Departments of 1 Physiology and Biophysics and 2 Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970
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ABSTRACT |
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Recently, we
reported the cloning and expression of the rat renal electrogenic
Na+-HCO3
cotransporter (rkNBC) in Xenopus
oocytes [M. F. Romero, P. Fong, U. V. Berger, M. A. Hediger, and
W. F. Boron. Am. J. Physiol. 274 (Renal Physiol. 43): F425-F432,
1998]. Thus far, all NBC cDNAs are at least 95% homologous.
Additionally, when expressed in oocytes the NBCs are
1) electrogenic,
2)
Na+ dependent,
3)
HCO
3 dependent, and
4) inhibited by stilbenes such as
DIDS. The apparent
HCO
3:Na+
coupling ratio ranges from 3:1 in kidney to 2:1 in pancreas and brain
to 1:1 in the heart. This study investigates the cation and voltage
dependence of rkNBC expressed in
Xenopus oocytes to better understand
NBC's apparent tissue-specific physiology. Using two-electrode voltage clamp, we studied the cation specificity, Na+ dependence, and the
current-voltage
(I-V)
profile of rkNBC. These experiments indicate that
K+ and choline do not stimulate
HCO
3-sensitive currents via rkNBC, and
Li+ elicits only 3 ± 2% of
the total Na+ current. The
Na+ dose response studies show
that the apparent affinity of rkNBC for extracellular
Na+ (~30 mM
[Na+]o)
is voltage and HCO
3 independent,
whereas the rkNBC
I-V
relationship is Na+ dependent. At
[Na+]o
vmax (96 mM), the
I-V
response is approximately linear; both inward and outward
Na+-HCO
3
cotransport are observed. In contrast, only outward cotransport occurs
at low
[Na+]o
(<1 mM
[Na+]o).
All rkNBC currents are inhibited by extracellular application of DIDS,
independent of voltage and
[Na+]o.
Using ion-selective microelectrodes, we monitored intracellular pH and
Na+ activity. We then calculated
intracellular [HCO
3] and,
with the observed reversal potentials, calculated the stoichiometry of
rkNBC over a range of
[Na+]o
values from 10 to 96 mM at 10 and 33 mM
[HCO
3]o. rkNBC stoichiometry is 2 HCO
3:1
Na+ over this entire
Na+ range at both
HCO
3 concentrations. Our results indicate that rkNBC is highly selective for
Na+, with transport direction and
magnitude sensitive to
[Na+]o
as well as membrane potential. Since the rkNBC protein alone in oocytes
exhibits a stoichiometry of less than the 3 HCO
3:1 Na+ thought necessary for
HCO
3 reabsorption by the renal
proximal tubule, a control mechanism or signal that alters its in vivo
function is hypothesized.
sodium/bicarbonate cotransport; NBC; Xenopus oocyte expression; intracellular pH; sodium transport; bicarbonate transport; kinetics; voltage clamp
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INTRODUCTION |
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THE ELECTROGENIC
Na+-HCO3
cotransporter was first described in the renal proximal tubule (8) and
later cloned from the salamander kidney by functional expression using Xenopus oocytes (34). By homology,
several
Na+-HCO
3
cotransporter (NBC) cDNA isoforms have been cloned from different
mammalian tissues including rat kidney (33), human kidney (9), human
heart (13), human pancreas (2, 29), and brain (5). These proteins are
either 1,035 (kidney) or 1,079 (other organs) amino acids in length and
are at least 95% identical to one another. Variation in sequence
occurs predominantly in the
NH2-terminal 45-85 amino
acids. Expression studies in oocytes (13, 33, 34) show that the basic
functions of these NBC isoforms are similar: they are
1) electrogenic,
2) Na+ dependent,
3)
HCO
3 dependent, and
4) inhibited by stilbenes such as DIDS.
Although
Na+-HCO3
cotransport systems are physiologically implicated in many tissues,
these cotransporters appear to function differently depending on the
tissue. NBC is located at the basolateral membrane of renal proximal
tubule cells (37) and electrogenically moves
Na+/HCO
3
out of the cell into the blood. This mechanism is responsible for
80-90% of HCO
3 reabsorbed in the
kidney. However, in pancreatic ductal cells,
Na+/HCO
3
influx is thought to occur (19). In cultured hippocampal glial cells
(6, 36) and in the eye (21, 23), both
HCO
3 influx and efflux have been measured and are electrogenic (6, 26, 27). Yet recovery from an acid
load elicited by increasing heart rate in cat papillary muscle is also
attributed to electrogenic
Na+/HCO
3
influx (3, 10, 11), whereas experiments in guinea pig ventricular
myocytes indicate that the
Na+-HCO
3
cotransport is electroneutral (24).
Electrogenic NBC transport moves a net negative charge in the direction
of transport (i.e., a ratio of
HCO3:Na+ > 1:1). Both electrochemical gradients and cell membrane potential dictate the direction of ion flux. The
HCO
3:Na+
coupling ratio has been used as a predictor of transport direction. The
larger this ratio, the more effective the cell potential acts as a
driving force to move
Na+/HCO
3
out of the cell against the Na+
gradient. Studies of the electrogenic
Na+/HCO
3
cotransporter in renal tubules (48), proximal tubule cell lines (16),
and vesicles made from rabbit basolateral membranes (39) predict a
HCO
3:Na+
coupling of 3:1. It is thought that this ratio is
necessary to move HCO
3 out of the
proximal tubule cell across the basolateral membrane. However, the
Na+-HCO
3
cotransporter in the pancreas and brain is thought to have a coupling
ratio of 2:1 (6, 15, 19) and the heart may either be 2:1 (11) or 1:1
(14, 24).
Although different physiological characteristics have been attributed
to these different tissues, the NBC clones are greater than 95%
homologous. Thus the purpose of the present study was to elucidate NBC
transport characteristics to understand how and if NBC can fulfill all
of these roles. Specifically, we expressed rkNBC in
Xenopus oocytes to
1) determine the monovalent cation specificity, 2) define the voltage
dependence, and 3) determine apparent affinity for extracellular
Na+
([Na+]o).
For rkNBC specificity, all ionic and current changes attributed to
rkNBC are inhibitable by the stilbene, DIDS. From measurements of the
[Na+]o
dose response of rkNBC-stimulated HCO3
current, and with measurements of intracellular pH
(pHi) and sodium activity (aNai),
we directly calculate the stoichiometry of rkNBC-mediated Na+-HCO
3
cotransport over a range of
[Na+]o
levels. Interestingly, our studies indicate that
the stoichiometry of rkNBC is 2 HCO
3:1
Na+, rather than 3:1 as previously
reported for in vitro tissue studies, and is independent of the
Na+ gradient and
[HCO
3]o.
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METHODS |
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Oocyte Experiments
Oocyte isolation and injection. Xenopus laevis were purchased from Xenopus Express (Beverly Hills, FL). Oocytes were removed and collagenase dissociated as previously described (32, 33). To optimize rkNBC expression, we used a rkNBC-cDNA construct in the Xenopus expression vector pTLN2 (33). Capped cRNA was synthesized using a linearized cDNA template and the SP6 mMessage mMachine kit (Ambion, Austin, TX). Oocytes were injected with 50 nl of rkNBC cRNA (0.2 µg/µl) or water and incubated at 18°C in OR3 media (32). Oocytes were studied 3-10 days after injection. Each experimental procedure was studied on at least two batches of oocytes from different Xenopus to account for possible biologic variations between animals.Electrophysiology
Solutions. Experimental solutions are detailed in Table 1.
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Two-electrode voltage clamp. Oocyte
currents were recorded with OC-720C voltage clamp (Warner Instruments,
Hamden, CT). Electrodes were fashioned from borosilicate glass using a
model P-97 puller (Sutter, Novato, CA). Electrode tips were filled with
1% agarose/3 M KCl and backfilled with 3 M KCl. Current and voltage
electrodes had resistances of 0.5-1 M. Current signals were
filtered with an eight-pole Bessel filter (
3-dB cutoff,
frequency of 2-5 kHz) and digitized at 10 kHz.
Current and voltage signals were acquired via an EPC-16 I/O interface
using Pulse software, and data were analyzed using the PulseFit program
(HEKA). Oocytes were clamped at a holding potential
(Vh) of
60 mV; and current was constantly monitored and recorded at 1 Hz. Initial experiments determined that currents elicited by voltage
steps saturated in 50 ms to steady-state levels. Thus current-voltage
(I-V)
protocols consisted of 70-ms steps from
Vh to potentials
from
160 mV to 60 mV in 20-mV steps. The mean
steady-state current is plotted against voltage (Fig. 2,
A-E).
Ion-selective microelectrode.
Ion-selective microelectrodes were used to monitor
pHi and
aNai
of rkNBC and water-injected oocytes as previously described (33).
Intracellular ion activity was measured as the difference between the
ion-selective electrode (pH or
Na+) and a KCl voltage electrode
impaled into the oocyte; membrane potential
(Vm) was
measured as the potential difference between the KCl microelectrode and
an extracellular calomel (33, 34). Briefly, ion-selective
microelectrodes were fabricated using filamented borosilicate glass
pulled to 0.5-µm tips and silanized at 210°C with
bis-(dimethylamino)dimethylsilane
(Fluka, Ronkonkoma, NY), and the shanks were coated with Sylgard (Dow
Corning, Midland, MI). Micropipettes were cooled under vacuum, and the
tips were filled with either Na+
ionophore cocktail A or H+
ionophore I-cocktail B ion-selective resin (Fluka
Chemical). Na+
electrodes were backfilled with 150 mM NaCl.
H+ electrodes were backfilled with
(in mM) 40 KH2PO4,
23 NaOH, and 15 NaCl, pH 7.0. pH electrodes were calibrated using pH
6.0 and 8.0 (traceable National Bureau Standards; Fisher Scientific,
Pittsburgh, PA) followed by point calibration in ND96 (pH 7.50, solution 1, Table 1).
Na+ electrodes were calibrated
with 10 mM and 100 mM NaCl, and the specificity was checked using 100 mM KCl, followed by point calibration in ND96 (96 mM
Na+).
Na+ electrodes had a
selectivity1
of 52 ± 5 for Na+ over
K+ (calculated as described by
Abdulnour-Nakhoul and coworkers Ref. 1). Both types of
ion-selective electrodes had slopes of at least 56 mV/decade change.
Buffering power was calculated as previously reported (35). Briefly,
the total apparent buffering power
(T, see Table
2) is defined as the change in
[HCO
3] before and after
application of
CO2/HCO
3
(once steady-state is reached) divided by the change in
pHi elicited from
the same solution changes, i.e.,
T =
[HCO
3]steady-state/
pHi.
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Cation selectivity and apparent extracellular
Na+
vmax.
The cation selectivity of rkNBC-mediated cotransport and the apparent
maximal extracellular
Na+-stimulated
HCO3 current
(vmax) were
measured using the bath solution protocol shown in Fig.
4A. The ability of
K+,
Li+, and choline to stimulate
HCO
3-dependent current via rkNBC is
tested by measuring cation current responses ± HCO
3. Briefly, an oocyte was perfused
with a test cation (solution 3, 4, or
5, Table 1)
non-HCO
3 solution and an I-V
relation was recorded. The bath solution was switched to the respective
HCO
3/test cation solution (1.5%
CO2/10 mM
HCO
3/pH 7.5, solutions 8-12 and
5, Table 1), i.e.,
Li+/ND96
(solution 3, Table 1) to
Li+/1.5%
CO2/HCO
3
(solution 10, Table 1). An
I-V relation was measured at the peak
HCO
3-stimulated current. The
HCO
3-dependent response was calculated as the difference between the two
I-V
relationships (Fig. 4). The
[Na+]o
at which rkNBC-mediated
HCO
3 current saturates (i.e., attains
an apparent current
vmax) was
determined using the solution protocol in Fig.
4A. The
HCO
3-mediated I-V
responses for
[Na+]o
ranging from 84 to 120 mM were tested. Solution osmolalities were
matched with choline (solutions 2,
6, 9,
13, Table 1).
[Na+]o
concentration dependence.
Figure
1A shows
a schematic of the experimental protocol used to study the
extracellular Na+ dependence of
rkNBC. Our protocol was designed to monitor
Na+-induced currents while
maintaining pHi and
aNai
at steady state. An oocyte was put into an ~100-µl perfusion
chamber and perfused at 8-10 ml/min. Dye exchange experiments
showed that the entire chamber volume was exchanged in <1 s.
Therefore, the initial current response of rkNBC cotransport can be
measured without cell rundown due to long mixing times. The
Vh (60 mV)
current of rkNBC-expressing oocytes was recorded for the duration of
the experiment at 1 Hz. A baseline
non-HCO
3
I-V
relation was recorded once current stabilized after initial electrode
impalements. The bath solution was changed to 1.5%
CO2/10 mM
HCO
3/96 mM
Na+ (solution
8, Table 1) or 5%
CO2/33 mM
HCO
3/96 mM
Na+ (solution
14, Table 1) for 10 min to allow a steady-state current and pHi to be reached (compare
Figs. 1B and
3B). Unless otherwise specified,
Na+ replacement was with choline.
The bath solution was changed to 0 Na+ (solution
12, Table 1) for 24 s, pulsed for 8 s to a test
Na+ (in mM 0, 1, 10, 24, 36, 48, 72, 84, 96), then to 96 mM Na+ for
24 s. An
I-V
relation was recorded at the peak current induced by each
Na+ change. This quick pulse
protocol maintains a steady-state
pHi and
aNai,
and baseline 0 Na+ current,
allowing three to six randomly ordered test measurements of
[Na+]o
per oocyte to be taken and ensures that test
Na+ responses are measured from
the same "fully outward" transport state of rkNBC (Figs.
1B and
2B).
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RESULTS |
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Voltage Dependence of rkNBC
In the presence of extracellular CO2/HCOFigure 2, A and
B, illustrates that addition of
extracellular
CO2/HCO3
stimulates a DIDS-inhibitable current in rkNBC oocytes not observed in
water-injected oocytes (Fig. 2C). In
the presence of 96 mM extracellular
Na+, the
I-V
response of rkNBC oocytes is almost linear, i.e., the direction and
magnitude of current
(Na+/HCO
3
transport) depends on
Vm. Positive
currents represent the net inward movement of
Na+/HCO
3
(Fig. 2F), whereas negative currents
represent the outward movement of
Na+/HCO
3.
At the reversal potentials,
Erev, there is no
net current (transport).
Erev values for
10 and 33 mM extracellular HCO
3 and
[Na+]o
from 10 to 96 mM are listed in Table 3.
These data show that decreasing extracellular
Na+ shifts
Erev more
positive. The positive shift of
Erev does not mirror the calculated shift in the Nernst potential for
Na+ (Table 3),
indicating that transport direction is not dependent on the
Na+ gradient alone. Furthermore,
for our conditions (Fig. 1),
pHi and thus
[HCO
3]i
do not change (Fig.
3B),
indicating that for the brief solution changes, the
HCO
3 gradient is approximately static.
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When Na+ is removed from the bath
solution, only negative current is observed (Fig. 2,
B and
E). These data indicate that
NBC-mediated influx of negative charge does not occur without
extracellular Na+. Additionally,
without extracellular Na+, the
negative currents are augmented, indicating increased outward Na+-HCO3
cotransport. Taken together, these data indicate that neither
Na+ nor
HCO
3 alone is capable of inducing
rkNBC currents. Moreover, all of these currents are inhibited by 200 µM bath DIDS (Fig. 2, A and
B), independent of extracellular
Na+ and
Vm.
Intracellular Ion Effects of rkNBC Expression
CO2/HCOCO2/HCO3
addition to rkNBC-oocytes also results in an
aNai
increase in concert with the hyperpolarization (Fig.
3D) but not in water-injected
controls (Fig. 3C). Nominal air
CO2 over several days
(days 0-3) provides sufficient
solution HCO
3 to raise initial
aNai
by ~2 mM in rkNBC oocytes over water controls. rkNBC oocytes exhibit
a pHi recovery (HCO
3 influx) over 5-10 min in
the continued presence of
CO2/HCO
3.
Removal of extracellular Na+
causes an immediate and reversible depolarization
(Na+/HCO
3
efflux), followed by a delayed fall in pHi (33) and
aNai
(Fig. 3D). Both
pHi and
aNai
responses of rkNBC-expressing oocytes are blocked by 200 µM DIDS (not shown).
Figure 3B also illustrates that
pHi can remain at steady state
upon short and repeated removals and replacement of extracellular Na+ (every 30 s). Thus we can
measure changes in whole cell current on the second time scale without
significantly disturbing the steady-state
[HCO3]i
or
aNai.
Cation Dependence of rkNBC
Li+ appears to substitute for Na+ for transport via the Na+HCO
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Extracellular Na+ Dependence of rkNBC
Determination of experimental
vmax.
We next determined the maximal Na+
response (vmax)
of rkNBC-expressing oocytes in constant to
CO2/HCO3.
Recording the
I-V
responses after varying
[Na+]o
between 84 and 120 mM (osmolalities matched with
choline), we found no difference between the 96 and 120 mM
Na+
I-V
response (Fig. 5,
top). Thus we conclude that the
[Na+]o
at which vmax
current is observed is ~96 mM. Even at supermaximal Na+ concentrations, DIDS inhibits
all
Na+/HCO
3
current responses (Fig. 5, top).
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Extracellular
Na+
concentration dependence.
We developed the solution protocol in Fig.
1A to study the extracellular
Na+ dose response profile of
rkNBC. As described above, a rkNBC oocyte reaches a
pHi and voltage steady state
within 8-10 min perfusion with
CO2/HCO3
at saturating
[Na+]o.
Figure 1B illustrates that the
resulting current, at
Vh =
60
mV, also plateaus within the 10-min initial perfusion. Figure 5,
middle, shows a set of
I-V
relations measured for a dose response experiment. The magnitude of
outward current (NaHCO3 influx) is decreased, and inward current
(NaHCO3 efflux) is augmented with decreasing extracellular Na+. As
noted above, Erev
shifts in the positive direction, but is not equated with the
Na+ reversal potential (Table 3).
We also found that 100 µM DIDS inhibits rkNBC current at all
Na+ concentrations tested, 0 to
120 mM (Fig. 5, top and
bottom), independent of
Na+ and voltage.
Calculation of apparent
K0.5.
Figures 5, middle, and Fig.
6A
illustrate that rkNBC transport direction is voltage and extracellular
Na+ dependent. From these
experiments, we calculated the apparent affinity coefficient of rkNBC
for extracellular Na+
(K0.5) of
Na+-HCO3
cotransport at each voltage measured. To plot the rkNBC-specific
currents, we subtracted the current at a given
[Na+]o
and Vm from the
current elicited at that same
Vm by the 0 Na+/CO2/HCO
3
solution, i.e., I
I0 Na+
(Fig. 6B). This subtraction yields
the full range of rkNBC activity (maximal outward
I to maximal inward
I). We fit these data at each pulse
Vm with a right
rectangular hyperbolic
function2
(Michaelis-Menten) and calculated the apparent
K0.5 as ~30 mM (Fig. 6). Remarkably, these data indicate that at every test
Vm (
160 to
+60 mV) that the apparent
K0.5 for
extracellular Na+ is ~30 mM.
Since all curves were similar, after normalizing currents, we grouped
all voltages for each test
[Na+] to generate a
composite graph (Fig. 6C).
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HCO3:Na+
stoichiometry of rkNBC.
Cotransport of Na+ and
HCO
3 through rkNBC can be described as
a coupled transport process by the chemical equation
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DISCUSSION |
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The process of electrogenic
Na+-HCO3
cotransport is functionally described in a variety of tissues, e.g., kidney, brain, heart, eye, and pancreas. Cloning of these NBC isoforms
reveals that they are 95% homologous at the amino acid level (30).
However, function in these tissues ranges from strict HCO
3 excretion (kidney), to mainly
HCO
3 influx
(pancreas),3
and both modes of transport (eye and heart). To begin to define the
determinants of NBC function, we studied the voltage- and cation-dependent properties of
Na+-HCO
3
cotransport expressed in Xenopus
oocytes. Our studies indicate that rkNBC is specific for
Na+ over
K+ and choline. These results are
consistent with studies of the human kidney isoform, indicating that
K+ is not a substrate for NBC (4).
However, our Li+ results differ,
i.e., Li+ minimally stimulates
rkNBC-mediated HCO
3 transport. Yet the
hkNBC and rkNBC isoforms are 97% homologous. Of the 22 amino acid
difference between the two clones, 10 alter charge. H502
in rat is switched to N502 in human and has the predicted location at
the putative extracellular portion of transmembrane span 3 (TM-3, L482
to F498). Such charge switches may sufficiently alter a
Na+ selectivity filter of NBC in
the human clone to allow Li+ but
not K+ to be transported. This
amino acid is conserved between the human kidney (9, 13, 29), human
heart (13), and human pancreas (2) NBC isoforms. The ability of human
NBC to cotransport Li+ could have
therapeutic consequences resulting from
Li+ administration. Such
Li+ cotransport capacity might not
be predicted to alter NBC renal function, but might affect both the
brain and the heart where NBC-mediated cotransport is bidirectional.
Since extracellular Na+ is high
and cytosolic Na+ is low, the
normal, physiological, electrochemical gradient for
Na+ is inward. Under this
condition, we find the
I-V
response of rkNBC-expressing oocytes is roughly linear. Extracellular
Na+ of 96 mM is the experimentally
determined saturation point of rkNBC current with bath
CO2/HCO3,
or the functional vmax for our
conditions. At this apparent
vmax, the
Erev is
94 mV and
100 mV for 10 and 33 mM extracellular
HCO
3, respectively. Only at voltages
more negative than
Erev is outward cotransport observed. At
73 mV (49), the
approximate basolateral membrane potential of mammalian, renal proximal
tubule cells, rkNBC-mediated cotransport in oocytes is inward (raising
both pHi and
aNai
compared with controls), not outward as expected for electrogenic
Na+-HCO
3
cotransporter function of the proximal tubule basolateral membrane. The
observed rkNBC function in oocytes is more comparable to that described
in tissues where NBC functions as an acid extruder, such as the brain
and pancreas. Our results suggest that a mechanism of directional
control for the cotransporter is present in the kidney and not found in
these other tissues. Additionally, our data predict that
variable modes of cotransport would occur with changes in cell membrane
potential, physiological or pathological. For example, Camilion de
Hurtado and coworkers (10, 11) reported that cat papillary muscle could
recover from an acid load caused by increased heart rate. These authors argued that depolarization during cardiac contraction increased the
activity of the electrogenic
Na+-HCO
3
cotransporter causing HCO
3 influx.
This finding is consistent with our data which show a depolarization
could move the membrane potential through
Erev of rkNBC
reversing the cotransport mode from outward to inward and thus increase
pHi.
Our data indicate that addition of the stilbene DIDS to
the bath solution inhibits both the inward and outward
Na+-HCO3
cotransport modes of rkNBC. Blockade is not overcome by increasing
extracellular Na+ or by altering
Vm, and with time
the blockade is irreversible (not shown). In the murine band 3 protein
(AE1), H2-DIDS covalently binds to
Lys539 and
Lys851 (28). The NBCs and the
anion exchangers (AE1-3) are the initial members of a
HCO
3 superfamily of transporters (30,
33) for which a predicted DIDS binding motif [K-(Y)-(X)-K for Y = M, L, and X = I, V, Y] seems to exist (30). rkNBC
has two putative motifs: 1) KMIK at
558-561 (near H502N described above) and
2) KLKK at 768-771. Perhaps
these sites are close to a substrate binding site or permeant path.
Upon DIDS binding, this path may be occluded or the regional structure
altered, leading to complete abolition of transport.
Using BSC1 cells to examine the native electrogenic
Na+-HCO3
cotransporter, Jentsch and associates (22) found that for 10 mM
HCO
3, the apparent
K0.5 for
extracellular Na+ was between 35 and 45 mM. To study the native electrogenic
Na+-HCO
3
cotransporter, presumably NBC, Gross and Hopfer (17) used a rat
proximal tubular cell line (SKPT-0193). Employing a
"zero-trans"
condition (15 mM intracellular
HCO
3 with no basolateral
Na+ present), these investigators
calculated the apparent
K0.5 for intracellular Na+ binding as ~18
mM. Nevertheless, it is unclear whether intra- and
extracellular Na+ affinities of
NBC are similar. For rkNBC expressed in
Xenopus oocytes, we calculate the
apparent K0.5 for
extracellular Na+ to be ~30 mM
at a "normal"
Vm (
60
mV). The Na+ sensitivity of rkNBC
cotransport appears unaffected by membrane potential. That is, over the
entire voltage, range the apparent K0.5 for
extracellular Na+ is ~30 mM
(Fig. 6). However, although the apparent
K0.5 is stable over the entire voltage range tested (
160 to +60 mV), the
current magnitude increases as
Vm becomes more
positive. Thus either more rkNBC protein is inserted into the plasma
membrane or the rate of cotransport is increasing. Since insertion and
retrieval of protein is unlikely to occur in the millisecond-to-second
time scale, the cotransport-mediated current increase likely indicates that the overall transport rate is voltage sensitive, yet extracellular Na+ binding is not. Moreover, with
physiological Na+ concentration
greater than 30 mM, it is unlikely that extracellular Na+ binding is a rate-limiting
step of cotransport.
This study was designed to elucidate some of the fundamental properties
of rkNBC in an isolated system. A
HCO3:Na+
stoichiometry of 3:1 is thought to be necessary for
HCO
3 efflux from renal proximal tubule
cells. This stoichiometry is necessary to overcome the
Na+ gradient and use the favorable
movement of charge down the voltage gradient. However, here we have
determined the stoichiometry of rkNBC expressed in
Xenopus oocytes as 2:1. This
stoichiometry is
[Na+]o
independent (Fig. 7), as evidenced by a stoichiometry of 2:1 between 10 and 96 mM extracellular Na+. For
normal physiological conditions (high extracellular
Na+) and
Vm more positive
than
80 mV, we find only inward
Na+-HCO
3
cotransport (outward current). This same stoichiometry is found at both
10 and 33 mM HCO
3 (Fig. 7). Taken
together, these results indicate that rkNBC alone can mediate
HCO
3 influx as described for some tissues (e.g., brain, heart, liver, and pancreas). However, these data
alone cannot explain the HCO
3
reabsorption by the renal proximal tubule cell. A recent report
examining rkNBC function in giant patches has also found
a 2 HCO
3:1 Na+ stoichiometry (18).
Additionally, several groups have measured or calculated a
stoichiometry of 3 HCO
3:1
Na+ for native membranes of
basolateral membrane vesicles (39), intact proximal tubules (48), or
proximal tubular cell monolayers (16). Although formally one could
hypothesize another NBC isoform in the kidney, Schmitt and associates
(37) in a recent immunolocalization study have found that two different
antibodies, specific for NBC, recognize a major protein at the
basolateral membrane of mammalian proximal tubules. The same study
explicitly demonstrated that these antibodies recognize the functional
and recombinant rkNBC expressed in
Xenopus oocytes. Moreover, in the
several years since NBC was cloned by expression (31, 34), no other
electrogenic Na+-HCO
3
cotransporter has been reported for the kidney nor have similar renal
clones appeared in the Expressed Sequence Tag (EST)
databases. Consequently, although formally possible, it seems unlikely
that another (novel and major protein) electrogenic
Na+-HCO
3
cotransporter mediating HCO
3 reabsorption in the mammalian proximal tubule, will be
found. It is possible that a factor(s) is absent in the
Xenopus oocyte but present
endogenously in HEK-293 cells or native proximal tubule membranes. Such
a factor might be regulated such that it, in turn, could shift the
activity of NBC from an acid extruder to that of an acid loader, i.e.,
HCO
3 reabsorption. Alternatively, an
as yet unknown endogenous protein of the
Xenopus oocyte could interact with
rkNBC to mask the "true" stoichiometry of rkNBC transport.
Additional studies using other heterologous expression systems or
planar lipid bilayers will be required to entirely rule out this latter possibility.
Our data suggest that in the intact proximal tubule or vesicles there
are likely other factors, such as binding partners or cellular factors,
which modify the rkNBC function allowing
NaHCO3 efflux
(HCO3 reabsorption). Regulation of
rkNBC activity or coupling by protein kinase A (PKA) and/or PKC might be important, since there is a predicted PKA phosphorylation site and
seven PKC consensus phosphorylation sites (30, 33). Such activation
could shift the cotransporter voltage dependence such that
Na+/HCO
3
efflux occurs. Currently, we have no information indicating that other
proteins associate with rkNBC. However, accessory proteins, such as
those recently illustrated with the Na/H exchangers (25, 46, 47), could
modify rkNBC function. Moreover, AE1 has long been known to form dimers
(7, 20, 43-45), associate with cytoskeletal proteins (12), and has
recently been shown to associate with carbonic anhydrase II (42).
Although it is unclear whether such modifications or protein
associations alter the transport function of either the AEs or the
NBCs, it is attractive speculation. By extension, tissue-specific
expression of a modulator could allow the same NBC protein to be used
for both HCO
3 influx and efflux in
different locations. The specific regulator(s) of rkNBC function that
allow HCO
3 efflux in the renal
proximal tubule is (are) yet unknown. Further studies that explore the
isolated protein properties will help to determine whether factors or
binding partners can alter NBC function.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Eberhard Frömter, Ulrich Hopfer, and Suzanne Müller-Berger for helpful suggestions on the manuscript. We thank Dr. Stephen Jones for suggesting how to present the Na+ current response data.
![]() |
FOOTNOTES |
---|
Portions of this work have been presented in preliminary form (38).
This work was supported by a grant from the American Heart Association (to M. F. Romero) and a HHMI-institutional grant (to Case Western Reserve University). C. M. Sciortino was supported by National Institute of Diabetes and Digestive and Kidney Diseases Predoctoral Fellowship DK-07678.
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
Selectivity for the ion of interest (i) over
the interfering ion (j) is calculated as
Kij = ai/[ajexp(zi/zj)] × 10exp[(Ej Ei)/m],
where a is activity an
m is the slope of the electrode. Na+ electrodes maintain their
calibrated slope even at 5 mM NaCl.
2 Note that similar results are obtained if the data are fit by a second order polynomial.
3
A preliminary report from our laboratory (41)
indicates that models of pancreatic
HCO3 absorption and secretion may not
include all of the relevant transporters. In rat pancreatic ductal
epithelia, NBC protein is mainly basolateral but is also present
apically. Moreover, the basolateral membranes of rat pancreatic acinar
cells also contain NBC protein.
Address for reprint requests and other correspondence: M. F. Romero, Dept. of Physiology & Biophysics, Case Western Reserve Univ. School of Medicine, 2119 Abington Rd., Cleveland, OH 44106-4970 (E-mail: mfr2{at}po.cwru.edu).
Received 17 December 1998; accepted in final form 18 May 1999.
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