screening a human heart cDNA library and subsequently employing a PCR
approach, we cloned a full-length cDNA, hhNBC. The open reading frame
of hhNBC encodes a protein of 1,079 amino acids, with a predicted
molecular mass of 121 kDa. As shown in Fig.
1A, this
clone has a 5' coding region that is different from that of the
previously published human kidney clone, hkNBC (14). The first 255 bp
(i.e., 85 amino acids) of the coding region of hhNBC replaces the first
123 bp (41 amino acids) of hkNBC (Fig.
1B). In addition, hhNBC has a
phenylalanine at amino acid position 255 instead of the serine reported
for hkNBC. We obtained the portion of hhNBC that encodes the F255 by
screening a cDNA library. Except for these differences, hhNBC is
identical to hkNBC, including the 3'-UTR.
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The unique amino terminus of hhNBC consists mainly of charged amino acids, such as aspartate, glutamate, histidine, lysine, and arginine. These charged residues comprise ~50% of the unique 85 amino acids in hhNBC, considerably higher than the ~22% of residues that are charged among the corresponding 41 amino acids of hkNBC. Thus the amino-terminal region of hhNBC is very hydrophilic (Fig. 1C). This region is reminiscent of AE2 and AE3, which also have large, amino-terminal domains consisting of highly charged amino acids. There is 20-30% sequence similarity among the amino-terminal charged domains of hhNBC, AE2, and AE3, but not AE1. Finally, the unique amino terminus of hhNBC contains a second putative consensus protein kinase A (PKA) phosphorylation at amino acid position 49, in addition to a conserved PKA site at amino acid position 1023.
Tissue distribution of hhNBC mRNA.
Using the unique 5' region (nucleotide positions 7-271 of
the coding regions) of hhNBC as a probe, we performed a high-stringency
Northern blot analysis in various human tissues. After a 1-day
exposure, the probe showed substantial hybridization to an ~9-kb
transcript in pancreas but not to any of the other tissues
on the blot (Fig.
2A).
Because we expected to detect hhNBC mRNA in the heart, having cloned
hhNBC from human heart tissues, we exposed the film for 7 days to
determine whether the signal could be detected in heart. We found that
hhNBC mRNA is strongly expressed in pancreas and weakly expressed in heart and brain (Fig. 2B). hhNBC
mRNA is undetectable in the kidney. It is interesting to note that, in
Ambystoma (i.e., tiger salamander) tissues, Romero et al. (32) did not detect signals in pancreas, even
though they probed with a full-length cDNA.
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Abuladze et al. (2) probed a Northern blot, obtained from the same source as ours, with part of the unique 5' region of hhNBC. They detected strong expression in pancreas and moderate expression in brain, but not heart. This disparity may be due to the probe specificity and/or stringency. We used nearly the entire unique nucleotide (7-271) as a probe, whereas Abuladze et al. used a synthetic oligonucleotide probe corresponding to nucleotides 118-212 (95 bp).
Immunoblot experiments on expression of hhNBC in
oocytes. To study the functional
properties of hhNBC, we expressed hhNBC in
Xenopus oocytes by injecting them with
hhNBC cRNA. We first tested whether the open reading frame of hhNBC
could be translated into protein with the expected molecular mass. We
used a rabbit reticulocyte system (without microsomes) to perform an in
vitro translation of hhNBC cRNA and observed a 120-kDa product (not shown), as expected from the deduced amino acid sequence of hhNBC cDNA,
which predicts a molecular mass of 121 kDa. We then injected the same
cRNA into the oocytes and tested the expression of hhNBC by Western
blot analysis, using an antibody against the carboxy terminus of rkNBC
(36). Because the deduced amino acid sequences of hhNBC and rkNBC are
~98% identical at their carboxy termini, we expected the antibody
against rkNBC to react with the hhNBC protein. Figure
3 shows that the antibody reacted with a
130-kDa protein in oocytes injected with rkNBC cRNA
(lane 3). The same antibody also
recognized an ~135-kDa band in oocytes injected with hhNBC cRNA
(lane 2). The slightly greater
molecular mass is expected because the 85 amino acids at the amino
terminus of hhNBC replace the 41 amino acids at the amino terminus of
rkNBC. In addition to the major band, the antibody detects a smaller band (~130 kDa), which may be the result of proteolysis or a less extensive glycosylation. Water-injected control oocytes showed no
immunoreactivity (lane 1).
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Functional properties of hhNBC expressed in Xenopus
oocytes. To study the functional properties of
hhNBC, we used microelectrodes to monitor
pHi and membrane potential
(Vm) in control
oocytes as well as in oocytes expressing hhNBC. Figure
4A
illustrates the effects of exposing a water-injected oocyte to a
solution buffered with 1.5%
CO2/10 mM
HCO3. Figure
4B is a similar experiment, but on an
oocyte expressing hhNBC. In both oocytes, applying
CO2/HCO
3
caused a decrease in pHi. In the
case of the control oocyte, pHi
fell rapidly and by a relatively large amount and then recovered very
slowly. In the case of the hhNBC oocyte, the
pHi decrease was smaller and slower. Moreover, pHi recovered
rather rapidly. Thus the pHi
profile in the hhNBC oocyte is consistent with an extremely robust
expression of the cotransporter, which not only produced a
pHi recovery but also blunted the
initial CO2-induced acidification.
Applying
CO2/HCO
3 also caused characteristic changes in
Vm, a slowly
developing depolarization in the water-injected oocyte but a very large
and rapid hyperpolarization in the hhNBC oocyte. The hyperpolarization wanes in the case of the hhNBC oocyte because, for three reasons, the hhNBC-mediated inward transport of
Na+ and
HCO
3 gradually slows down:
1) CO2 diffuses into the cell and produces HCO
3,
2) hhNBC actively transports
HCO
3 into the cell, and
3) hhNBC transports
Na+ into the oocyte. In each
case, an hhNBC substrate slowly builds up inside the cell,
slowing inward transport. Note, however, that Vm is always more negative in the presence
than in the absence of
CO2/HCO
3.
Thus, as previously shown for aNBC (32) and rkNBC (33), the direction
of electrogenic
Na+-HCO
3
cotransport by hhNBC is inward in oocytes.
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To test whether the expressed transporter is coupled to
Na+, we next removed
Na+ from the bath in the continued
presence of
CO2/HCO3. Removing extracellular Na+
(replacing Na+ with choline) had
little effect on pHi in the
control oocyte (Fig. 4A) but
produced a pHi decrease in the
hhNBC oocyte. This intracellular acidification is consistent with the
"reversal" of HCO
3 transport
(i.e., Na+-coupled
HCO
3 efflux rather than influx).
Removing extracellular Na+ also
produced characteristic
Vm changes in the
two oocytes. In the control oocyte,
Na+ removal elicited a small
hyperpolarization, reflecting a small Na+ conductance, as previously
observed (32, 33). In the hhNBC oocyte, however,
Na+ removal produced a large
depolarization (from
80 to
10 mV), consistent with the
electrogenic efflux of Na+ with
more than one HCO
3. Returning
Na+ to the outside of the oocytes
reversed the pHi and
Vm changes.
To test the unlikely possibility that hhNBC encodes a
Na+-driven
Cl/HCO
3
exchanger, we removed Cl
from the bath (replacing Cl
with gluconate). As shown in Fig. 4C,
removing extracellular Cl
in the presence of
CO2/HCO
3
had no significant effect on either the rate of
pHi recovery or the membrane
potential (n = 4). If the
pHi recovery had been due to a
Na+-driven
Cl
/HCO
3
exchanger, then Cl
removal
should have speeded up the pHi
recovery, at least transiently. Thus our results indicate that hhNBC
does not transport Cl
,
although we cannot rule out the unlikely possibility that hhNBC may
require intracellular Cl
.
Finally, we addressed the question of whether this electrogenic,
Na+-dependent (and
Cl-independent)
HCO
3 cotransport process can be blocked by the stilbene derivative DIDS, which is known to inhibit electrogenic
Na+-HCO
3
cotransport in other systems (11). Before exposing the oocyte to DIDS,
we briefly pulsed the cell with
CO2/HCO
3 and then returned it to the
CO2/HCO
3-free
ND96 media (arrow in Fig. 4D). This
brief
CO2/HCO
3 pulse produced a hyperpolarization of nearly 60 mV in this experiment. We then applied 200 µM DIDS in the absence of
CO2/HCO
3. In the continued presence of DIDS, the second exposure to
CO2/HCO
3 elicited a hyperpolarization of >20 mV. Three other similar
experiments showed that 200 µM DIDS blocked, on average, 70 ± 4%
of the voltage change elicited by
CO2/HCO
3
(P < 0.01). Figure 4D also shows that DIDS failed to
block completely the depolarization and acidification elicited by
removing extracellular Na+. In
contrast, 200 µM DIDS completely blocks aNBC expressed in oocytes
(31, 32). Not shown are experiments in which we found that 500 µM
DIDS reduced the hyperpolarization by ~90%
(n = 3).
Effect of substituting the amino terminus of hkNBC for
the amino terminus of hhNBC. Our functional studies
described above indicate that, despite its unique amino terminus, hhNBC
is an electrogenic
Na+-HCO3
cotransporter, similar to rat kidney NBC or salamander kidney NBC.
However, the electrogenicity had yet to be examined for human kidney
NBC, which is nearly identical to hhNBC, except for a different amino
terminus. To address the functional impact of the charged amino
terminus of hhNBC, we therefore constructed hkNBC by performing RT-PCR
with primers specific to the amino terminus of hkNBC and replacing the
unique amino terminus of the hhNBC cDNA with the one for hkNBC. We then
expressed the hkNBC in oocytes and monitored the
pHi and
Vm as described above.
Figure 5 shows that applying
CO2/HCO3
caused an acidification, followed by a
pHi recovery. The application of
CO2/HCO
3
also causes a rapid hyperpolarization that slowly wanes
(n = 5), as in hhNBC-expressing
oocytes. Removing extracellular
Na+ (replacing
Na+ with choline) produced an
intracellular acidification, as well as a rapid depolarization. The
pHi and
Vm effects of
adding
CO2/HCO
3 and removing Na+ were fully
reversible. Thus, as is the case for the other NBCs, hkNBC is
electrogenic.
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We noticed that the
CO2/HCO3
pulse generally produced a smaller hyperpolarization in
hkNBC-expressing oocytes than hhNBC-expressing oocytes. hhNBC and hkNBC
differ in the nucleotide sequence surrounding the initiator methionine.
The hhNBC cDNA has a perfect Kozak sequence
(A
G),
whereas hkNBC cDNA has an imperfect sequence
(A
T). The highly conserved G in position +4 immediately following the ATG
codon in hhNBC is critically important for halting the scanning 40S
ribosomal subunit and thus to initiate translation (26). Thus hhNBC and
hkNBC may be translated with different efficiencies.
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DISCUSSION |
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Cloning of hhNBC from human heart. In
this study, we report the cloning of an NBC-related cDNA from human
heart tissues. Studies on pHi
regulation of heart cells have provided conflicting evidence for both
an electroneutral
Na+-HCO3
cotransporter (18, 27) and an electrogenic Na+-HCO
3
cotransporter (15). The evidence for the electroneutral cotransporter
was obtained in guinea pig myocytes and sheep Purkinje fibers, whereas
the data supporting the presence of an electrogenic
Na+-HCO
3
cotransporter was obtained in cat myocardium. Thus it is possible that
the conflicting conclusions of the two groups reflect a species
difference. Another possibility is that both transporters are present
in the same tissue.
We cloned hhNBC from heart tissue of humans. hhNBC has a unique
amino-terminal domain, which consists mainly of charged or polar amino
acid residues, and is definitely electrogenic. Because there are no
functional studies of the
Na+-HCO3
cotransporter in human cardiac tissue, it is possible that hhNBC
represents the (or at least one of the) Na+-HCO
3
cotransporters in human heart tissue. A question that arises is why the
level of mRNA is apparently so low in cardiac tissue.
One possibility is that the heart mRNA used in our Northern blot was partially degraded. Another possibility is that hhNBC mRNA levels are indeed low in human heart. However, even low mRNA levels may be sufficient to maintain high levels of the hhNBC protein. On the other hand, even if the hhNBC protein levels are low, the activity of each hhNBC molecule may be sufficiently high to produce a high functional level of hhNBC activity in human cardiac cells. Yet a third possibility is that hhNBC is expressed at high levels, but only in specialized cell types (e.g., endothelium, vascular smooth muscle cells, sinoatrial nodal cells). Finally, it is possible that hhNBC is normally expressed at relatively low levels but is upregulated in response to certain stimuli (e.g., training, hypoxia).
hhNBC is an isoform of electrogenic renal NBC. Except for the first 85 amino acid residues at the cytoplasmic amino terminus of hhNBC, hhNBC and hkNBC are identical at the amino acid level. It is likely that hhNBC and hkNBC represent alternative splice products. Supporting this hypothesis is the observation that the partial fragment (1.6 kb) of the 3'-UTR of hhNBC is identical to the corresponding region of hkNBC (data not shown). The UTRs are under less selective pressure during evolution and thus are more prone to mutations. If hhNBC and hkNBC originate from separate genes, we should expect sequence variations between hhNBC and hkNBC in the 3'-UTR, which is not the case.
Abuladze et al. (2) reported a cDNA from pancreas (hpNBC) that, in its coding region, has a sequence that is identical to that of hhNBC. Interestingly, the 5'-UTR of hhNBC is different from that reported for hpNBC. One possible explanation for this difference is that hhNBC and hpNBC are encoded by the same gene but have different tissue-specific 5'-UTRs and thus different regulation at the mRNA level. There is evidence that alternatively spliced 5'-UTRs can play a role in posttranscriptional regulation in other genes, including the N-methyl-D-aspartate receptor subunit 2 (25, 35), human erythrocyte protein 4.1 (7), human inducible nitric oxide synthase (17), and the excitatory amino acid transporter EAAT2 (29). The genomic structure(s) of NBC should clarify this issue. It is interesting to note that the 5'-UTR of hpNBC is 100% identical to the known 29 bp of the 5'-UTR of the NBC cloned from mouse pancreas (GenBank accession no. AF020195, cited in Ref. 2). Moreover, the 5'-UTR of hpNBC is 88% identical to the 5'-UTR of a rat NBC-related cDNA (accession no. AI144995).
In the present study, we report that both hhNBC and hkNBC are
electrogenic. Because both cotransporters are electrogenic, despite the
differences in their amino termini, our results indicate that different
regions of the respective amino termini are not responsible for the
electrogenicity of NBC. However, hhNBC appears to be less sensitive to
DIDS than Ambystoma
Na+-HCO3
cotransport (31, 32) or rkNBC (M. F. Romero, unpublished results). This
result suggests that the amino terminus, which is believed to be
cytoplasmic, may be able to influence the binding of DIDS to the
extracellular surface.
Electrogenicity of hkNBC. Burnham et
al. (14) cloned hkNBC from human kidney and verified that
it cotransports Na+ and
HCO3 and that it is blocked by DIDS.
We have now demonstrated that hkNBC, like the renal NBC clones from salamander and rat, is electrogenic. Our results on hhNBC and hkNBC
thus provide the first molecular evidence for the existence of an
electrogenic
Na+-HCO
3
cotransporter from a human source. Moreover, our data demonstrate that
more than one variant of an electrogenic Na+-HCO
3
cotransporter exists in humans.
Possible physiological function of
hhNBC. We found that the unique amino terminus of
hhNBC, which encodes 85 amino acids, is strongly expressed in the
pancreas, as shown by Northern blot analysis. Indeed, Abuladze et al.
cloned from the pancreas a cDNA that is identical to hhNBC (2). In the
amino terminus of hkNBC, 41 amino acids replace the 85 amino acids of
hhNBC. What might be the significance for the pancreas of the unique
amino terminus of hhNBC? The pancreatic ducts rapidly secrete
HCO3 into the duct lumen, producing a
luminal HCO
3 concentration that is
fivefold higher than the HCO
3 concentration in the blood plasma. This
HCO
3 secretion neutralizes a large
portion of the gastric acid that reaches the duodenum. Physiological
experiments have shown that pancreatic duct cells have a
Na+-dependent
HCO
3 transporter at the basolateral membrane that mediates net HCO
3 uptake
into the duct cells (23, 24). This pancreatic transporter differs functionally from hkNBC in at least one crucial respect: the renal cotransporter mediates net HCO
3
efflux, whereas the pancreatic cotransporter mediates net
HCO
3 influx. The difference between
net HCO
3 efflux (kidney) and influx
(pancreas) could be dictated simply by the ion and voltage gradients in
the respective cell type or by differences in posttranslational
modification. However, it is possible that the unique 85 amino acids of
hhNBC change the stoichiometry of the cotransporter from 1:3 to 1:2.
Another possible role for the unique 85 amino acids at the amino
terminus of hhNBC is suggested by the observation that these 85 amino
acids include a novel consensus phosphorylation site for PKA. Both
hhNBC and hkNBC have consensus PKA sites near the carboxy terminus, but
only hhNBC has such a site near the amino terminus. It is interesting
to recall that secretin strongly stimulates both net
HCO3 secretion by pancreatic ducts (5)
as well as HCO
3 uptake across the
basolateral membrane via the putative pancreatic NBC (23). Secretin
interacts with specific membrane receptors on pancreatic duct cells and activates the cAMP/PKA cascade. Thus the presence of a second PKA site
in the amino terminus of hhNBC could be important for activation of
HCO
3 transport in the pancreas by secretin.
In summary, an NBC isoform was cloned from human heart tissues (hhNBC).
The hhNBC clone, when expressed in
Xenopus oocytes, reveals physiological
properties similar to the renal electrogenic Na+-HCO3 transporter.
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ACKNOWLEDGEMENTS |
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We thank Duncan Wong for computer programming support and Dr. Gritchenko for technical support.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30344. For part of the time, I. Choi was supported by a postdoctoral fellowship from the American Heart Association. M. F. Romero was supported by a National Research Service Award (DK-09342) and a grant from the American Heart Association.
Portions of this work have been published in preliminary form (16).
Present address of M. F. Romero: Dept. of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 2119 Abington Rd., Cleveland, OH 44106-4790.
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 We used 35 cycles of PCR, with denaturation at 94°C for 1 min, annealing and extension at 68°C for 3 min, and 7 min at 68°C for the last extension.
2 In the first 10 PCR cycles, we denatured at 94°C for 30 s, annealed at 65°C for 1 min, and extended at 68°C for 2 min. The second 20 cycles were identical, except that we lengthened the duration of the extension by 20 s in each cycle. After the 30th cycle, we allowed an additional 7 min for extension at 68°C.
Address for reprint requests: I. Choi, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520 (E-mail: inyeong.choi{at}qm.yale.edu).
Received 8 July 1998; accepted in final form 17 November 1998.
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