Functional characterization of human NBC4 as an electrogenic
Na+-HCO
cotransporter (NBCe2)
Leila V.
Virkki1,
Darren A.
Wilson2,
Richard D.
Vaughan-Jones2, and
Walter F.
Boron1
1 Department of Cellular and Molecular Physiology,
Yale University School of Medicine, New Haven, Connecticut 06520;
and 2 University Laboratory of Physiology,
University of Oxford, Oxford OX1 3PT, United Kingdom
 |
ABSTRACT |
We have
functionally characterized Na+-driven bicarbonate
transporter (NBC)4, originally cloned from human heart by Pushkin et
al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four
NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no.
AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function with
microelectrodes. Exposing an NBC4c-expressing oocyte to a solution
containing 5% CO2 and 33 mM HCO
elicited a large hyperpolarization, indicating that the transporter is
electrogenic. The initial CO2-induced decrease in
intracellular pH (pHi) was followed by a slow recovery that
was reversed by removing external Na+. Two-electrode
voltage clamp of NBC4c-expressing oocytes revealed large
HCO
- and Na+-dependent currents. When we
voltage clamped Vm far from NBC4c's estimated
reversal potential (Erev), the pHi
recovery rate increased substantially. Both the currents and
pHi recovery were blocked by 200 µM
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated
the transporter's HCO
:Na+ stoichiometry
by measuring Erev at different extracellular
Na+ concentration ([Na+]o)
values. A plot of Erev against
log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as
the absolute Erev values, are consistent with a
2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we
propose to call NBCe2-c, is similar to that of NBCe1, the first
electrogenic NBC.
intracellular pH; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; microelectrodes; stoichiometry; Xenopus laevis oocytes
 |
INTRODUCTION |
THE EXPRESSION
CLONING of the first Na+-driven bicarbonate
transporter (NBC) by Romero et al. (36) led to the cloning
of many other electrogenic and electroneutral Na+-driven
HCO
transporters. The original electrogenic NBC in
mammals (NBCe1) is currently represented by three splice variants:
NBCe1-A, which is found predominantly in kidney (8, 35);
NBCe1-B, which is found in pancreas, heart, and many other tissues
(1, 12); and NBCe1-C, which is found predominantly in
brain (4).
The electroneutral Na+-driven HCO
transporters in mammals share ~70% sequence identity on the amino acid level and appear to fall into two functional groups. The first
group mediates Cl
-independent
Na+-HCO
cotransport. The first member of
this group has been cloned under various names, including NBC2 from
human retina (22), NBC3 from human skeletal muscle
(31), and NBCn1 from rat smooth muscle (11).
However, the NH2-terminal 90 amino acids of NBC2 are
missing, and the next 28 represent a cloning artifact.
The second group of electroneutral Na+-driven
HCO
transporters mediates the
Cl
-dependent transport of Na+ and
HCO
, functionally described as Na+-driven Cl
/HCO
exchange. In mammals, this transporter is represented by a clone from
human brain, NDCBE1 (18). A partial clone had been named
NBC3 (3), representing degeneracy in the nomenclature. A
related Drosophila clone (NDAE) encodes a
Na+-driven anion exchanger (37).
NBC4, originally cloned by Pushkin et al. (32), is a new
member of the HCO
transporter superfamily. It is
~60% identical at the amino acid level to the three electrogenic NBCe1 splice variants and 40-50% identical to the electroneutral Na+-driven HCO
transporters. A Northern blot (32) and a similar mirror image (33)
showed high levels of NBC4 mRNA in liver, spleen, and testes and
moderate levels in heart, kidney, placenta, and stomach.
We mapped the NBC4 sequence onto a recent topology model developed for
the anion exchanger AE1 (Fig.
1A), which is in the same
superfamily as the Na+-coupled HCO
transporters. Pushkin et al. (32, 33) described two
variants of NBC4, NBC4a and NBC4b, and have submitted two additional
sequences, NBC4c and NBC4d, to GenBank. As shown in Fig. 1B,
NBC4a and NBC4b each contain, beginning just after the predicted
transmembrane segment (TM) 11, a putative exon not present in either
NBC4c or NBC4d. NBC4b has a unique 16-bp insertion in its last TM. This
insertion/frameshift introduces a premature stop codon and causes the
deduced amino acid sequence to exhibit little homology with other
members of the HCO
transporter (BT) superfamily. NBC4d is missing two exons present in NBC4c, resulting in the elimination of all amino acids between the putative end of TM 10 and
near the beginning of TM 14. To date, no functional data have been
reported for any of the four NBC4 variants.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Na+-driven bicarbonate transporter (NBC)4 sequence
analysis and topology. A: using sequence alignment analysis,
we mapped the NBC4c amino acid sequence on a recent topology model
developed for anion exchanger (AE)1 (43). The putative
NH2 and COOH termini are intracellular. Gray cylinders
indicate transmembrane segments (TMs) 1-14, of which TM 12 is
nonhelical (stippled). Four glycosylation consensus motifs are located
on the large extracellular loop connecting TMs 5 and 6. B:
gray bars indicate the relative length of the amino acid sequence of
the 4 NBC4 variants. The position of the putative TMs are indicated by
numbered vertical light gray bars. Variant b has a 16-bp insertion
(cross-hatching on dark background) in the middle of the last TM, which
causes a frameshift and results in a unique COOH terminus
(cross-hatching on white background). Variant c lacks a 48-bp exon
(between TMs 11 and 12) present in variants a and b. Variant d lacks 2 exons in addition to the 1 missing in variant c, resulting in the loss
of the region between TMs 10 and 14.
|
|
We have undertaken the present study to investigate the functional
properties of NBC4. Using PCR, we obtained nine clones corresponding to
the NBC4c coding region but none corresponding to the other NBC4
variants. We have expressed NBC4c in Xenopus laevis oocytes
and assayed for pH regulatory function as well as membrane potential
and ionic currents. The results show that NBC4c is an electrogenic
Na+-HCO
cotransporter that, at least in
the oocyte, appears to operate with a
HCO
:Na+ stoichiometry of 2:1.
Portions of this work have been published in abstract form
(44).
 |
METHODS |
cDNA Cloning
On the basis of the published cDNA sequence for NBC4a (GenBank
accession no. AF243499; Ref. 33), we designed
oligonucleotide primers corresponding to the 5' and 3' regions of the
open reading frame to this clone. We performed PCR with sense primer
5'-CCTCGAGTCATGAAGGTGAAGGAGGAGAAGC-3' and antisense primer
5'-CCCGGGAAGAATCAGAGTGAGTAACTCCAACTGG-3' using, as a
template, a mixture of human cDNAs from 37 different normal tissues,
including brain, heart, kidney, liver, lung, and testes (Human
Universal QUICK-Clone cDNA; Clontech Laboratories, Palo Alto, CA). The
underlined portions of the primer sequences correspond to the
engineered restriction enzyme sites for XhoI and
XmaI, respectively. We subcloned the PCR products into a TA cloning vector (pCR II TOPO; Invitrogen, Carlsbad, CA). Individual clones were sequenced by GeneWiz (New York, NY) or the Keck
Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine,
Yale University. The consensus cDNA sequence contained a 3,366-bp open reading frame that encodes 1,121 amino acids, which is identical to the
c variant of human NBC4 (GenBank accession no. AF293337).
Expression in Oocytes
We subcloned the cDNA fragment into the KSM Xenopus
oocyte expression vector by excising the insert from pCR II TOPO with XhoI and XmaI. The KSM expression vector is a
derivative of pBluescript, in which the entire polylinker was replaced
by a PCR product encoding (from 5' to 3') the 5' untranslated region
(UTR) of the Xenopus
-globin gene, a series of
restriction sites for subcloning, the 3' UTR of the Xenopus
-globin gene, a poly-A tail, and several additional restriction
sites for 3' linearization of cDNA before in vitro transcription. The
vector was a kind gift from Dr. William Joiner (Yale University).
Capped mRNA was synthesized in vitro with the T3 Message Machine kit
(Ambion, Austin, TX).
Stage V-VI oocytes from Xenopus laevis were isolated as
described previously (35). One day after isolation, the
oocytes were injected with 50 nl of a solution containing 0.5 ng/nl of mRNA encoding NBC4c. Control oocytes were injected with 50 nl of
sterile water. The oocytes were used in experiments 2-5 days after
injection. All experiments were performed at room temperature (~22°C).
Solutions
Nominally HCO
-free ND96 solution contained
(in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and
5 mM HEPES at a pH of 7.50. HCO
-containing solutions
were prepared by replacing 33 mM NaCl with 33 mM NaHCO3 in
ND96 and equilibrating the solution with 5% CO2-balance
oxygen. Na+-free ND96 and HCO
solutions
were prepared by substituting
N-methyl-D-glucammonium (NMDG+) for
Na+. Cl
-free solutions were prepared by
substituting gluconate for Cl
. Osmolality of all
solutions was ~200 mosmol/kgH2O. For
butyrate-containing solutions, 30 mM Na-butyrate replaced 30 mM NaCl in
ND96. Assuming that butyrate has a pKa of 4.8 and that the buffering power of an oocyte is 13.5 mM/pH,1 we calculated that 30 mM extracellular butyrate would give approximately the same degree of
intracellular acidification as 5% CO2.
Electrophysiological Measurements
An oocyte was placed in a perfusion chamber and constantly
superfused at a solution flow of 4 ml/min. Bath solutions were delivered with syringe pumps (Harvard Apparatus, South Natick, MA), and
solutions were switched with pneumatically operated valves (Clippard
Instrument Laboratory, Cincinnati, OH). In all experiments, the oocyte
was initially superfused with the ND96 solution, which is nominally
CO2/HCO
free.
Measurement of intracellular pH.
We assayed pH regulatory function of oocytes expressing NBC4c by
measuring intracellular pH (pHi) with pH-sensitive
microelectrodes. The electrodes were fabricated and used as described
previously (36, 40). Briefly, the oocyte was impaled with
two microelectrodes, one for measuring the membrane potential
(Vm) and the other for measuring
pHi. The tip of the pH electrode contained a liquid membrane across which a pH-dependent voltage was generated.
pHi was obtained by subtracting the signal of the
Vm electrode from that of the pH electrode. A
calomel electrode was used as the reference in the bath. Voltages were
measured with an FD 223 electrometer (World Precision Instruments,
Sarasota, FL), and data were acquired with software written in-house.
The system was calibrated with buffered pH standards at pH 6.0 and 8.0. An additional single-point calibration was performed with the standard
ND96 solution of pH 7.50 in the bath before the oocyte was impaled.
Two-electrode voltage clamp.
We used two-electrode voltage clamp to measure whole cell ionic
currents in oocytes expressing NBC4c or injected with water (control).
Oocyte currents and voltages were recorded with a model OC-725C oocyte
clamp (Warner Instruments, Hamden, CT) controlled by the Clampex module
of pCLAMP software (Version 8; Axon Instruments, Foster City, CA).
Electrodes were pulled from thin-walled borosilicate glass and had
resistances of 0.4-1.0 M
when filled with 3 M KCl. Oocytes were
held at a potential close to the spontaneous Vm
until the initiation of the voltage-clamp protocol. Current-voltage (I-V) relationships were generated by stepping the holding
potential from
140 mV to +40 mV in 20-mV increments, each of which
lasted 200 ms. Data were analyzed with the Clampfit module of pCLAMP.
Stoichiometry
To probe the HCO
:Na+
stoichiometry of NBC4, we used two-electrode voltage clamp to determine
zero-current potential (reversal potential,
Erev) in oocytes expressing NBC4c. We began by
applying a solution containing 98.5 mM Na+ and 5%
CO2/33 mM HCO
. In separate experiments, we measured pHi to verify that, after 5 min in
this solution, pHi had reached its minimum value and would
thereafter change only very slowly. The oocytes subsequently were
exposed to CO2/HCO
-containing solutions
with one of four different concentrations of extracellular
Na+ (9.8, 29.6, 69.0, or 98.5 mM). After completing the
voltage-clamp protocol at one Na+ concentration, we turned
off the voltage clamp, switched to a Na+-free
HCO
solution for 2-3 min, then switched to
another of the Na+-containing test solutions, waited
2-3 min for Vm to stabilize, clamped the
oocyte to this new spontaneous Vm, and then
performed the voltage-clamp protocol once again. We bracketed the
entire procedure with I-V curves obtained at 98.5 mM
Na+. We discarded the data if the spontaneous
Vm values differed by >5 mV for the bracketing
periods in 98.5 mM Na+. With these criteria, the bracketing
I-V curves in 98.5 mM Na+ were virtually
identical except for small shifts (<5 mV) in
Erev.
The reason for returning to Na+-free solutions for 2-3
min between I-V curves was to minimize changes in
intracellular Na+ concentration
([Na+]i) and
[HCO
]i due to NBC4 transport activity;
these Na+-free periods prompted NBC4 to run backwards,
presumably depleting the Na+ and HCO
that had built up as NBC4 was running in the forward direction in the
presence of Na+. We obtained
HCO
-dependent Erev from the
above I-V curves by subtracting the HCO
curve measured in ND96 (CO2/HCO
free) from each of the I-V curves measured in the presence of
CO2/HCO
at different Na+ concentrations.
 |
RESULTS |
Nine Consecutive PCR Products Represented NBC4c
Our cloning strategy was designed to pick up any of the four NBC4
variants currently in GenBank. After performing end-to-end PCR and
subcloning the PCR product into a cloning vector, we sequenced nine
independent clones. All of these clones corresponded to the c variant
of NBC4.
NBC4 is Electrogenic, Na+
Dependent, and Cl
Independent
pHi data.
Figure 2A shows a recording of
pHi and Vm for an oocyte injected
with cRNA encoding NBC4c, and Fig. 2B shows a similar
recording for a water-injected (control) oocyte. The initial
pHi of the NBC4c-expressing oocytes was slightly higher
(7.34 ± 0.01; n = 18) than in the controls
(7.22 ± 0.03; n = 9), suggesting that the ambient
HCO
in the incubation medium had provided enough
substrate for at least some NBC4c activity since the time of the cRNA
injection. We then switched the superfusate from ND96 to 5%
CO2/33 mM HCO
, which caused a dramatic
hyperpolarization of the NBC4c-expressing oocyte, followed by a slower
relaxation of Vm toward more positive values. The rapid hyperpolarization is consistent with the hypothesis that
NBC4c mediates the uptake of Na+ and the equivalent of two
or more HCO
ions. In addition, pHi began
to fall rapidly, reflecting the entry of CO2 into the
oocyte, the subsequent hydration to form H2CO3, and dissociation to liberate HCO
and H+.
In the continued presence of CO2/HCO
, pHi eventually started to recover slowly (Table
1), consistent with the NBC4c-mediated
uptake of HCO
. When we removed extracellular
Na+ (replacing it with NMDG+), the cell rapidly
depolarized and began to acidify, indicating that the direction of
transport had been reversed. Reintroducing Na+ caused the
opposite set of Vm and pHi changes.
At the end of the experiments, removing
CO2/HCO
caused the cell to depolarize
and alkalinize rapidly. The rapid depolarization probably reflects the
temporary reversal of NBC4c caused by the removal of extracellular
HCO
while [HCO
]i is
still high. The rise in pHi reflects the exit of
CO2, which produces the depletion of HCO
that is reflected by the slower negative drift of
Vm.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Recording of intracellular pH (pHi) and membrane
potential (Vm). A: representative
pHi and Vm trace of an oocyte
expressing NBC4c. The oocyte was initially superfused with ND96. During
the indicated period, the oocyte was superfused with the 5%
CO2/33 mM HCO solution.
CO2/HCO -induced hyperpolarization
averaged 90 ± 6 mV in 6 experiments. While in the
CO2/HCO solution, extracellular
Na+ was removed as indicated. This resulted in a marked
depolarization, averaging +95 ± 10 mV in the same experiments.
B: data obtained with a similar protocol on a water-injected
oocyte. Arrowheads indicate when Na+ was removed or
reintroduced. The experiment was performed on 5 water-injected
oocytes.
|
|
In the control oocyte in Fig. 2B, the exposure to
CO2/HCO
did not cause the rapid, initial
hyperpolarization. Also, we observed little pHi recovery
during the prolonged exposure to CO2/HCO
(Table 1). Moreover, removing extracellular Na+ from the
control oocyte caused a slight hyperpolarization (Fig. 2B),
reflecting the native Na+ permeability of the oocyte
membrane. This native Na+ permeability is completely masked
in the NBC4c-expressing oocyte by the overwhelming NBC4c-dependent depolarization.
Voltage-clamp data.
Using a two-electrode voltage clamp, we further explored the
electrogenic nature of NBC4c. Figure
3A shows representative current records obtained in an NBC4c-expressing oocyte under three conditions: the nominal absence of
CO2/HCO
, the presence of 5%
CO2/33 mM HCO
, and the absence of
Na+ in the continued presence of
CO2/HCO
. Introducing
CO2/HCO
elicits a large increase in the
whole cell current, whereas removing Na+ virtually
abolishes the NBC4c-dependent outward currents.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Voltage-clamp experiments. A: representative current
recordings obtained from an NBC4c-expressing oocyte. The top
tracing was acquired in ND96, and the middle and
bottom tracings were acquired in 5% CO2/33 mM
HCO . In the bottom tracing, external
Na+ was removed. B: representative
current-voltage (I-V) curves for NBC4c-expressing oocytes
generated from current tracings similar to those in A. Data
were acquired in ND96 solution ( ),
CO2/HCO solution ( ),
Cl -free CO2/HCO solution
( ), and Na+-free
CO2/HCO solution ( ).
C: representative I-V curves generated from
water-injected oocytes (dashed lines) and NBC4c-expressing oocytes
(solid lines), all in the nominal absence of
CO2/HCO . Data from water-injected
oocytes were acquired in ND96 solution ( ) and
CO2/HCO solution ( ).
Data from NBC4c-expressing oocytes were acquired in ND96 solution
( ), Na+-free ND96 solution
( ), and Cl -free ND96 solution
( ). We performed a total of 8 such experiments for
water-injected oocytes and 7 for NBC4c-injected oocytes. Results were
similar to those shown.
|
|
Figure 3B shows representative I-V relationships
for an NBC4c-expressing oocyte. Replacing extracellular
Cl
with the impermeant gluconate does not change
Erev and has little effect on either the inward
or outward currents, indicating that NBC4c does not require external
Cl
to operate in either direction. In contrast, replacing
extracellular Na+ with NMDG+ shifts
Erev to more positive values and virtually
abolishes outward current (i.e., inward movement of the negative net
charge carried by 1 Na+ and 2 or more
HCO
). In other experiments (not shown), we observed
that the CO2/HCO
-dependent current
increased during the first 3-4 min of exposure to
CO2/HCO
but remained stable for several
minutes afterwards. We therefore performed all ionic substitution
experiments after at least 5 min of
CO2/HCO
exposure.
Figure 3C shows that, in water-injected control oocytes,
there is little difference between currents obtained in the presence and absence of 5% CO2/33 mM HCO
,
demonstrating the absence of HCO
-dependent currents in native oocytes. Figure 3C also shows that in nominally
HCO
-free solutions, the whole cell conductance of
NBC4c-expressing oocytes is markedly larger than in water-injected
controls. It is conceivable that the small amount of
HCO
present in air-equilibrated ND96 solutions
(estimated at ~200 µM) is sufficient to carry measurable current in
NBC4c-expressing oocytes. However, our observation that the current is
unaffected by removal of external Na+ makes this
explanation unlikely. Removal of external Cl
slightly
reduced the outward current (i.e., inward movement of Cl
)
but did not bring the residual outward currents to the same levels as
in control oocytes. The increased "background" conductance seen in
the present study is not unique to NBC4c. Compared with control
oocytes, those expressing either rat kidney NBCe1-A (39) or rat NBCn1 (11) exhibit increased background conductances.
Electrical Gradient Can Drive Substantial Acid-Base Transport
Through NBC4c
In the experiment shown in Fig. 2A, the
pHi recoveries in the presence of Na+ and
CO2/HCO
, as well as the pHi
decline in the absence of Na+, were quite slow despite
marked changes in Vm. We hypothesized that the
reason for this apparent discrepancy was that the spontaneous Vm was only 3-4 mV more positive than the
Erev for NBC4c. This would be the case if NBC4c
were the dominant current carrier in the oocyte (i.e., if the oocytes
had minimal background ion conductance). For example, during the slow
pHi recovery in the presence of Na+ and
CO2/HCO
in Fig. 2A,
Vm was about
120 mV. This voltage is nearly
the same as Erev for the I-V curve in
Fig. 3B, which was obtained under similar conditions.
To test this hypothesis, we performed an experiment similar to that
shown in Fig. 2A except that, at times, we not only
monitored pHi but voltage clamped as well. The oocyte had a
spontaneous Vm of about
50 mV when impaled
only with the voltage electrode. Vm shifted to
about
45 mV when we introduced the current electrode and initially
fell to about
25 when we introduced the pH electrode. By the
beginning of the record in Fig. 4,
Vm had recovered somewhat, but this oocyte was
still rather "leaky." Introducing
CO2/HCO
caused Vm
to shift only to
100 mV (compared with about
155 mV for the tighter
oocyte in Fig. 2A). At the times indicated in Fig. 4, we
acquired I-V curves by using the same voltage protocol as in
Fig. 3. We calculated Erev for the
NBC4c-dependent currents by subtracting the I-V curve
acquired in ND96 from the I-V curves acquired in the
CO2/HCO
solutions. For the first
I-V curve, Erev was
110 mV,
compared with a spontaneous Vm of about
95 mV.
This ~15-mV difference between Erev and
Vm would explain why the spontaneous
pHi recovery is faster in Fig. 4 than in Fig. 2. When we
then voltage-clamped the oocyte in Fig. 4 to
30 mV, pHi
increased approximately six times more rapidly than when
Vm was free-floating at about
95 mV. This
large increase in the pHi recovery rate is consistent with
the large increase in the difference between
Erev (
90 mV) and the clamped
Vm of
30 mV. When we reversed the transporter
by removing extracellular Na+ in Fig. 4
(Vm =
30 mV), pHi initially
decreased more rapidly than in Fig. 2 (Vm = +2 mV). Thus a large electrical gradient can substantially increase
acid-base transport through NBC4c. The rates of pHi change
in oocytes clamped to
30 mV are compared with those of unclamped
oocytes in Table 1.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Measurement of pHi in a voltage-clamped
oocyte. The oocyte was initially superfused with ND96 without clamping
the voltage. During the indicated period, the oocyte was superfused
with the 5% CO2/33 mM HCO solution.
While in the CO2/HCO solution, first
extracellular Na+ and then also extracellular
Cl were removed as indicated. Gray area indicates the
period during which the oocyte was voltage-clamped to 30 mV.
Asterisks indicate the time points at which an I-V curve was
acquired. We performed a total of 3 such experiments on different
NBC4c-expressing oocytes, obtaining results similar to those shown.
|
|
While continuing to voltage clamp the oocyte in Fig. 4 at
30 mV, we
removed extracellular Cl
in the absence of
Na+ to revisit the question of whether acid-base transport
by NBC4c requires extracellular Cl
. If NBC4c were
a Na+-driven Cl
-HCO
exchanger, then it should require extracellular Cl
when
running in reverse. However, even at a relatively high rate of
pHi decrease in Fig. 4, Cl
removal had no
effect on the pHi trajectory. Thus NBC4c does not mediate
Na+-driven Cl-HCO
exchange.
NBC4c is HCO
Dependent
In Fig. 2A, we saw that exposing an NBC4c-expressing
oocyte to CO2/HCO
caused an abrupt
hyperpolarization as well as a pHi decrease from which the
cell slowly recovered. To address the question of whether the
Vm change and the pHi recovery depended on the application of CO2/HCO
, and not merely the intracellular acidification induced by
CO2, we used butyrate to acidify oocytes in the nominal
absence of HCO
. We calculated that a butyrate concentration of 30 mM would produce about the same fall in
pHi as 5% CO2 (see Solutions). As
shown in Fig. 5A, exposing an
NBC4c-expressing oocyte to a solution containing 30 mM butyrate causes
virtually no change in Vm and a large, rapid
acidification. However, in the absence of HCO
,
pHi fails to recover from the acid load. After the butyrate
is removed, a subsequent exposure to
CO2/HCO
causes the usual hyperpolarization as well as the pHi recovery from the acid
load. In the control oocyte, neither butyrate nor
CO2/HCO
elicited large changes in
Vm or a pHi recovery from the acid
loads (Fig. 5B). Thus both the voltage changes and the
pHi recovery require HCO
per se and not
merely an acidification of the cytoplasm.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
HCO dependence of NBC4. A:
representative pHi and Vm traces of
an oocyte expressing NBC4c. The oocyte was initially superfused with
ND96. During the indicated time period, the oocyte was superfused with
30 mM butyrate solution at a constant extracellular pH of 7.50. After
washout of the butyrate-containing solution, the oocyte was reacidified
with 5% CO2-33 mM HCO solution.
B: data obtained with a similar protocol on a water-injected
oocyte. We performed a total of 3 such experiments on different
water-injected oocytes and 3 on NBC4c-expressing oocytes. Results were
similar to those shown.
|
|
NBC4c Is Blocked by DIDS
pHi data.
To determine whether NBC4c is sensitive to
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), we
exposed NBC4c-expressing oocytes and control oocytes to 200 µM DIDS
during the plateau phase of a CO2/HCO
pulse. Figure 6A shows that in
an NBC4c-expressing oocyte, application of DIDS markedly reduced the
rate of pHi recovery and also caused a modest depolarization. DIDS reduced the mean rate of pHi recovery
from 21.5 ± 4.2 to 3 ± 1.2 × 10
5 pH units/s
(n = 5), an inhibition of ~80%. Thus DIDS strongly inhibits the transport function of
NBC4c.2 Although the
inhibition of some HCO
transporters by DIDS is
poorly reversible, the pHi recovery in oocytes expressing
NBC4c resumed on washout of DIDS. In the control oocyte (Fig.
6B), pHi did not recover after the initial
acidification and application of DIDS had no effect on pHi.
The mean rate of pHi change was 0.7 ± 2.2 × 10
5 pH units/s before application of DIDS and
1.7 ± 0.9 × 10
5 pH units/s after application of DIDS
(n = 3).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)
on pHi recovery from an acid load. A:
representative pHi and Vm traces of
an oocyte expressing NBC4c. The oocyte was initially superfused with
ND96. During the indicated time period, the oocyte was superfused with
the 5% CO2/33 mM HCO solution. DIDS
(200 µM) was applied during the
CO2/HCO exposure. B: data
obtained with a similar protocol on a water-injected oocyte. We
performed a total of 3 such experiments on different water-injected
oocytes and 5 on different NBC4c-expressing oocytes. Results were
similar to those shown.
|
|
Voltage-clamp data.
Using two-electrode voltage clamp, we investigated the effect of 200 µM DIDS on the current carried by NBC4c. Because work by Diakov et
al. (14) has shown that DIDS (at a concentration of 250 µM) can activate endogenous ion channels in the oocyte over a period
of 15-60 s, we exposed the oocytes to DIDS for no more than ~20
s, which we found to be sufficient for maximal inhibition.
Figure 7A summarizes data from
a representative experiment in which we obtained sequential
I-V curves at four times, with the oocyte exposed to
1) ND96, 2)
CO2/HCO
, 3)
CO2/HCO
+ DIDS after a ~20-s
pretreatment with DIDS, and 4) in
CO2/HCO
after washout of DIDS for 2 min.
DIDS reduced the currents to nearly the levels observed in ND96.
Despite the short treatment period, the inhibitory effect of DIDS was
not completely reversible.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of DIDS on ionic currents. A:
representative I-V curves for NBC4c-expressing oocytes. Data
were acquired in ND96 solution ( ),
CO2/HCO solution
( ),CO2/HCO with 200 µM
DIDS ( ), and CO2/HCO
solution after washout of DIDS ( ). B:
representative I-V curves generated from control oocytes
(dashed lines) and NBC4c-expressing oocytes (solid lines), all in the
nominal absence of CO2/HCO . Data from
water-injected oocytes were acquired in ND96 solution
( ) and ND96 solution with DIDS ( ). Data
from NBC4-expressing oocytes were acquired in ND96 solution
( ), ND96 with 200 µM DIDS ( ), and
ND96 solution after washout of DIDS ( ). C:
average DIDS-sensitive currents in NBC4c-expressing oocytes.
DIDS-sensitive current in ND96 solutions ( ) is the
average obtained by subtracting data from 5 experiments similar to
those shown in B ( and ).
DIDS-sensitive current in CO2/HCO
solutions ( ) is the comparable average for 5 different
experiments similar to those shown in A ( and ). Vertical bars indicate SE; bars are omitted when
they would have been smaller than the symbol.
|
|
Figure 7B summarizes the results of an experiment in which
we exposed either an NBC4c-expressing oocyte or a control oocyte to
DIDS in ND96 solution (i.e., in the nominal absence of
CO2/HCO
). In the NBC4c-expressing oocyte
DIDS blocked some of the inward and outward currents, whereas in the
control oocyte the effect of DIDS was very small. The very small
DIDS-sensitive currents in the nominal absence of
CO2/HCO
are not due to residual NBC4c
activity; recall that in Fig. 3C, we saw that removing
external Na+ in the nominal absence of
CO2/HCO
did not affect the currents in
an NBC4c-expressing oocyte.
Figure 7C shows the DIDS-sensitive component of the currents
in NBC4c-expressing oocytes, averaged from five different experiments. The open circles in Fig. 7C represent data obtained in the
presence of CO2/HCO
(e.g., the
difference between closed and open circles in Fig. 7A). The
closed diamonds in Fig. 7C represent data obtained in the
nominal absence of CO2/HCO
(e.g., the
difference between closed diamonds and open circles in Fig.
7B). It is clear that the DIDS-sensitive currents are far
larger in the presence of CO2/HCO
.
Apparent
HCO
:Na+
Stoichiometry of NBC4c is 2:1
Slope of Erev vs. log of extracellular
Na+ concentration.
To determine the HCO
:Na+ stoichiometry
of NBC4c, we used a two-electrode voltage clamp to measure the reversal
potentials of HCO
-dependent currents in
NBC4c-expressing oocytes at four different values of extracellular
Na+ concentration ([Na+]o) (see
METHODS). Erev of an electrogenic
Na+-HCO
cotransporter is given by the following equation (5)
|
(1)
|
where q is the
HCO
:Na+ stoichiometry,
[Na+]i,
[Na+]o,
[HCO
]i, and
[HCO
]o are the respective
intracellular and extracellular concentrations, R is the gas
constant, T is temperature, and F is the Faraday constant. This equation predicts that a plot of
Erev vs. log [Na+]o
should be a straight line, whose slope is
ln(10) × RT/[F(q
1)]. Thus the higher
the HCO
:Na+ stoichiometry, the less
steep the slope of the line. Thus, for a
HCO
:Na+ stoichiometry of 2:1, the
expected slope at 22°C would be
58.5 mV/log
[Na+]o, whereas for a stoichiometry of 3:1,
the slope would be only half as steep,
29.3 mV/log
[Na+]o. The results of five experiments (each
including all 4 [Na+]o values) are summarized
in Fig. 8, which is a plot of
Erev against [Na+]o.
Our experimental data are fitted well by a straight line with a slope
of
54.8 mV/log [Na+]o, consistent with the
hypothesis that NBC4c operates with a 2:1 stoichiometry.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 8.
Dependence of the NBC4 reversal potential
(Erev) on extracellular Na+
concentration ([Na+]o). Using a 2-electrode
voltage-clamp technique, we measured the Erev of
NBC4c-expressing oocytes in CO2/HCO
solutions at 4 different values of [Na+]o.
The plot of Erev vs.
log[Na+]o was fitted with a straight line
(R2 = 0.9356), the slope of which is 54.8
mV/log[Na+]o. This slope is consistent with a
HCO :Na+ stoichiometry of 2:1. Each
symbol represents the mean ± SE. We performed a total of 5 experiments on different NBC4c-expressing oocytes.
|
|
The advantage of calculating transporter stoichiometry from the slope
of the line in Fig. 8 is that one does not need to know any of the
absolute concentrations in Eq. 1, only the fractional change
in the concentration of the varied ion (i.e.,
[Na+]o in this case). However, in using this
approach, one must assume that the values of the other three
concentration terms are constant. In Fig. 8, we used only tight
oocytes, like the one in Fig. 2, for which the spontaneous
Vm was very close to
Erev. When Vm
Erev, the transporter has a low activity, and
one would expect [Na+] and [HCO
] in
the intra- and extracellular unstirred layers near the membrane to be
very close to the respective concentrations in the bulk fluids.
Moreover, we know from Fig. 2 that pHi does not change
appreciably in a tight oocyte, even when one shifts
[Na+]o between 98.5 and 0 mM. We have no
information on how [Na+]i might have varied
during the 2-3 min during which we altered [Na+]o. However, in oocytes expressing rat
kidney NBCe1-A, Sciortino and Romero (39) found that
removing extracellular Na+ caused
[Na+]i
measured with an intracellular
microelectrode
to fall from ~9 mM to only ~8 mM over ~2 min.
Moreover, if [Na+] near the two unstirred layers in our
experiments had changed appreciably, we would not have obtained the
linear relationship shown in Fig. 8.
Absolute value of Erev.
Another approach for estimating stoichiometry is to compute
q directly from each Erev value.
Solving Eq. 1 for q yields
|
(2)
|
We calculated [HCO
]i in our
experiments from average pHi data and the
Henderson-Hasselbalch equation, obtaining a mean value of 7.6 mM.
Sciortino and Romero (39) found that oocytes expressing
rat kidney NBCe1 and bathed in 5% CO2/33 mM
HCO
had a mean [Na+]i of
10.4 mM. Using these values for [HCO
]i and [Na+]i, 33 mM for
[HCO
]o, and the four different
[Na+]o values from our experiments, we
computed the four q values summarized in Table
2. The mean value for all data, 2.1 ± 0.1, is consistent with a 2:1 stoichiometry.
 |
DISCUSSION |
The aim of this study was to determine the functional properties
of NBC4, originally cloned from human heart by Pushkin et al.
(32) but uncharacterized physiologically. Currently
GenBank contains the sequences for four different NBC4 variants (NBC4a, b, c, and d). Our own cloning efforts resulted in the cloning of the
NBC4c variant, which we have expressed in Xenopus oocytes. We report here that NBC4c carries out electrogenic
Na+-HCO
cotransport. Furthermore, NBC4c is independent of extracellular Cl
, is blocked by DIDS,
and operates with an HCO
:Na+
stoichiometry of 2:1 when expressed in oocytes. These results correlate
well with sequence alignment analysis, which shows that, on the amino
acid level, NBC4 is most closely related to electrogenic NBCe1. On the
basis of the function of NBC4c, we propose to call it NBCe2-c.
Different NBC4 Variants
Considerable progress has been made in the last few years on the
topology of anion exchangers (AEs), which are ~30% identical to the
NBCs on the amino acid level. The membrane domain of AE1 (i.e., the
portion lacking the presumably cytoplasmic NH2 and COOH
termini) is ~40% identical to that of the Na+-coupled
HCO
transporters and is, by itself, sufficient to
carry out transport (23, 26, 27). Reasoning that the
topology of the membrane domains of the BTs are probably conserved, in
Fig. 1A we mapped NBC4c onto a recent folding model
developed in the laboratory of Joe Casey (43). The model
predicts 14 membrane-spanning segments, of which the twelfth segment is
proposed to be nonhelical.
Comparing the cDNA sequence of NBC4c with the human genome database
reveals the presence of 25 exons that span the length of the coding
region. Variants a and b share an additional exon not present in c and
d. The presence of this additional exon results in a 16-amino acid
insertion into the very short region between putative TMs 11 and 12, a
region that is highly conserved among all superfamily members. In
addition, the third-to-last exon in variant b is longer than in the
other variants because, at its 5' end, it contains 16 bp of intronic
sequence (Fig. 1B). This 16-bp insertion creates a
frameshift in the middle of the last TM, resulting in a truncated COOH
terminus (Fig. 1B) that has no homology to other members of
the BT superfamily. Variant d is lacking two additional exons between
TMs 10 and 14, resulting in a 294-bp deletion in a region that is very
highly conserved in all members of the BT superfamily. Thus variant c
is the only one of the four NBC4 variants whose deduced amino acid
sequence maps onto the superfamily consensus sequence without major
gaps or insertions. Indeed, nine consecutive cDNA clones in our
study
obtained by PCR using, as a template, a mixture of human cDNAs
from different tissues
proved to be of the c variety. If the four
original NBC4 mRNA species had been evenly distributed, and if the PCR
efficiencies for the respective cDNA species were identical, then the
odds of picking the c variant nine times in a row would be 1 in
49. It will be informative to learn whether variants a, b,
and d encode functional proteins.
DIDS
Our results show that 200 µM DIDS strongly blocks both the
pHi recovery and ionic currents mediated by NBC4c. DIDS is
a classic inhibitor of anion transport via AEs, various
Na+-coupled HCO
transporters, and anion channels. Cabantchik and Rothstein (9) established that
DIDS interacts with the protein now known to be AE1 in two steps: a rapid ionic interaction that is reversed by scavenging the DIDS with
albumin and a slower covalent reaction. However, whether DIDS binds
ionically or covalently, it blocks transport. Aligning the amino acid
sequences of AE1, AE2, and AE3, Kopito et al. (23) proposed a consensus motif at the extracellular end of TM 5 for the
covalent interaction of DIDS with the AEs: KLXK (X = I, Y). After
cloning the first Na+-HCO
cotransporter,
Romero et al. (36) pointed out that NBCe1 has KMIK at the
site homologous to KLXK in the AEs. Therefore, they suggested a
consensus DIDS-reaction motif of KX1X2K
(X1 = M, L; X2 = I, V, Y). Indeed,
NBCn1, which is not sensitive to DIDS, has a disrupted consensus motif:
KLFH (11).
In NBC4c, the sequence at the homologous TM 5 site is KMIG. Thus DIDS
blocks the function of NBC4c even though NBC4c lacks the second K of
the consensus motif. However, NBC4c is not the first DIDS-sensitive BT
family member with a disrupted consensus motif. NDAE1 from
Drosophila (37) and human NDCBE
(18) are both DIDS sensitive, even though the sequences at
their respective homologous sites are NVMV and KLIH. One explanation
for these observations is that DIDS may inhibit transport by NBC4c (and also by NDAE and NDCBE1) via an ionic interaction that does not require
the consensus DIDS reaction motif. Alternatively, DIDS may covalently
react at another site on the transporter molecule. Distinguishing among
these possibilities will require a structure-function analysis of the
three different aspects of the interaction of DIDS with the
Na+-coupled HCO
transporters:
1) inhibition of transport, 2) ionic or
reversible binding, and 3) covalent or irreversible binding.
NBC4 in Liver
Probing human Northern blots with NBC4, Pushkin et al.
(32) found that the strongest signal appears in the liver.
So far, no other Na+-coupled HCO
transporters have been shown to be strongly expressed in liver: NBCe1
(1, 35), NBCn1 (11, 22, 31), NDCBE1 (3,
18), and NCBE (45). Hepatocytes carry out a
diversity of metabolic and transport functions that may severely
challenge pHi homeostasis in these cells. To counteract the
effects of changes in pHi produced by metabolism and
transport, hepatocytes have evolved canalicular (apical)
Cl
/HCO
exchangers as well as
sinusoidal (basolateral) Na+/H+ exchangers and
electrogenic Na+-HCO
cotransporters
(reviewed in Ref. 7). The importance of
Na+-HCO
cotransport for hepatocyte
pHi regulation is underscored by three observations:
1) steady-state pHi is lower in isolated
hepatocytes incubated in the absence of
CO2/HCO
(17); 2)
the acid extrusion rate during pHi recovery from an acid
load is markedly lower in the absence of
CO2/HCO
(16); and 3) at the higher pHi prevailing in the presence
of CO2/HCO
, the
Na+/H+ exchanger is virtually inactive
(34, 42). In addition, intracellular acidification in
hepatocytes produces a depolarization (presumably via pH-sensitive
K+ channels) that would increase the driving force for
HCO
entry via an electrogenic
Na+/HCO
cotransporter (15).
Inasmuch as NBC4 is electrogenic, DIDS sensitive, and strongly
expressed in liver, it is a good candidate for the major hepatic
Na+-HCO
cotransporter.
NBC4 in Kidney
The vast majority of renal HCO
reabsorption is
mediated by electrogenic Na+-HCO
cotransport in the proximal tubule. Using light microscopy, Schmitt et
al. (38) showed that NBCe1 is restricted to the proximal
tubule, specifically to the basolateral membrane of the S1 and S2
segments of the rat and rabbit proximal tubule. Maunsbach et al.
(28) confirmed and extended these observations on the rat
with immunoelectron microscopy, demonstrating that the antibody (raised
to the COOH terminus of NBCe1) labels an epitope on the cytoplasmic
side of the membrane. According to thermodynamic calculations, the
stoichiometry of the Na+-HCO
cotransporter would have to be at least 3:1 for the transporter to
mediate the net efflux of Na+ and HCO
across the basolateral membrane of the proximal tubule and oppose the
large electrochemical gradient favoring the entry of Na+
into the cell (6, 46). Indeed, Soleimani et al.
(41) determined that in isolated rabbit renal membrane
vesicles the stoichiometry of Na-HCO3 cotransport is 3:1.
However, when the kidney variant of NBCe1 is expressed in
Xenopus oocytes, it appears to operate with a 2:1
stoichiometry (21). There is, however, evidence that the
stoichiometry of NBCe1 is not fixed at a particular ratio but can
change (30). Gross et al. presented evidence that the HCO
:Na+ stoichiometry might shift from
2:1 to 3:1, depending on the cell type in which it is expressed
(19) and on the phosphorylation state of the protein
(20). In addition, the conditions within a particular cell
(such as intracellular Ca2+) may affect the stoichiometry
(29).
The only thing that is known about the renal localization of NBC4 is
that the message is present in the kidney lane of human Northern blots.
Thus it is not clear whether NBC4 participates in
HCO
reabsorption. Our results suggest that NBC4c
operates with a 2:1 stoichiometry in oocytes, so that NBC4c would be
expected to mediate net HCO
uptake (i.e., acid
extrusion) in most cells. In this context, NBC4c could help to protect
against intracellular acidification in cells throughout the kidney. If
present at an apical membrane, and operating with a 2:1 stoichiometry,
NBC4c could thus contribute to HCO
reabsorption. If
NBC4c were present at the basolateral membrane and functioned with a
3:1 stoichiometry in the proximal tubule, then it could contribute to
HCO
reabsorption in that nephron segment. In
principle, even with a 2:1 stoichiometry, basolateral NBC4 could
contribute to HCO
reabsorption elsewhere in the
nephron, provided that [Na+]i and/or
[HCO
]i were sufficiently high and/or
provided that the basolateral membrane potential were sufficiently negative.
NBC4 in Heart
Na+-HCO
cotransport plays an
important role in pHi regulation in mammalian cardiac
myocytes, in which it mediates the equivalent of HCO
uptake (2, 10, 24, 25). In guinea pig ventricular myocytes (24) and sheep cardiac Purkinje fibers (13),
this HCO
uptake appears to be voltage insensitive,
consistent with an electroneutral cotransporter. However, other reports
on rat myocytes (2) and cat papillary muscle
(10) indicate a strong voltage sensitivity, consistent
with an electrogenic cotransporter, possibly with a HCO
:Na+ stoichiometry of 2:1. The
molecular identity of the cotransporter(s) that mediate pHi
regulation in these different cell types and species is not known. At
the mRNA level, the two electrogenic transporters, NBCe1-B
(12) and NBC4 (32), as well as the
electroneutral NBCn1 (31) are all present in human heart.
At least NBCn1 is expressed in rat heart (11, 22). Thus
further work will be required to correlate functional
Na+-HCO
cotransport with specific NBC proteins in different cardiac cell types from various species.
 |
ACKNOWLEDGEMENTS |
We thank Dr. William Joiner for the gift of the Xenopus
expression vector.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-30344. L. V. Virkki was supported by
a fellowship from the American Heart Association.
1
The buffering power
of an oocyte was
calculated from the change in [HCO
]i
produced in control oocytes by exposure to 5% CO2/33 mM
HCO
, divided by the change in pHi, so
that
=
[HCO
]/
pHi.
2
A question that arises is why DIDS inhibits
acid-base transport by ~80% but has only a modest effect on
Vm. Because the oocyte is very "tight"
electrically, a small NBC4c current can drive Vm
very close to Erev. Thus, whereas the NBC4c
current and the rate of pHi recovery depend linearly on the
turnover rate of NBC4c, Vm does not
because of
the tightness of the oocyte, only a relatively small turnover is
required to drive Vm very close to
Erev.
Address for reprint requests and other correspondence:
L. V. Virkki, Dept. of Cellular and Molecular Physiology,
Yale Univ. School of Medicine, PO Box 208026, 333 Cedar St., New Haven,
CT 06520-8026 (E-mail:
leila.virkki{at}yale.edu).
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.
First published January 16, 2002;10.1152/ajpcell.00589.2001
Received 12 December 2001; accepted in final form 9 January 2002.
 |
REFERENCES |
1.
Abuladze, N,
Lee I,
Newman D,
Hwang J,
Boorer K,
Pushkin A,
and
Kurtz I.
Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter.
J Biol Chem
273:
17689-17695,
1998[Abstract/Free Full Text].
2.
Aiello, EA,
Petroff MG,
Mattiazzi AR,
and
Cingolani HE.
Evidence for an electrogenic Na-HCO3 symport in rat cardiac myocytes.
J Physiol
512:
137-148,
1998[Abstract/Free Full Text].
3.
Amlal, H,
Burnham CE,
and
Soleimani M.
Characterization of the Na+/HCO
cotransporter isoform NBC-3.
Am J Physiol Renal Physiol
276:
F903-F913,
1999[Abstract/Free Full Text].
4.
Bevensee, MO,
Schmitt BM,
Choi I,
Romero MF,
and
Boron WF.
An electrogenic Na+-HCO
cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain.
Am J Physiol Cell Physiol
278:
C1200-C1211,
2000[Abstract/Free Full Text].
5.
Boron, WF,
and
Boulpaep EL.
Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO
transport.
J Gen Physiol
81:
53-94,
1983[Abstract].
6.
Boron, WF,
and
Boulpaep EL.
The electrogenic Na/HCO3 cotransporter.
Kidney Int
36:
392-402,
1989[ISI][Medline].
7.
Boyer, JL,
Graf J,
and
Meier PJ.
Hepatic transport systems regulating pHi, cell volume, and bile secretion.
Annu Rev Physiol
54:
415-438,
1992[ISI][Medline].
8.
Burnham, CE,
Amlal H,
Wang Z,
Shull GE,
and
Soleimani M.
Cloning and functional expression of a human kidney Na+:HCO
cotransporter.
J Biol Chem
272:
19111-19114,
1997[Abstract/Free Full Text].
9.
Cabantchik, ZI,
and
Rothstein A.
The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives.
J Membr Biol
10:
311-328,
1972[ISI][Medline].
10.
Camilion de Hurtado, MC,
Perez NG,
and
Cingolani HE.
An electrogenic sodium-bicarbonate cotransport in the regulation of myocardial intracellular pH.
J Mol Cell Cardiol
27:
231-242,
1995[ISI][Medline].
11.
Choi, I,
Aalkjaer C,
Boulpaep EL,
and
Boron WF.
An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel.
Nature
405:
571-575,
2000[ISI][Medline].
12.
Choi, I,
Romero MF,
Khandoudi N,
Bril A,
and
Boron WF.
Cloning and characterization of a human electrogenic Na+-HCO
cotransporter isoform (hhNBC).
Am J Physiol Cell Physiol
276:
C576-C584,
1999[Abstract/Free Full Text].
13.
Dart, C,
and
Vaughan-Jones RD.
Na+-HCO
symport in the sheep cardiac Purkinje fibre.
J Physiol
451:
365-385,
1992[Abstract].
14.
Diakov, A,
Koch JP,
Ducoudret O,
Muller-Berger S,
and
Fromter E.
The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes.
Pflügers Arch
442:
700-708,
2001[ISI][Medline].
15.
Fitz, JG,
Lidofsky SD,
and
Scharschmidt BF.
Regulation of hepatic Na+-HCO
cotransport and pH by membrane potential difference.
Am J Physiol Gastrointest Liver Physiol
265:
G1-G8,
1993[Abstract/Free Full Text].
16.
Fitz, JG,
Lidofsky SD,
Xie MH,
Cochran M,
and
Scharschmidt BF.
Plasma membrane H+-HCO
transport in rat hepatocytes: a principal role for Na+-coupled HCO
transport.
Am J Physiol Gastrointest Liver Physiol
261:
G803-G809,
1991[Abstract/Free Full Text].
17.
Gleeson, D,
Smith ND,
and
Boyer JL.
Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes.
J Clin Invest
84:
312-321,
1989[ISI][Medline].
18.
Grichtchenko, II,
Choi I,
Zhong X,
Bray-Ward P,
Russell JM,
and
Boron WF.
Cloning, characterization, and chromosomal mapping of a human electroneutral Na+-driven Cl-HCO3 exchanger.
J Biol Chem
276:
8358-8363,
2001[Abstract/Free Full Text].
19.
Gross, E,
Hawkins K,
Abuladze N,
Pushkin A,
Cotton CU,
Hopfer U,
and
Kurtz I.
The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent.
J Physiol
531:
597-603,
2001[Abstract/Free Full Text].
20.
Gross, E,
Hawkins K,
Pushkin A,
Sassani P,
Dukkipati R,
Abuladze N,
Hopfer U,
and
Kurtz I.
Phosphorylation of Ser982 in the sodium bicarbonate cotransporter kNBC1 shifts the HCO
:Na+ stoichiometry from 3:1 to 2:1 in murine proximal tubule cells.
J Physiol
537:
659-665,
2001[Abstract/Free Full Text].
21.
Heyer, M,
Muller-Berger S,
Romero MF,
Boron WF,
and
Frömter E.
Stoichiometry of the rat kidney Na+-HCO
cotransporter expressed in Xenopus laevis oocytes.
Pflügers Arch
438:
322-329,
1999[ISI][Medline].
22.
Ishibashi, K,
Sasaki S,
and
Marumo F.
Molecular cloning of a new sodium bicarbonate cotransporter cDNA from human retina.
Biochem Biophys Res Commun
246:
535-538,
1998[ISI][Medline].
23.
Kopito, RR,
Lee BS,
Simmons DM,
Lindsey AE,
Morgans CW,
and
Schneider K.
Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger.
Cell
59:
927-937,
1989[ISI][Medline].
24.
Lagadic-Gossmann, D,
Buckler KJ,
and
Vaughan-Jones RD.
Role of bicarbonate in pH recovery from intracellular acidosis in the guinea-pig ventricular myocyte.
J Physiol
458:
361-384,
1992[Abstract].
25.
Leem, CH,
Lagadic-Gossmann D,
and
Vaughan-Jones RD.
Characterization of intracellular pH regulation in the guinea-pig ventricular myocyte.
J Physiol
517:
159-180,
1999[Abstract/Free Full Text].
26.
Lepke, S,
Becker A,
and
Passow H.
Mediation of inorganic anion transport by the hydrophobic domain of mouse erythroid band 3 protein expressed in oocytes of Xenopus laevis.
Biochim Biophys Acta
1106:
13-16,
1992[ISI][Medline].
27.
Lepke, S,
and
Passow H.
Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts.
Biochim Biophys Acta
455:
353-370,
1976[ISI][Medline].
28.
Maunsbach, AB,
Vorum H,
Kwon TH,
Nielsen S,
Simonsen B,
Choi I,
Schmitt BM,
Boron WF,
and
Aalkjaer C.
Immunoelectron microscopic localization of the electrogenic Na/HCO3 cotransporter in rat and ambystoma kidney.
J Am Soc Nephrol
11:
2179-2189,
2000[Abstract/Free Full Text].
29.
Muller-Berger, S,
Ducoudret O,
Diakov A,
and
Frömter E.
The renal Na-HCO
cotransporter expressed in Xenopus laevis oocytes: change in stoichiometry in response to elevation of cytosolic Ca2+ concentration.
Pflügers Arch
442:
718-728,
2001[ISI][Medline].
30.
Planelles, G,
Thomas SR,
and
Anagnostopoulos T.
Change of apparent stoichiometry of proximal-tubule Na+-HCO
cotransport upon experimental reversal of its orientation.
Proc Natl Acad Sci USA
90:
7406-7410,
1993[Abstract].
31.
Pushkin, A,
Abuladze N,
Lee I,
Newman D,
Hwang J,
and
Kurtz I.
Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family.
J Biol Chem
274:
16569-16575,
1999[Abstract/Free Full Text].
32.
Pushkin, A,
Abuladze N,
Newman D,
Lee I,
Xu G,
and
Kurtz I.
Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family.
Biochim Biophys Acta
1493:
215-218,
2000[ISI][Medline].
33.
Pushkin, A,
Abuladze N,
Newman D,
Lee I,
Xu G,
and
Kurtz I.
Two C-terminal variants of NBC4, a new member of the sodium bicarbonate cotransporter family: cloning, characterization, and localization.
IUBMB Life
50:
13-19,
2000[ISI][Medline].
34.
Renner, EL,
Lake JR,
Persico M,
and
Scharschmidt BF.
Na+-H+ exchange activity in rat hepatocytes: role in regulation of intracellular pH.
Am J Physiol Gastrointest Liver Physiol
256:
G44-G52,
1989[Abstract/Free Full Text].
35.
Romero, MF,
Fong P,
Berger UV,
Hediger MA,
and
Boron WF.
Cloning and functional expression of rNBC, an electrogenic Na+-HCO
cotransporter from rat kidney.
Am J Physiol Renal Physiol
274:
F425-F432,
1998[Abstract/Free Full Text].
36.
Romero, MF,
Hediger MA,
Boulpaep EL,
and
Boron WF.
Expression cloning and characterization of a renal electrogenic Na+/HCO
cotransporter.
Nature
387:
409-413,
1997[ISI][Medline].
37.
Romero, MF,
Henry D,
Nelson S,
Harte PJ,
Dillon AK,
and
Sciortino CM.
Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter.
J Biol Chem
275:
24552-24559,
2000[Abstract/Free Full Text].
38.
Schmitt, BM,
Biemesderfer D,
Romero MF,
Boulpaep EL,
and
Boron WF.
Immunolocalization of the electrogenic Na+-HCO
cotransporter in mammalian and amphibian kidney.
Am J Physiol Renal Physiol
276:
F27-F36,
1999[Abstract/Free Full Text].
39.
Sciortino, CM,
and
Romero MF.
Cation and voltage dependence of rat kidney electrogenic Na+-HCO
cotransporter, rkNBC, expressed in oocytes.
Am J Physiol Renal Physiol
277:
F611-F623,
1999[Abstract/Free Full Text].
40.
Siebens, AW,
and
Boron WF.
Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubules.
J Gen Physiol
90:
799-831,
1987[Abstract].
41.
Soleimani, M,
Grassl SM,
and
Aronson PS.
Stoichiometry of Na+-HCO
cotransport in basolateral membrane vesicles isolated from rabbit renal cortex.
J Clin Invest
79:
1276-1280,
1987[ISI][Medline].
42.
Stewart, DJ.
Sodium-proton exchanger in isolated hepatocytes exhibits a set point.
Am J Physiol Gastrointest Liver Physiol
255:
G346-G351,
1988[Abstract/Free Full Text].
43.
Taylor, AM,
Zhu Q,
and
Casey JR.
Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface.
Biochem J
359:
661-668,
2001[ISI][Medline].
44.
Virkki LV, Wilson D, Vaughan-Jones RD, and Boron WF.
Characterization of "NBC4" as an electrogenic NBC (Abstract).
FASEB J. In press.
45.
Wang, CZ,
Yano H,
Nagashima K,
and
Seino S.
The Na+-driven Cl
/HCO
exchanger: cloning, tissue distribution, and functional characterization.
J Biol Chem
275:
35486-35490,
2000[Abstract/Free Full Text].
46.
Yoshitomi, K,
Burckhardt BC,
and
Frömter E.
Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule.
Pflügers Arch
405:
360-366,
1985[ISI][Medline].
Am J Physiol Cell Physiol 282(6):C1278-C1289
0363-6143/02 $5.00
Copyright © 2002 the American Physiological Society