(Received for publication, April 24, 1997, and in revised form, June 3, 1997)
From the Departments of Internal Medicine and
§ Molecular Genetics, Biochemistry, and Microbiology,
University of Cincinnati School of Medicine, Cincinnati, Ohio
45267-0585 and the ¶ Veterans Affairs Medical Center,
Cincinnati, Ohio 45267
Several modes of HCO3
transport occur in the kidney, including
Na+-independent Cl/HCO3
exchange
(mediated by the AE family of
Cl
/HCO3
exchangers),
sodium-dependent Cl
/HCO3
exchange, and Na+:HCO3
cotransport. The functional similarities between the
Na+-coupled HCO3
transporters and
the AE isoforms (i.e. transport of
HCO3
and sensitivity to inhibition by
4,4
-diisothiocyanatostilbene-2,2
-disulfonic acid) suggested a
strategy for cloning the other transporters based on structural
similarity with the AE family. An expressed sequence tag encoding part
of a protein that is related to the known anion exchangers was
identified in the GenBankTM expressed sequence tag data base and used
to design an oligonucleotide probe. This probe was used to screen a
human kidney cDNA library. Several clones were identified,
isolated, and sequenced. Two overlapping cDNA clones were spliced
together to form a 7.6-kilobase cDNA that contained the entire
coding region of a novel protein. Based on the deduced amino acid
sequence, the cDNA encodes a protein with a
Mr of 116,040. The protein has 29% identity
with human brain AE3. Northern blot analysis reveals that the
7.6-kilobase mRNA is highly expressed in kidney and pancreas, with
detectable levels in brain. Functional studies in transiently
transfected HEK-293 cells demonstrate that the cloned transporter
mediates Na+:HCO3
cotransport.
More than 85% of the filtered load of
HCO3 is reabsorbed in the proximal
tubule of the kidney (1, 2). This transepithelial flux is accomplished
predominantly via a luminal membrane Na+/H+
exchanger and a basolateral
Na+:HCO3
cotransporter
(1-5). The Na+:HCO3
cotransporter (3-5) mediates an electrogenic process with an apparent
stoichiometry of 3 HCO3
ions per
Na+ ion (6-8). In addition to the
Na+:HCO3
cotransporter and
Cl
/HCO3
exchangers, a
Na+-dependent
Cl
/HCO3
exchange system
has been described in the kidney, which exchanges Cl
for
Na+ and HCO3
(4). The
Na+:HCO3
cotransporter,
the Na+-dependent
Cl
/HCO3
exchanger, and
the electroneutral
Cl
/HCO3
exchangers (AE
isoforms) share similar pharmacological and functional properties,
including sensitivity to inhibition by disulfonic stilbenes and
transport of HCO3
(3-5, 7-11). This
suggested that the Na+-dependent
HCO3
transporters might be related to
the AE family of Cl
/HCO3
exchangers. In fact during the revision of this manuscript the sequence
of an amphibian Na+:HCO3
cotransporter (NBC)1 that is
related to the AE family was
reported2 (12). We report
here the isolation and functional characterization of a human kidney
NBC using a cloning strategy based on its similarity to the known AE
isoforms.
The GenBankTM nonredundant EST data base was queried against the three known anion exchangers AE1, AE2, and AE3. A sequence with a score of 172, from a human pancreatic islet cell line (GenBankTM accession number W39298) was a close, but not an identical, match to rat AE3.
A sense-stranded oligonucleotide, 5-AGG GAG CAA AGA GTC ACT GGA
ACC-3
, from W39298 was synthesized, biotinylated and used in the
GeneTrapperTM cDNA positive selection system (Life Technologies,
Inc.) to screen a SuperScriptTM human (38-year-old Caucasian male)
kidney cDNA library (Life Technologies, Inc.) directionally cloned
in pCMV·SPORT1. The GeneTrapperTM system uses streptavidin-linked
magnetic beads to enrich for sequences complementary to the
biotinylated oligonucleotide. After plating, the library was screened
by hybridization with 32P-end-labeled oligonucleotide, and
21 positive clones were selected.
The three largest unique clones were chosen for sequencing. Two of the
three clones (one of approximately 5 kb and another of approximately
1.6 kb) contained the EST sequence (W39298) and also contained
sequences of perfect homology to each other. An open reading frame
analysis suggested that the entire coding region was not contained
between the two clones. Therefore, a second oligonucleotide was
synthesized (which was 5 to the EST sequence) for a second round of
GeneTrapperTM cDNA selection. The sequence of the second
oligonucleotide was: 5
-CAA GCC AAC AAG TCC AAA CCG AGG-3
. A
polymerase chain reaction analysis was performed using the T7 and SP6
primers to reveal the size of the cDNA inserts of 48 randomly
chosen clones. The largest (with a 3.5-kb insert) overlapped the 5-kb
clone by 828 base pairs, included the EST clone W39298, and contained
the remainder of the open reading frame (as well as 149 base pairs of
the 5
-noncoding region). Two Sse8387I restriction sites,
one in the region of overlap and another in the polylinker, allowed the
construction of the full-length Na+:HCO3
cotransporter
clone.
HEK-293 cells, grown for 24 h on fibronectin-coated glass coverslips (in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) were transfected with 8 µg of the full-length cDNA construct (in the cloning/expression vector pCMV·SPORT1) by calcium phosphate-DNA coprecipitation (13), and cells were studied 44-52 h after transfection.
Intracellular pH MeasurementChanges in intracellular pH
(pHi) were monitored in cells using BCECF (14, 15). HEK-293
cells were loaded with 5 µM BCECF and monitored for
changes in pHi using a Delta Scan dual excitation
spectrofluorometer (14, 15). The pHi calibration was generated
using the KCl/nigericin technique. To test for the presence of
Na+:HCO3 cotransport,
cells were acid loaded by NH4+-prepulse (16) using
solution B and subsequently, solution A (Table I). The initial rate of
pHi recovery was monitored in the presence of 1 mM
amiloride in a solution containing both HCO3
and Na+ (solution C).
To test for the presence of
Cl
/HCO3
exchange, cells
were switched from a chloride-containing solution (solution C) to a
chloride-free solution (solution D) and examined for cell
alkalinization (17). All solutions were gassed with 95%
O2, 5% CO2.
|
Fig. 1 shows the nucleotide sequence
and conceptual translation of the open reading frame of the 7.6-kb
Na+:HCO3 cotransporter
cDNA. The nucleotide sequence encodes a protein of 116 kDa. Three
potential N-linked glycosylation sites are found at amino
acid numbers 592, 597, and 617. A single cAMP-dependent protein kinase phosphorylation site (Lys-Lys-Gly-Ser) occurs at amino
acid number 979. Protein kinase C and casein kinase II phosphorylation sites are indicated in Fig. 1. 149 nucleotides precede the coding region on the 5
-end and are also indicated in Fig. 1. The 4.3-kb 3
-untranslated region is not shown, but is included in the sequence as
submitted to GenBankTM.
An amino acid comparison between the
Na+:HCO3 cotransporter and
AE3 illustrates significant similarity (29% identity). A hydropathy plot of the Na+:HCO3
cotransporter indicates at least nine transmembrane domains (Fig. 2, lower panel). The carboxyl
terminus contains a highly hydrophilic stretch of 15 amino acids (12 of
which are lysine) corresponding to the extreme hydrophilic region in
the plot.
Multiple Tissue Northern Blots
A human multiple tissue
Northern blot was purchased from Clontech and
probed with a 32P-labeled polymerase chain reaction product
containing nucleotides 2737-2973 (the homologous region of W39298,
which was common to both cDNA clones used in the construct). Fig.
3 (upper panel) shows a 7.6-kb
mRNA in human kidney and pancreas hybridized strongly with the
probe, indicating that these tissues express relatively high amounts of
the Na+:HCO3 cotransporter
under basal conditions. A faint band can also be detected in human
brain. The lower panel in Fig. 3 demonstrates the expression
of GADPH in all lanes of the multiple tissue Northern blot, indicating
that intact mRNA was present in all lanes.
Functional Expression of the Cloned cDNA
To determine the
functional identity of the protein encoded by the cDNA, transiently
transfected cells were assayed for the presence of
Na+:HCO3 cotransport,
Na+-dependent
Cl
/HCO3
exchange, or
Na+-independent
Cl
/HCO3
exchange.
Accordingly, cells were acidified using an NH-pulse and allowed to
recover in the presence of Na+ and 1 mM
amiloride (to block Na+/H+ exchange). In the
absence of HCO3
, transfected cells
showed negligible recovery from intracellular acidosis (data not
shown). In the presence of both HCO3
and sodium, control cells showed no pHi recovery from the
intracellular acidification (Fig.
4A). However, in transfected cells, switching from the Na+-free solution (solution A,
Table I) to the
Na+-containing solution (solution C) in the presence of
HCO3
resulted in a rapid recovery from
acidic pHi (Fig. 4B), with the recovery of 0.225 pH
(
pHi of 0.225 ± 0.035 in transfected cells
versus almost 0 in nontransfected cells) (n = 5). The recovery from cell acidification was observed only in the
presence Na+ and HCO3
and
was completely inhibited by 300 µM DIDS (Fig.
4B), consistent with the presence of
Na+:HCO3
cotransport.
Depleting the intracellular Cl
(18) by incubating the
cells in Cl
-free media (only Cl
-free
solutions were used for the duration of the experiment) did not reduce
the rate of Na+-dependent
HCO3
movement into acid-loaded cells
(Fig. 4C), indicating that the cloned transporter is not the
Na+-dependent
Cl
/HCO3
exchanger
(
pHi was 0.225 ± 0.035 in chlorine-containing cells
(n = 5) and 0.28 ± 0.018 in chlorine-depleted
cells (n = 4) (p > 0.05).
Transfected cells, switched from a Cl
-containing solution
(solution C) to Cl
-free media (solution D), demonstrated
little cell alkalinization (Fig. 4D), indicating that under
physiological conditions, the cloned transporter does not function in
Cl
/HCO3
exchange
mode.
To determine whether the cloned transporter can mediate
HCO3-dependent 22Na influx, HEK 293 cells were grown in 24-well plates, acidified with NH4
pulse in a manner similar to Fig. 4A and assayed for 22Na influx in the presence of
HCO3. The results showed that
transfected cells mediated significant acid-stimulated DIDS-sensitive
22Na influx in the presence of
HCO3
, whereas nontransfected cells had
no DIDS-sensitive 22Na influx (7.15 ± 0.5 nmol/mg of
protein/4 min in transfected versus 0.25 ± 0.1 nmol/mg
of protein/4 min in nontransfected cells, p < 0.001, n = 4).
The Na+:HCO3 cotransporter
can mediate the movement of HCO3
out
of or into the cell, depending on the ionic composition of experimental
solutions (3-5). In the present study, switching the
Na+-containing solution to Na+-free media did
not result in significant cell acidification, as would have been the
case if the transporter were functioning in efflux mode. Rather, the
transporter functioned only in an uptake mode. Whether lack of efflux
mode was caused by low intracellular Na+ concentration,
decreased membrane potential, or other mechanisms is not clear at
present.
The cDNA clone was identified by virtue of its significant
homology with, but divergence from, the anion exchanger family. Both
the homology with, and the divergence from AE3, are apparent in Fig. 2.
Fig. 1 shows the nucleotide sequence and conceptual translation of the
open reading frame of the
Na+:HCO3 cotransporter.
The close relationship between the size of the cDNA clone (7586 base pairs, not including the poly(A) tail) and the mRNA found in
human kidney indicate that the full sequence has been obtained.
Tissue distribution studies show high expression levels in kidney and
pancreas, with lower levels of expression in the brain. Expression in
the kidney (3-5), pancreas (19), and brain is consistent with
functional studies (20). Heart and lung do not appear to express this
transporter under basal conditions despite the fact that functional
studies demonstrate the presence of
Na+:HCO3 cotransport in
these tissues (18, 21). This finding raises the possibility that the
Na+: cotransporter in the heart and lung is another isoform
from this family.
Functional studies upon transient expression in HEK293 cells showed
that this transporter causes recovery from acute intracellular acidosis
only in the presence of sodium and bicarbonate. The
amiloride-insensitive pHi recovery in the presence of
bicarbonate was detectable only in the presence of Na+,
indicating the presence of a Na+ and
HCO3-dependent cotransport
process. The Cl
independence of this cotransporter and
sensitivity to inhibition by DIDS indicates that this transporter is
distinct from the Na+-dependent
Cl
/HCO3
exchange and
thus demonstrates the presence of
Na+:HCO3
cotransport
(Figs. 4, B and C).
Functional studies have shown inhibition of
Na+:HCO3 cotransport by
protein kinase C and by cAMP-dependent protein kinase (22). Hence, it is not surprising to find consensus sites for phosphorylation by these two enzymes. A closer look at regulation of
Na+:HCO3
cotransport by
casein kinase II may be warranted, since a large number of potential
phosphorylation sites for this enzyme were found.
In certain epithelia, such as kidney and colon, this transporter
mediates the exit of HCO3 from the
cell to the blood (3-5), whereas in other epithelial tissues, as well
as in non-epithelial tissues, sodium bicarbonate is transported from
blood to the cell (18-21). Whether the difference in the direction of
the Na+:HCO3
cotransporter
movement in kidney and other tissues is due to differences in the
membrane potential, cellular ionic composition in these tissues, or
whether it suggests the presence of other isoforms of this transporter
remains to be determined.
In conclusion, a cDNA encoding a
Na+:HCO3 cotransporter was
cloned based on similarity of the anion exchangers to an expressed sequence tag. The Na+:HCO3
cotransporter cDNA encodes an mRNA of 7.6 kb and a protein
molecular mass of 116 kDa. The
Na+:HCO3
cotransporter
mRNA is expressed in several tissues, including kidney, pancreas,
and brain, indicating its pivotal role in cell pH regulation in
mammalian tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF007216.