INVITED REVIEW
Structural determinants and significance of regulation of
electrogenic Na+-HCO
cotransporter
stoichiometry
Eitan
Gross1 and
Ira
Kurtz2
1 Departments of Urology and Physiology and
Biophysics, Case Western Reserve University, and Veterans Affairs
Medical Center, Cleveland, Ohio 44106; and 2 Division of
Nephrology, David Geffen School of Medicine, University of
California, Los Angeles, California 90095
 |
ABSTRACT |
Na+-HCO
cotransporters play an important role in intracellular pH regulation
and transepithelial HCO
transport in various
tissues. Of the characterized members of the HCO
transporter superfamily, NBC1 and NBC4 proteins are known to be electrogenic. An important functional property of electrogenic Na+-HCO
cotransporters is their
HCO
:Na+ coupling ratio, which sets the
transporter reversal potential and determines the direction of
Na+-HCO
flux. Recent studies have shown that the HCO
:Na+ transport stoichiometry
of NBC1 proteins is either 2:1 or 3:1 depending on the cell type in
which the transporters are expressed, indicating that the
HCO
:Na+ coupling ratio can be regulated.
Mutational analysis has been very helpful in revealing the molecular
mechanisms and signaling pathways that modulate the coupling ratio.
These studies have demonstrated that PKA-dependent phosphorylation of
the COOH terminus of NBC1 proteins alters the transport
stoichiometry. This cAMP-dependent signaling pathway provides
HCO
-transporting epithelia with an efficient
mechanism for modulating the direction of
Na+-HCO
flux through the cotransporter.
bicarbonate; transport; sodium
 |
INTRODUCTION |
THE
BICARBONATE/CARBON DIOXIDE
(HCO
/CO2) system is the most important
buffer system in the extracellular fluid space (8).
CO2 and HCO
are in equilibrium according
to the following relationship
|
(1)
|
The uniqueness of this reaction is derived from the fact that the
components of the HCO
buffer system are under the
physiological control of two organs: the lungs, which modulate the
level of PCO2 in the blood by altering the
CO2 excretion rate, and the kidneys, which control the
HCO
concentration by absorbing the filtered
HCO
load and maintaining whole body proton balance.
The pKa of the H2CO3
HCO
+ H+ reaction is 3.57, and it would
appear that HCO
would not be an effective buffer.
However, because dissolved CO2 is in equilibrium with
H2CO3, Eq. 1 can be simplified to
CO2 + H2O
HCO
+ H+ (pK'a 6.1). Although the
pK'a is still significantly less than the
normal extracellular pH of 7.4, this system is an effective buffer
because the PCO2 can be regulated by changes in
alveolar ventilation. An additional reaction HCO
H+ + CO
can occur; however, the
pKa of this reaction is 10.1; therefore, the
CO
concentration is ~1:500 the
HCO
concentration at a pH of 7.4.
An essential role of the kidney in maintaining the blood
HCO
concentration at ~25 mM is to reclaim HCO
filtered through the glomeruli. The renal
proximal tubule is the site in the nephron where the greatest quantity
of HCO
is reabsorbed into the peritubular blood.
Proximal tubule transepithelial HCO
absorption is
mediated by the coupled secretion of protons into the lumen and the
transport of an equal number of base equivalents across the basolateral
membrane into the peritubular blood. Apical proton secretion is
mediated by an apical membrane Na+/H+ exchanger
(NHE3) and to a lesser degree, a vacuolar H+-ATPase
(8). The majority of basolateral plasma membrane
HCO
absorption is mediated by electrogenic
Na+-HCO
cotransport (7, 20, 45, 113, 125).
Unlike the proximal tubule, which absorbs HCO
from
the glomerular filtrate, the direction of transepithelial HCO
transport is in the secretory mode in several
organs, including the duodenum (5), pancreas (35,
51, 57, 66, 109), airway epithelium (111), and salivary glands (72, 126). In the pancreatic duct, the
concentration of HCO
can reach 140 mM in humans and
guinea pigs (12, 13). Given a total fluid secretion rate of ~1 liter/day, the human pancreas therefore secretes ~ 140 meq/day of HCO
, which drives sodium and water into
the lumen by electroosmotic coupling (30, 56, 127). Pancreatic and duodenal HCO
secretion play an
important role in buffering the hydrochloric acid load from the stomach
(5). Furthermore, recent evidence suggests that the
duodenum has a basolateral electrogenic Na+-
HCO
cotransport process (5, 53, 59, 92)
that mediates HCO
influx and protects the epithelium
from gastric acid-induced injury (5). In addition, by
elevating pancreatic ductal pH, HCO
secretion may
have an additional role in activating pancreatic enzymes
(35).
In contrast to the proximal tubule, the cellular model for pancreatic
duct transepithelial HCO
transport is less well
understood. In pancreatic ducts, a basolateral Na+-HCO
cotransport process plays a
major role in mediating basolateral HCO
influx, with
subsequent HCO
secretion across the apical membrane
(2, 41, 56, 57, 118, 127). This basolateral
Na+-HCO
cotransport process was also found to be electrogenic (41). The apical transport
mechanism(s) responsible for apical HCO
secretions have not been completely delineated. The traditional model for apical
HCO
exit in exchange for Cl
entry via
the anion exchanger is still questionable, as none of the known anion
exchanger (AE) isoforms has been found in the apical membrane of these
cells, nor has the obligatory dependence on apical Cl
been demonstrated. Greeley et al. (39) reported
stimulation of downregulated in adenoma (DRA; SLC26A3) and the anion
transporter PAT1 (SLC26A6) in cultured pancreatic duct cells (CFPAC-1)
transfected with CFTR. They also found high levels of apical DRA in
native mouse pancreatic duct cells by immunocytochemistry. While the presence of DRA may account for HCO
/Cl
exchange activity in some species, it does not explain the independence of HCO
exit on the presence of apical Cl
in guinea pigs and possibly humans (55).
Alternatively, it is conceivable that PAT1 plays a role in apical
HCO
exits in these cells. More recently, Ishiguro et
al. (55), using a Cl
-sensitive fluorophore
to measure intracellular Cl
concentrations, proposed a
model in which the luminal HCO
exit mode switches
from a mixed mechanism involving both anion exchange and an anion
conductance (most probably CFTR) at a high luminal Cl
concentration to a single mechanism involving only CFTR at a low
luminal Cl
concentration. A similar mechanism was
proposed to account for HCO
secretion in the rat
pancreatic duct (38, 87) and in airway submucosal gland
cells (31). An important role for CFTR in
HCO
secretion is also implicated by the finding of
impaired HCO
secretion in the pancreas of patients
with cystic fibrosis (27). Shumaker et al.
(110) have suggested that PKA stimulates basolateral HCO
influx in pancreatic duct cells through the
basolateral electrogenic Na+-HCO
cotransporter by activating the apical CFTR Cl
channel
and depolarizing the basolateral membrane potential. It was speculated
that the reduced number of functional CFTR Cl
channels in
the cystic fibrotic pancreas is responsible for impaired HCO
secretion due to an insufficient depolarization
of the basolateral membrane potential.
 |
PROXIMAL TUBULE AND PANCREATIC DUCT CELL MODELS |
Figure 1 illustrates the relative
roles that the electrogenic Na+-HCO
cotransporter plays in HCO
transport in the renal
proximal tubule and the pancreatic duct. These models represent the
presently accepted cellular mechanisms for transepithelial
HCO
transport in these two epithelia. In the renal
proximal convoluted tubule (PCT), the cotransporter supports
transepithelial HCO
absorption by mediating
basolateral efflux of Na+ and HCO
,
whereas in the pancreas the cotransporter supports transepithelial
HCO
secretion by mediating basolateral influx of
these ions. There are several questions that arise with regard to these
transport models. First, how does the cotransporter in the proximal
tubule transport Na+ and HCO
across the
membrane against their respective concentration gradients? Second, does
the same electrogenic Na+-HCO
cotransporter contribute to transepithelial Na+-HCO
cotransport in both epithelia? Third, what is the mechanism(s) for the difference in
Na+:HCO
stoichiometry in renal proximal tubule vs. pancreatic duct cells?

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Fig. 1.
Cellular models for transepithelial
HCO transport. In the renal proximal tubule
(A), kNBC1 mediates Na+ and
HCO efflux by coupling the transport of 3 HCO and 1 Na+ to the membrane potential.
In the exocrine pancreas (B), pNBC1 mediates Na+
and HCO influx by coupling the transport of 2 HCO to the downhill flux of Na+.
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 |
ELECTROGENIC NBC PROTEINS AND MEMBERS OF THE
HCO TRANSPORTER SUPERFAMILY |
NBC1 is a member of the HCO
transporter
superfamily (BTS), which includes the
Cl
/HCO
exchanger proteins AE1-AE3
(6, 24); the Na+-driven
Cl
/HCO
exchangers (40, 100,
124); the electroneutral Na+-HCO
cotransporter NBC3 (93, 94) [splice variant NBC2
(54); rat orthologue NBCn1(25)]; and the second known electrogenic Na+-HCO
cotransporter isoform NBC4 (95, 96, 104). AE4, which was
initially reported to be a Cl
/HCO
exchanger (118), may function as an electroneutral
Na+-HCO
cotransporter (88,
89). kNBC1, encoded by an alternate promoter in the NBC1
(SLC4A4) gene (3), is the main NBC1 variant expressed in
the basolateral membrane of the renal proximal tubule (2, 22, 99,
105), where it normally operates with a 3 HCO
:1 Na+ stoichiometry. pNBC1 [also
referred to as hhNBC (26), dNBC1 (59), and
NBC1b (114)] is the major NBC1 variant expressed in the
basolateral membrane of pancreatic ducts (1, 41, 73), where it normally operates with a 2:1 stoichiometry. Sequence alignment
of kNBC1 and pNBC1 reveals that the two variants are 93% identical to
each other, except that the 41 NH2-terminal amino acids of
kNBC1 are replaced by 85 in pNBC1. An additional NBC1 COOH-terminal
variant (rb2NBC) cloned from rat brain mediates electrogenic
Na+-HCO
cotransport; however, its
transport stoichiometry has not been measured (17).
Finally, the recently cloned NBC4c splice variant of NBC4, which shares
the greatest similarity at the amino acid level with NBC1 proteins,
functions as an electrogenic Na+-HCO
cotransporter in mammalian epithelial cells and is expressed in several
tissues, including brain, heart, kidney, testis, pancreas, liver, and
muscle (95, 96, 104).
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THERMODYNAMICS OF ELECTROGENIC NA+-COUPLED
HCO TRANSPORT |
Electrogenic Na+-HCO
cotransporters
are an example of a secondary active cotransporter, which couples the
transport of one solute(s) against its concentration gradient to the
electrochemical gradient of another solute(s). In the proximal tubule,
kNBC1 couples the uphill transport of Na+ and
HCO
to the membrane potential. The latter is
determined mainly by the concentration gradient of potassium, generated
by the action of the Na+-K+-ATPase.
Thermodynamics dictates that for the membrane potential to drive the
transport in the efflux direction, under average steady-state intra-
and extracellular concentrations of Na+ and
HCO
found in the proximal tubule, the cotransporter
should carry a net charge of
2 equivalents, e.g., 3 HCO
+ 1 Na+ or 1 CO
+ 1 HCO
+ 1 Na+. These stoichiometries
place the reversal potential of the cotransporter positive relative to
the membrane potential and therefore ensures efflux of these ions (Fig.
2). A 3 HCO
:1 Na+ stoichiometry was reported by us and by other groups
for basolateral Na+-HCO
cotransport
in the proximal tubule, although there are also reports of a
2:1 stoichiometry (see Table 1).

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Fig. 2.
Plot of the reversal potential
(Erev) vs. the stoichiometry (n) for
an electrogenic Na+-HCO cotransporter.
The plot was generated using Eq. 5 with a 10-fold
Na+ concentration gradient (high outside) and no
HCO gradient. The plot illustrates that a
basolateral cotransporter with a 2 HCO :1
Na+ stoichiometry such as pNBC1 in the pancreatic ducts
will mediate the basolateral influx of HCO . A
transporter with a 3:1 stoichiometry such as kNBC1 in the renal
proximal tubule will mediate basolateral HCO efflux.
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Several methods have been described in the literature for measurements
of HCO
:Na+ stoichiometry. However,
before describing present techniques for measuring the stoichiometry,
we will briefly discuss the basic thermodynamics of the cotransport
process. The cotransport of Na+ and HCO
across a membrane can be described by
|
(2)
|
where the subscripts i and o stand for intracellular and
extracellular compartments, respectively. The transport stoichiometry of the Na+-HCO
cotransporter can be
determined by finding Na+ and/or HCO
concentration gradients and membrane potentials at which no flux
through the transporter occurs because electrical and chemical driving
forces balance each other. The transporter reaction (Eq. 2)
is at equilibrium and no net flux occurs when
|
(3)
|
where
µNai
o is the in-to-out electrochemical
potential difference for Na+,
n
µHCO3o
i is the out-to-in
electrochemical potential difference for HCO
, and
n is the number of HCO
anions cotransported with each Na+ cation. Expressing the
electrochemical potential differences in terms of the relevant ion
concentrations and the membrane potential, Vm,
yields
|
(4)
|
where F, R, and T have their
usual meaning, and brackets and subscripts i and o stand for the intra-
and extracellular concentration of the indicated ions, respectively.
Vm at which no flux (current) occurs is called
reversal potential (Erev), and can be
experimentally determined from the current-voltage
(I-V) relationship. Once determined, Erev can be used to evaluate the actual
HCO
-to-Na+ transport ratio,
n, according to
|
(5)
|
Equation 5 suggests that Erev
depends logarithmically on the intra-to-extracellular ratio of
Na+ and HCO
concentrations and inversely on the cotransport ratio (Fig. 2). The prelogarithmic factor in Eq. 5 equals 26 mV for n = 2. Thus a 10-fold
change in extracellular Na+ concentration will result in an
initial change in membrane potential of 26 mV, whereas a 10-fold change
in extracellular HCO
concentration will be expected
to result in a 120-mV change. Seki et al. (107) have
measured the cotransport stoichiometry in microperfused proximal
tubules from rabbits and found it to be 2:1. They also measured the
initial rates of HCO
and Na+ fluxes, by
measuring intracellular pH and Na+ concetnration with a
microelectrode, in response to a 10-fold step reduction of
extracellular HCO
concentration, and found the
former flux to be about twice as large as the latter, thus reconfirming
the 2:1 stoichiometry determined independently by the membrane
potential measurements. Soleimani et al. (112, 113) used a
variation of the flux ratio method in measuring the uptake of
22Na+ into vesicles made from the basolateral
membrane of rabbit proximal tubule cells. We have used an apically
permeabilized preparation of high-resistance epithelial monolayers
grown on filters, in combination with an ion-substitution protocol, to
measure the I-V relationship of electrogenic
Na+-HCO
cotransporters at defined
Na+ concentration gradients (41, 43-47,
104). We then determined the reversal potential of the
cotransporter from the I-V relationships and
used Eq. 5 to calculate the stoichiometry. Figure
3 illustrates the experimental protocol
used to measure the I-V relationships of
electrogenic Na+-HCO
cotransporters.
Because thermodynamics describes net charge movements but not the
chemical identity of the ions that carry the charge, none of the
methods described above can distinguish between a stoichiometry that
involves the transport of 2 HCO
anions vs. 1 CO
divalent anion. Indeed, a 3:1 stoichiometry
could result from the transport of 3 HCO
:1
Na+ or 1 CO
:1 HCO
:1 Na+.. Furthermore, a 2:1 stoichiometry could result from
either the loss of one HCO
binding site or a change
from the transport of 1 CO
and 1 HCO
to 2 HCO
anions (76,
77, 90), possibly due to a conformational change in the
cotransporter that then leads to altered substrate affinity. A
distinction between these various transport modes is not presently possible. Because at an extracellular pH of 7.4 the concentration of
CO
is ~500-fold lower than that of
HCO
and ~1,000-fold lower at an intracellular pH
of 7.1, it might be predicted that the flux of CO
would be negligible compared with that of HCO
.
However, this assumption would not be correct if the binding constant
of the cotransporter for CO
is ~500- to
1,000-fold higher then that for HCO
.

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Fig. 3.
Experimental protocol for measurement of
current-voltage (I-V) relationship of an
electrogenic Na+-HCO cotransporter in
epithelial monolayers. A 5-fold Na+ concentration gradient
was applied across apically permeabilized cell monolayers in an Ussing
chamber by perfusing the basolateral compartment with a modified
HCO Ringer solution containing 50 mM Na+
and the apical compartment with a solution containing 10 mM
Na+. A: voltage pulse protocol used to collect
I-V relationships in the absence (solid line)
and presence (dotted line) of 2 mM DNDS. Voltage was stepped from 100
mV to 100 mV with 10-mV steps. B: I-V
relationships in the absence (filled circles) and presence (open
circles) of DNDS obtained by averaging the current at each voltage over
5 s. C: I-V relationships in the absence
(filled circles) and presence (open circles) of DNDS for a 5-fold
Na+ concentration gradient but in the nominal absence of
HCO /CO2. The difference between the
currents in B and those measured in C at the
corresponding voltages represent the DNDS-sensitive currents.
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REGULATION OF NBC1
HCO :NA+ STOICHIOMETRY |
Table 1 summarizes the HCO
:Na+
stoichiometries reported in different cell types. As can be seen from
Table 1, different stoichiometries have been reported for different
species, different tissues/cell types in the same species, and even for
the same cell type in the same species. While it is possible that the
various stoichiometries might be due to differences in the techniques
utilized in some of these studies (e.g., isotope fluxes into
basolateral vesicles vs. basolateral membrane potential changes in
response to alterations in peritubular HCO
and
Na+ concentrations), additional data suggest that this
explanation is insufficient. Specifically, when proximal tubules were
incubated in HCO
-containing Ringer, the
HCO
:Na+ stoichiometry of the
cotransporter was 2:1 (67, 77). However, when the
incubation medium was changed to DMEM + norepinephrine, the
stoichiometry changed to 3:1. In addition, Planelles et al. (90) has shown that the stoichiometry shifts from 3:1 to
2:1 in Necturus proximal tubules exposed to peritubular
isohydric hypercapnia. It was unclear until recently whether the
shift in stoichiometry involved more then one cotransporter, each
operating with a different HCO
:Na+
stoichiometry, or whether the same cotransporter protein could change
its stoichiometry. This important question has been addressed by us at
the molecular level with the cloning of the NBC1 electrogenic Na+-HCO
cotransporters.
After the cloning of the pancreatic variant of the electrogenic
Na+-HCO
cotransporter (1),
pNBC1, we found that it operated with an
HCO
:Na+ stoichiometry of 2:1 in
pancreatic duct cells (41). This result suggested that the
stoichiometry shift observed previously in the proximal tubule
(67, 77, 90) may involve different NBC1 variants. However,
more recent data from our laboratories using heterologous mammalian
expression systems have challenged this view. We have expressed kNBC1
and pNBC1 in two different cell lines, mPCT cells derived from a murine
proximal tubule and mCD cells derived from the collecting duct (Ref.
43, Table 1). Both NBC1 variants exhibited a
3:1 stoichiometry in mPCT cells and a 2:1 stoichiometry in mCD cells.
These findings are significant in two ways. First, they suggest that
the difference in HCO
:Na+ stoichiometry
in the pancreas and the kidney is not related to the difference between
the NH2 termini of pNBC1 and kNBC1, respectively. Second,
the data indicate that the stoichiometry of each cotransporter can be
regulated by cells and is not an inherent property of the transporter.
 |
PHOSPHORYLATION OF NBC1 PROTEINS |
To address the possibility that a cell can regulate the transport
stoichiometry of NBC1 proteins, we have studied second messengers known
to regulate epithelial HCO
transport that could also
be potential candidates as modulators of the cotransporter's stoichiometry. cAMP is a strong modulator of HCO
transport in both the kidney and the exocrine pancreas. In the renal
proximal tubule, cAMP regulates HCO
absorption by
decreasing the rate of apical Na+/H+ exchange
and basolateral sodium HCO
efflux (68, 74,
102). In the guinea pig interlobular pancreatic duct, Ishiguro
et al. (57) found that secretin, acetylcholine, and
forskolin stimulate HCO
secretion. The effect was
mediated by stimulation of the basolateral
Na+-HCO
cotransporter and was
independent of Na+/H+ exchange or vacuolar
H+-ATPase activity. In mPCT cells transfected with kNBC1,
the cAMP agonist 8-bromoadenosine 3',5'-cyclic monophosphate caused the stoichiometry of kNBC1 to shift from 3 HCO
:1 Na+ to 2 HCO
:1 Na+. Using
single-site mutagensis and PKA pharmacological inhibitors, we found
that effect to be mediated by PKA phosphorylation of Ser982
at the COOH terminus of kNBC1 (44). pNBC1 has an
equivalent COOH-terminal consensus PKA phosphorylation site at
Ser1026 and an additional site in its NH2
terminus (Thr49). Work in our laboratories is presently
underway to determine the role of these sites in regulation of the
stoichiometry of this NBC1 variant. Recently, Muller-Berger et al.
(76) have reported that elevation of intracellular
Ca2+ in Xenopus laevis oocytes
expressing kNBC1 caused the HCO
:Na+
stoichiometry to shift from 2:1 to 3:1. In light of our finding that
phosphorylation of Ser982 shifts the stoichiometry of kNBC1
from 3:1 to 2:1, it is possible that elevation of intracellular
Ca2+ in the oocyte activates a protein phosphatase that
dephosphorylates kNBC1-Ser982. It would be interesting in
this regard to determine the phosphorylation state of NBC1 proteins in
X. laevis oocytes.
How does the proximal tubule cell maintain intracellular pH homeostasis
if PKA-dependent phosphorylation induces a switch in the direction of
kNBC1-mediated HCO
transport from efflux to influx?
One possible mechanism is a parallel decrease in luminal proton efflux
via downregulation of NHE3 activity (Fig. 1). Potential mediators of
the cross talk between the basolateral and luminal membrane domains
include the following. 1) Changes in intracellular pH:
intracellular pH is a very potent modulator of NHE3 activity in the
proximal tubule due to a proton binding regulatory site on the
cytoplasmic face of the protein (14). There is an
exponential increase in exchanger-mediated Na+ influx with
decreasing intracellular pH below 7.0 and complete inhibition at a pH
above 7.2. We found an ~2-fold increase in the current through the
Na+-HCO
cotransporter in renal proximal tubule cells at pH 7.25-7.50 compared with that at pH 6.5 (47). 2) Protein-protein interaction:
Kurashima et al. (68) and Zhao et al. (128)
studied protein-protein interaction between NHE3 and NHERF-1 in CHO
cells. PKA phosphorylated Ser605 in the intracellular COOH
terminus of the protein, which was associated with inhibition of the
exchanger. Using two-dimensional phosphopeptide maps of NHE3
immunoprecipitated from metabolically labeled cells and
back-phosphorylation assays of NHE3 from the same cells, NHERF-1 was
shown to be required for NHE3 phosphorylation by PKA. In a more recent
study, Weinman et al. (123) used an NHERF-1-deficient cell
line to show that cAMP-mediated inhibition of
Na+-HCO
cotransport requires the
presence of NHERF-1 protein in these cells, although neither the
cotransporter nor NHERF-1 were direct targets of PKA phosphorylation
(123). Furthermore, the cotransporter and NHERF-1 did not
associate with each other on yeast-two-hybrid or coimmunoprecipitation
assays nor did they colocalize on immunocytochemistry. It was
hypothesized that NHERF-1 plays an important role in cAMP-mediated
inhibition of the cotransporter in these cells by mediating PKA
phosphorylation of a presently unidentified third protein. Taken
together, these results suggest that in the proximal tubule cAMP may
serve as a second messenger to coordinate changes in acid-base flux
between the basolateral and apical membrane domains.
 |
MECHANISM OF PHOSPHORYLATION-INDUCED STOICHIOMETRY SHIFT |
How does phosphorylation of Ser982 at the COOH
terminus of kNBC1 shift its stoichiometry from 3:1 to 2:1? To address
this question, one requires a better understanding of how
HCO
interacts with the cotransporter. Previously, we
presented a mathematical model that describes the interaction of
HCO
and Na+ with the cotransporter
(46). The model consists of 6 states connected by 12 voltage-dependent rate constants and with separate binding and release
steps for Na+ and HCO
(Fig.
4). Na+ and
HCO
are assumed to bind to the cotransporter
on one side and are released on the other side in an ordered
(sequential) rather than a random manner. This simplification is
further supported by the finding that the current through the cotransporter as a function of either Na+ or
HCO
concentration can be described by a
Michaelis-Menten formalism (46). To further simplify the mathematical derivation of the model, we grouped the binding and release of all three negative charge equivalents into one step. Fitting
the model to a series of I-V relationships
obtained at different concentrations of Na+ and
HCO
indicated that the binding of 3 HCO
anions (or 1 CO
and 1 HCO
) to the cotransporter is voltage dependent, with
an electrical coefficient of 0.2 at pH 7.5. This indicates that, on
average, HCO
"senses" ~20% of the membrane's
electric field on binding to the cotransporter or that the binding site
for HCO
is located about one-fifth of the electrical
distance into the membrane. This result raises the possibility that the
binding of HCO
to the cotransporter might be
regulated by modifying the electric field around its binding site.

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Fig. 4.
Six-state ordered-binding transport model of NBC1. The
rate constants for the forward (fi) and backward
(bi) reactions are modulated by voltage and/or ligand
concentration as described previously (46). The binding of
3 HCO (Bic) anions to the transporter is described
as a single, lumped step (see text). The model does not distinguish the
binding of 3 HCO anions vs. 1 CO
and 1 HCO .
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Is it conceivable that phosphorylation of Ser982 shifts the
stoichiometry from 3:1 to 2:1 by modifying the local electric field around the HCO
binding site and perhaps disrupting
the binding of HCO
? Examination of the amino acid
sequence of kNBC1 reveals that the region flanking Ser982
is highly charged with an acidic cluster downstream of
Ser982 consisting of four aspartic acid residues,
Asp984, Asp986, Asp988 and
Asp989, and a poly-lysine cluster upstream of
Ser982. The presence of a highly charged segment in the
vicinity of an HCO
binding site might alter the electric field around it and interfere with HCO
binding to the cotransporter (Fig.
5A). Whatever the mechanism might be by which the COOH terminus "interferes" with
HCO
binding, this model requires that it can only
occur after phosphorylation of Ser982. This could occur if
phosphorylation induces a conformational change in the COOH terminus
that enables the "plug" domain to occlude an HCO
binding site. A similar concept was proposed to explain removal of fast
N-type inactivation of voltage-dependent potassium channels
(Kv) after phosphorylation by PKC (11). In
these studies, the authors used a synthetic inactivation domain to
demonstrate the loss of overall structural stability, and, after
phosphorylation of Ser8, Ser15 and
Ser21 resulted in the dissociation of the peptide from its
binding site on the channel and loss of channel inactivation.
Similarly, Hayashi et al. (48) found that
growth-associated protein-43 undergoes a conformational change from
random coil to
-helix on interaction with an acidic phospholipid
membrane phase. This change in conformation, which is required for the
binding of calmodulin, was abolished when the protein was
phosphorylated by PKC. Thus one may speculate that phosphorylation of
Ser982 in the COOH terminus of kNBC1 (and possibly
Ser1026 in pNBC1) renders this region more flexible and
capable of competing with HCO
for binding to the
cotransporter.

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|
Fig. 5.
Hypothetical model illustrating a potential mechanism for
the PKA-induced shift in the HCO :Na+
transport stoichiometry of NBC1. A: PKA-dependent
phosphorylation of kNBC1-Ser982 or
pNBC1-Ser1026 permits negatively charged aspartic acid
residues in the COOH terminus of NBC1 proteins to interact
electrostatically with one putative HCO binding site
on the transporter, resulting in a HCO :
Na+ transport stoichiometry of 2:1. B: when
kNBC1-Ser982 or pNBC1-Ser1026 is
unphosphorylated, the putative HCO binding site in
the transporter is unblocked and available to bind
HCO , resulting in an
HCO :Na+ transport stoichiometry of 3:1.
Also depicted in grey is a second putative protein such as carbonic
anhydrase II that may interact electrostatically with negatively
charged residues within the COOH terminus of unphosphorylated NBC1
proteins.
|
|
Alternatively, it is possible that phosphorylation of
Ser982 disrupts the interaction of kNBC1 with a second
protein. The COOH-terminal acidic aspartic acid residues could interact
electrostatically with one or more putative proteins in proximal tubule
cells, thereby preventing the plugging of one putative
HCO
binding site. The important structural features
of protein-protein recognition sites include the number of contact
residues involved (10), the interface area
(97), the chemical nature of the interfaces
(71), residue packing (61), electrostatic
interactions (86), and steric strain (101).
The hypothesis that a second protein can interact electrostatically
with the COOH terminus of kNBC1, thereby preventing this acidic
COOH-terminal region from plugging a putative HCO
transport site, is based in part on accumulating structural and
mutational evidence that reveals a central role for electrostatic
contributions to protein-protein interaction For example, in the
Ras-related protein Rap1A, Asp37, Asp38, and
Asp57 mediate its electrostatic interaction with the Ras
binding domain of the Ras effector molecule c-Raf1, a serine/threonine
kinase, the crystal structure of which was recently solved
(81). Dynamic electrostatic interactions involving
aspartic acid residues have been reported that can mediate important
biological effects. Specifically, agonist-dependent activation of the
1-adrenergic receptor is postulated to involve the
disruption of an interhelical electrostatic interaction between
Asp125 and Lys331 in transmembrane domains
three and seven, respectively (91). In addition, the
cAMP-specific phosphodiesterase (PDE4) contains unique signature
regions UCR1 and UCR2 that interact electrostatically (15). Importantly, the interaction is disrupted by
PKA-dependent phosphorylation of UCR1 Ser54 that results in
the activation of PDE4. Phospholamban PLB is a peptide that binds to
the Ca2+-ATPase in sarcoplsmic reticulum of cardiac
myocytes, thereby attenuating the pump rate (60).
Phosphorylation of PLB by PKA relieves this inhibition by disrupting
the interaction between the two proteins. Similarly, it is possible
that the interaction of acidic COOH-terminal aspartic acid residues
with one or more putative proteins in proximal tubule cells containing
a band of basic residues is modulated by the PKA-dependent
phosphorylation of Ser982 in the kNBC1 COOH terminus.
 |
ELECTROSTATIC PROTEIN-PROTEIN INTERACTIONS INVOLVING BTS PROTEINS |
AE1, a member of the BTS that mediates
Cl
/HCO
exchange, interacts
electrostatically via asparate residues in its COOH terminus with basic
residues in the NH2 terminus of carbonic anhydrase II
(119-121). Functional studies have demonstrated that
carbonic anhydrase II stimulates the transport function of AE1, AE2,
and AE3 (114). Free cytosolic carbonic anhydrase II is
apparently not sufficient to support maximal anion exchanger function.
The electrostatic interaction between carbonic anhydrase II and the
anion exchangers is thought to minimize the distance for
HCO
diffusion between the two proteins (63, 114). In
addition, the highly acidic NH2 terminus of AE1, which has
16 aspartic acid residues, is thought to interact strongly with basic
residues in the catalytic center of fructose 1,6-bisphosphate aldolase
(79). The first 31 residues of AE1 contain 5 aspartic acid
and 11 glutamic acid residues, whereas the catalytic region required
for interaction with aldolase is strongly basic. AE1 can be
specifically displaced from fructose 1,6-bisphosphate aldolase by the
anionic compounds ATP and 2,3-diphosphoglycerate (79). The
highly charged NH2 terminus of AE1 also interacts electrostatically with the basic residues Lys191 and
Lys212 in glyceraldehyde-3-phosphate dehydrogenase
(98). The binding reaction is rapidly reversible and
involves the anion NH2 terminus of AE1 and the basic active
site of the enzyme. In addition, the glycolytic enzyme
phosphofructokinase interacts electrostatically with the
NH2 terminus of AE1 (50). Sequence alignment
of kNBC1 with AE1 reveals a homologous acidic cluster at the COOH
terminus of human AE1 (D887ADD). This acidic cluster is critical for
binding of AE1 to carbonic anhydrase II (119).
Furthermore, Sterling et al. (114) found that inhibition
of carbonic anhydrase II with acetazolamide in HEK-293 cells
transfected with AE1 inhibited the anion exchanger by 50-60%.
Carbonic anhydrase II is expressed in the cytoplasm of proximal tubule
cells and intercalated cells, and the loss of function mutations in the
enzyme leads to proximal and distal renal tubular acidosis and
depletion of collecting duct intercalated cells (21, 70,
80). It is possible that carbonic anhydrase II binds to the COOH
terminus of kNBC1 in proximal tubule cells, creating a local increase
in the HCO
concentration in the vicinity of the
HCO
binding site of kNBC1. A local increase in
HCO
concentration might further stimulate the flux
of the transporter when it operates in the 3:1 stoichiometry mode (Fig.
5B). It is yet to be determined whether carbonic anhydrase
II binds to kNBC1 and whether such a functional interaction exists
between the two proteins. We have recently shown that an acidic cluster
in the COOH terminus of kNBC1 (D986NDD) is required for the
PKA-dependent stoichiometry shift (42). Whether
PKA-dependent phosphorylation of kNBC1-Ser982
disrupts the putative interaction between kNBC1 and carbonic anhydrase
II remains to be determined. Carbonic anhydrase II is also highly
expressed in pancreatic ducts (9, 36, 84). A similar model
whereby carbonic anhydrase II interacts with pNBC1 may play an
important role in modulating the rate of HCO
secretion in the pancreas.
 |
CHEMICAL PROBES FOR HCO /CO
BINDING SITES |
Stilbene derivatives block ion transport mediated by several
members of the BTS proteins (1, 6, 22, 40, 65, 99, 100, 103, 104,
108, 116, 117, 122), suggesting that these compounds might be
useful in probing specific HCO
binding sites in
these proteins. NBC1 has two putative DIDS binding motifs: KMIK, at a
site homologous to the KLXK (X = I, Y) in the AE proteins, and a KLKK
motif at a more COOH-terminal site. NBC3, an electroneutral
Na+-HCO
cotransporter (69, 93,
94), is missing the first motif but does contain the second
putative DIDS binding motif (944-KLKK). NBC4, a new member of the BTS, is also missing the first of the two putative DIDS binding motifs but
retains the second motif (863-KLKK). NBC4, cloned by Pushkin et al.
(95, 96, 104) is presently represented by four variants, NBC4a-NBC4d. When expressed in mPCT cells, NBC4c was found to be
electrogenic, with a stoichiometry of 3:1 (Table 1 and Ref. 104). These latter findings suggests that there is
probably no direct correlation between the
HCO
:Na+ stoichiometry and the number of
putative DIDS binding motifs. This conclusion is further augmented by
the finding that HCO
and DIDS do not compete on the
same binding site in AE1 (103). Recently, the
cyclooxygenase inhibitor tenidap has been shown to be a potent
inhibitor of kNBC1 expressed in X. laevis oocytes (32). The inhibitory potency of kNBC1 by tenidap is
similar to its inhibition of the
Cl
/HCO
exchange (75).
Whether tenidap can be utilized to probe the HCO
or CO
binding sites in NBC1 or other members of the
BTS is yet to be determined.
 |
SUMMARY AND FUTURE DIRECTIONS |
Significant progress has been made in characterizing the molecular
mechanisms involved in regulating the
HCO
:Na+ transport stoichiometry of NBC1.
The finding that phosphorylation of kNBC1 by PKA shifts its
HCO
: Na+ stoichiometry from 3:1 to 2:1
has important implications for HCO
transport in the
renal proximal tubule where kNBC1 normally mediates
HCO
reabsorption by operating with a 3:1
stoichiometry. The previous finding that PKA also modulates the
activity of NHE3 in these cells suggests that this second messenger may
also mediate cross talk between the basolateral and apical membrane
domains in proximal tubule cells. In the apical membrane, the
interaction between PKA and NHE3 is mediated by NHERF. Although
presently no NHERF isoforms have been localized to the basolateral
membrane of these cells, it is still possible that the interaction
between PKA and kNBC1 is also mediated by a third protein. PKA has been
shown to be compartmentalized in many cell types via A kinase anchoring proteins (28). It would thus be of interest to determine
whether A kinase anchoring proteins mediate PKA-kNBC1 or PKA-pNBC1
interaction. Additionally, kNBC1 and pNBC1 have several PKC consensus
phosphorylation sites whose role in modulating their transport
stoichiometry is presently being studied. The structural basis for the
unique coupling between the transport of Na+ and
HCO
by electrogenic
Na+-HCO
cotransporters is still largely unknown. Identification of the amino acids that participate in the
coordination of these ion ligands, and the distances between them in
three-dimensional space, could greatly assist in our
understanding of this question. Several photo- and chemically
labile probes have been previously used to identify amino acids that
participate in coordination of Na+ in other Na+
transport proteins such as NHE, ENaC, and the
Na+-K+-ATPase (33, 58, 64). A
similar approach may prove useful in identifying coordinating amino
acids in NBC1 proteins. Recent advances in crystallography of membrane
proteins (85) may also be applicable for structural
studies of electrogenic Na+-HCO
cotransporters. Finally, NMR and X-ray crystallography may also prove
useful in determining structural/conformational changes in the COOH
terminus of kNBC1 and pNBC1 after phosphorylation of Ser982
and Ser1026, respectively.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-46976, DK-58563, and DK-07789
(to I. Kurtz). E. Gross is supported by Cystic Fibrosis Foundation
Grant Gross01G0 and the American Heart Association Grant 9706507.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: I. Kurtz, Rm. 7-155 Factor Bldg., 10833 Le Conte Ave., Los Angeles, CA 90095 (E-mail:
Ikurtz{at}mednet.ucla.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.
10.1152/ajprenal.00148.2002
 |
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.
Abuladze, N,
Lee I,
Newman D,
Hwang J,
Pushkin A,
and
Kurtz I.
Axial heterogeneity of sodium-bicarbonate cotransporter expression in the rabbit proximal tubule.
Am J Physiol Renal Physiol
274:
F628-F633,
1998[Abstract/Free Full Text].
3.
Abuladze, N, M,
Song Pushkin A,
Newman D,
Lee I,
Nicholas S,
and
Kurtz I.
Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3.
Gene
251:
109-122,
2000[ISI][Medline].
4.
Aiello, EA,
Petroff MG,
Mattiazzi AR,
and
Cingolani HE.
Evidence for an electrogenic Na+-HCO
symport in rat cardiac myocytes.
J Physiol
512:
137-148,
1998[Abstract/Free Full Text].
5.
Akiba, Y,
Furukawa O,
Guth PH,
Engel E,
Nastaskin I,
Sassani P,
Dukkipatis R,
Pushkin A,
Kurtz I,
and
Kaunitz JD.
Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.
J Clin Invest
108:
1807-1816,
2001[Abstract/Free Full Text].
6.
Alper, SL.
The band 3-related anion exchanger (AE) gene family.
Annu Rev Physiol
53:
549-564,
1991[ISI][Medline].
7.
Alpern, RJ.
Mechanism of basolateral membrane H+/OH
/HCO
transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process.
J Gen Physiol
86:
613-636,
1985[Abstract].
8.
Alpern, R,
and
Rector RC, Jr.
Renal acidification mechanisms.
In: The Kidney, edited by Brenner BM,
and Rector FC, Jr.. Philadelphia, PA: Saunders, 1996, p. 408-471.
9.
Alvarez, L,
Fanjul M,
Carter N,
and
Hollande E.
Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1).
J Histochem Cytochem
49:
1045-1053,
2001[Abstract/Free Full Text].
10.
Amit, AG,
Mariuzza RA,
Phillips SE,
and
Poljak RJ.
Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution.
Science
233:
747-753,
1986[ISI][Medline].
11.
Antz, C,
Bauer T,
Kalbacher H,
Frank R,
Covarrubias M,
Kalbitzer HR,
Ruppersberg JP,
Baukrowitz T,
and
Fakler B.
Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.
Nat Struct Biol
6:
146-150,
1999[ISI][Medline].
12.
Argent, BE,
and
Case RM.
Pancreatic ducts. Cellular mechanism and control of bicarbonate secretion.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1994, p. 1473-1479.
13.
Argent, BE,
and
Gray MA.
Regulation and formation of fluid and electrolyte secretion by pancreatic ductal epithelium.
In: Biliary and Pancreatic Ductal Epithelia: Pathology and Pathophysiology, edited by Sirica AE,
and Longnecker DS.. New York: Dekker, 1997, p. 349-377.
14.
Aronson, PS,
Nee J,
and
Suhm MA.
Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles.
Nature
299:
161-163,
1982[ISI][Medline].
15.
Beard, MB,
Olsen AE,
Jones RE,
Erdogan S,
Houslay MD,
and
Bolger GB.
UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions.
J Biol Chem
275:
10349-10358,
2000[Abstract/Free Full Text].
16.
Bevensee, MO,
Apkon M,
and
Boron WF.
Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO
cotransport.
J Gen Physiol
110:
467-483,
1997[Abstract/Free Full Text].
17.
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].
18.
Bok, D,
Schibler MJ,
Pushkin A,
Sassani P,
Abuladze N,
Naser Z,
and
Kurtz I.
Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye.
Am J Physiol Renal Physiol
281:
F920-F935,
2001[Abstract/Free Full Text].
19.
Bonanno, JA,
and
Giasson C.
Intracellular pH regulation in fresh and cultured bovine corneal endothelium. II. Na+:HCO
cotransport and Cl
/HCO
exchange.
Invest Ophthalmol Vis Sci
33:
3068-3079,
1992[Abstract].
20.
Boron, WF,
and
Boulpaep EL.
Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO3 transport.
J Gen Physiol
81:
53-94,
1983[Abstract].
21.
Breton, S,
Alper SL,
Gluck SL,
Sly WS,
Barker JE,
and
Brown D.
Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F761-F774,
1995[Abstract/Free Full Text].
22.
Burnham, C,
Amlal H,
Wang Z,
Shull GE,
and
Soleimani M.
Cloning and functional expression of a human kidney Na+-HCO
cotransporter.
J Biol Chem
72:
19111-19114,
1997.
23.
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].
24.
Casey, JR,
and
Reithmeier RA.
Anion exchangers in the red cell and beyond.
Biochem Cell Biol
76:
709-713,
1998[ISI][Medline].
25.
Choi I, Aalkjaer C, Boulpaep EL, and Boron WF. An electroneutral
sodium/bicarbonate cotransporter NBCn1 and associated sodium channel.
Nature 405: 571-575.
26.
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].
27.
Choi, JY,
Muallem D,
Kiselyov K,
Lee MG,
Thomas PJ,
and
Muallem S.
Aberrant CFTR-dependent HCO
transport in mutations associated with cystic fibrosis.
Nature
410:
94-97,
2001[ISI][Medline].
28.
Coghlan, VM,
Perrino BA,
Howard M,
Langeberg LK,
Hicks JB,
Gallatin WM,
and
Scott JD.
Association of protein kinase A and protein phosphatase 2B with a common anchoring protein.
Science
267:
108-111,
1995[ISI][Medline].
29.
Deitmer, JW,
and
Schlue WR.
An inwardly directed electrogenic sodium-bicarbonate co-transport in leech glial cells.
J Physiol
411:
179-194,
1989[Abstract].
30.
De Ondarza, J,
and
Hootman SR.
Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts.
Am J Physiol Gastrointest Liver Physiol
272:
G124-G134,
1997[Abstract/Free Full Text].
31.
Devor, DC,
Singh AK,
Lambert LC,
DeLuca A,
Frizzell RA,
and
Bridges RJ.
Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.
J Gen Physiol
113:
743-760,
1999[Abstract/Free Full Text].
32.
Ducoudret, O,
Diakov A,
Muller-Berger S,
Romero MF,
and
Fromter E.
The renal Na-HCO
cotransporter expressed in Xenopus laevis oocytes: inhibition by tenidap and benzamil and effect of temperature on transport rate and stoichiometry.
Pflügers Arch
442:
709-717,
2001[ISI][Medline].
33.
Ellis-Davies, GC,
Kleyman TR,
and
Kaplan JH.
Photolabile amiloride derivatives as cation site probes of the Na,K-ATPase.
J Biol Chem
271:
10353-10358,
1996[Abstract/Free Full Text].
34.
Fitz, JG,
Persico M,
and
Scharschmidt BF.
Electrophysiological evidence for Na+-coupled bicarbonate transport in cultured rat hepatocytes.
Am J Physiol Gastrointest Liver Physiol
256:
G491-G500,
1989[Abstract/Free Full Text].
35.
Freedman, SD,
and
Scheele GA.
Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.
Ann N Y Acad Sci
713:
199-206,
1994[Abstract].
36.
Fujikawa-Adachi, K,
Nishimori I,
Sakamoto S,
Morita M,
Onishi S,
Yonezawa S,
and
Hollingsworth MA.
Identification of carbonic anhydrase IV and VI mRNA expression in human pancreas and salivary glands.
Pancreas
18:
329-335,
1999[ISI][Medline].
37.
Gleeson, D,
Smith ND,
and
Boyer JL.
Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO
cotransport.
J Clin Invest
84:
312-321,
1989[ISI][Medline].
38.
Gray, MA,
Greenwell JR,
and
Argent BE.
Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells.
J Membr Biol
105:
131-142,
1988[ISI][Medline].
39.
Greeley, T,
Shumaker H,
Wang Z,
Schweinfest CW,
and
Soleimani M.
Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells.
Am J Physiol Gastrointest Liver Physiol
281:
G1301-G1308,
2001[Abstract/Free Full Text].
40.
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].
41.
Gross, E,
Abuladze N,
Pushkin A,
Kurtz I,
and
Cotton CU.
The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO
:1 Na+.
J Physiol
531:
375-382,
2001[Abstract/Free Full Text].
42.
Gross, E,
Fedotoff O,
Pushkin A,
Abuladze N,
and
Kurtz I.
An aspartate cluster in the C-terminus of kNBC1 modulates the HCO
:Na+ stoichiometry (Abstract).
FASEB J
16:
A795,
2002.
43.
Gross, E,
Hawkins K,
Abuladze N,
Pushkin A,
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].
44.
Gross, E,
Hawkins K,
Pushkin A,
Abuladze N,
Hopfer U,
Sassani P,
Dukkipati R,
and
Kurtz I.
Phosphorylation of Ser982 in kNBC1 shifts the HCO
:Na+ stoichiometry from 3:1 to 2:1 in proximal tubule cells.
J Physiol
537:
659-665,
2001[Abstract/Free Full Text].
45.
Gross, E,
and
Hopfer U.
Activity and stoichiometry of Na:HCO3 cotransport in immortalized renal proximal tubule cells.
J Membr Biol
152:
245-252,
1996[ISI][Medline].
46.
Gross, E,
and
Hopfer U.
Voltage and co-substrate dependence of the Na-HCO3 cotransportrer kinetics in renal proximal tubule cells.
Biophys J
75:
810-824,
1998[Abstract/Free Full Text].
47.
Gross, E,
and
Hopfer U.
Effects of pH on kinetic parameters of the Na-HCO3 cotransporter in renal proximal tubule.
Biophys J
76:
3066-3075,
1999[Abstract/Free Full Text].
48.
Hayashi, N,
Matsubara M,
Titani K,
and
Taniguchi H.
Circular dichroism and 1H nuclear magnetic resonance studies on the solution and membrane structures of GAP-43 calmodulin-binding domain.
J Biol Chem
272:
7639-7645,
1997[Abstract/Free Full Text].
49.
Heyer, M,
Muller-Berger S,
Romero MF,
Boron WF,
and
Fromter E.
Stoichiometry of the rat kidney Na-HCO
cotransporter expressed in Xenopus laevis oocytes.
Pflügers Arch
438:
322-329,
1999[ISI][Medline].
50.
Higashi, T,
Richards CS,
and
Uyeda K.
The interaction of phosphofructokinase with erythrocyte membranes.
J Biol Chem
254:
9542-9550,
1979[ISI][Medline].
51.
Hootman, SR,
and
de Ondarza J.
Overview of pancreatic duct physiology and pathophysiology.
Digestion
54:
323-330,
1993[ISI][Medline].
52.
Hughes, BA,
Adorante JS,
Miller SS,
and
Lin H.
Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium.
J Gen Physiol
94:
125-150,
1989[Abstract].
53.
Isenberg, JI,
Ljungstrom M,
Safsten B,
and
Flemstrom G.
Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl
-HCO
exchange and NaHCO3 cotransport.
Am J Physiol Gastrointest Liver Physiol
265:
G677-G685,
1993[Abstract/Free Full Text].
54.
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].
55.
Ishiguro, H,
Naruse S,
Kitagawa M,
Mabuchi T,
Kondo T,
Hayakawa T,
Case RM,
and
Steward MC.
Chloride transport in microperfused interlobular ducts isolated from guinea-pig pancreas.
J Physiol
539:
175-189,
2002[Free Full Text].
56.
Ishiguro, H,
Steward MC,
Lindsay AR,
and
Case RM.
Accumulation of intracellular HCO
by Na+-HCO
cotransport in interlobular ducts from guinea-pig pancreas.
J Physiol
495:
169-178,
1996[Abstract].
57.
Ishiguro, H,
Steward MC,
Wilson RW,
and
Case RM.
Bicarbonate secretion in interlobular ducts from guinea-pig pancreas.
J Physiol
495:
179-191,
1996[Abstract].
58.
Ismailov, II,
Kieber-Emmons T,
Lin C,
Berdiev BK,
Shlyonsky VG,
Patton HK,
Fuller CM,
Worrell R,
Zuckerman JB,
Sun W,
Eaton DC,
Benos DJ,
and
Kleyman TR.
Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel.
J Biol Chem
272:
21075-21083,
1997[Abstract/Free Full Text].
59.
Jacob, P,
Christiani S,
Rossmann H,
Lamprecht G,
Vieillard-Baron D,
Muller R,
Gregor M,
and
Seidler U.
Role of Na+HCO
cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion.
Gastroenterology
119:
406-419,
2000[ISI][Medline].
60.
James, P,
Inui M,
Tada M,
Chiesi M,
and
Carafoli E.
Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum.
Nature
342:
90-92,
1989[ISI][Medline].
61.
Janin, J,
and
Chothia C.
The structure of protein-protein recognition sites.
J Biol Chem
265:
16027-16030,
1990[Free Full Text].
62.
Jensen, LJ,
Schmitt BM,
Berger UV,
Nsumu NN,
Boron WF,
Hediger MA,
Brown D,
and
Breton S.
Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis.
Biol Reprod
60:
573-579,
1999[Abstract/Free Full Text].
63.
Kifor, G,
Toon MR,
Janoshazi A,
and
Solomon AK.
Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase.
J Membr Biol
134:
169-179,
1993[ISI][Medline].
64.
Kleyman, TR,
and
Cragoe EJ.
Amiloride and its analogs as tools in the study of ion transport.
J Membr Biol
105:
1-21,
1988[ISI][Medline].
65.
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].
66.
Kuijpers, GA,
Van Nooy IG,
De Pont JJ,
and
Bonting SL.
The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors.
Biochim Biophys Acta
778:
324-331,
1984[ISI][Medline].
67.
Kunimi, M,
Seki G,
Hara C,
Taniguchi S,
Uwatoko S,
Goto A,
Kimura S,
and
Fujita T.
Dopamine inhibits renal Na+:HCO
cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats.
Kidney Int
57:
534-543,
2000[ISI][Medline].
68.
Kurashima, K,
Yu FH,
Cabado AG,
Szabo EZ,
Grinstein S,
and
Orlowski J.
Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms.
J Biol Chem
272:
28672-28679,
1997[Abstract/Free Full Text].
69.
Kwon, TH,
Pushkin A,
Abuladze N,
Nielsen S,
and
Kurtz I.
Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney.
Am J Physiol Renal Physiol
278:
F327-F336,
2000[Abstract/Free Full Text].
70.
Lai, LW,
Chan DM,
Erickson RP,
Hsu SJ,
and
Lien YH.
Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy.
J Clin Invest
101:
1320-1325,
1998[Abstract/Free Full Text].
71.
Lee, B,
and
Richards FM.
The interpretation of protein structures: estimation of static accessibility.
J Mol Biol
55:
379-400,
1971[ISI][Medline].
72.
Luo, X,
Choi JY,
Ko SB,
Pushkin A,
Kurtz I,
Ahn W,
Lee MG,
and
Muallem S.
HCO
salvage mechanisms in the submandibular gland acinar and duct cells.
J Biol Chem
276:
9808-9816,
2001[Abstract/Free Full Text].
73.
Marino, CR,
Jeanes V,
Boron WF,
and
Schmitt BM.
Expression and distribution of the Na+-HCO
cotransporter in human pancreas.
Am J Physiol Gastrointest Liver Physiol
277:
G487-G494,
1999[Abstract/Free Full Text].
74.
McKinney, TD,
and
Myers P.
Bicarbonate transport by proximal tubules: effect of parathyroid hormone and dibutyryl cyclic AMP.
Am J Physiol Renal Fluid Electrolyte Physiol
238:
F166-F174,
1980[Free Full Text].
75.
McNiff, P,
Robinson RP,
and
Gabel CA.
Reduction of intracellular pH by tenidap. Involvement of cellular anion transporters in the pH change.
Biochem Pharmacol
50:
1421-1432,
1995[ISI][Medline].
76.
Muller-Berger, S,
Ducoudret O,
Diakov A,
and
Fromter 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].
77.
Muller-Berger, S,
Nesterov VV,
and
Fromter E.
Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide.
Pflügers Arch
434:
383-391,
1997[ISI][Medline].
78.
Munsch, T,
and
Deitmer JW.
Sodium-bicarbonate cotransport current in identified leech glial cells.
J Physiol
474:
43-53,
1994[Abstract].
79.
Murthy, SN,
Liu T,
Kaul RK,
Kohler H,
and
Steck TL.
The aldolase-binding site of the human erythrocyte membrane is at the NH2 terminus of band 3.
J Biol Chem
256:
11203-11208,
1981[Abstract/Free Full Text].
80.
Nagai, R,
Kooh SW,
Balfe JW,
Fenton T,
and
Halperin ML.
Renal tubular acidosis and osteopetrosis with carbonic anhydrase II deficiency: pathogenesis of impaired acidification.
Ped Nephrol
11:
633-636,
1997[ISI][Medline].
81.
Nassar, N,
Horn G,
Herrmann C,
Scherer A,
McCormick F,
and
Wittinghofer A.
The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue.
Nature
375:
554-560,
1995[ISI][Medline].
82.
Newman, EA.
Sodium-bicarbonate cotransport in retinal Muller (glial) cells of the salamander.
J Neurosci
11:
3972-3983,
1991[Abstract].
83.
Newman, EA,
and
Astion ML.
Localization and stoichiometry of electrogenic sodium bicarbonate cotransport in retinal glial cells.
Glia
4:
424-428,
1991[ISI][Medline].
84.
Nishimori, I,
FujikawaAdachi K,
Onishi S,
and
Hollingsworth MA.
Carbonic anhydrase in human pancreas: hypotheses for the pathophysiological roles of CA isozymes.
Ann NY Acad Sci
880:
5-16,
1999[Abstract/Free Full Text].
85.
Nollert, P,
Navarro J,
and
Landau EM.
Crystallization of membrane proteins in cubo.
Methods Enzymol
343:
183-199,
2002[ISI][Medline].
86.
Norel, R,
Sheinerman F,
Petrey D,
and
Honig B.
Electrostatic contributions to protein-protein interactions: fast energetic filters for docking and their physical basis.
Protein Sci
10:
2147-2161,
2001[Abstract/Free Full Text].
87.
Novak, I,
and
Greger R.
Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport.
Pflügers Arch
411:
546-553,
1988[ISI][Medline].
88.
Parker, MD,
Boron WF,
and
Tanner MJ.
Characterization of human "AE4" as an electroneutral, sodium-dependent bicarbonate transporter (Abstract).
FASEB J
16:
A796,
2002.
89.
Parker, MD,
Ourmozdi EP,
and
Tanner MJ.
Human BTR1, a new bicarbonate transporter superfamily member and human AE4 from kidney.
Biochem Biophys Res Commun
282:
1103-1109,
2001[ISI][Medline].
90.
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].
91.
Porter, JE,
and
Perez DM.
Characteristics for a salt-bridge switch mutation of the
1b adrenergic receptor. Altered pharmacology and rescue of constitutive activity.
J Biol Chem
274:
34535-34538,
1999[Abstract/Free Full Text].
92.
Praetorius, J,
Hager H,
Nielsen S,
Aalkjaer C,
Friis UG,
Ainsworth MA,
and
Johansen T.
Molecular and functional evidence for electrogenic and electroneutral Na+-HCO
cotransporters in murine duodenum.
Am J Physiol Gastrointest Liver Physiol
280:
G332-G343,
2001[Abstract/Free Full Text].
93.
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].
94.
Pushkin, A,
Abuladze N,
Lee I,
Newman D,
Hwang J,
and
Kurtz I.
Mapping of the human NBC3 (SLC4A7) gene to chromosome 3p22.
Genomics
57:
321-322,
1999[ISI][Medline].
95.
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].
96.
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.
Int Un Biochem Mol Biol
50:
13-19,
2000.
97.
Rees, DC,
and
Lipscomb WN.
Refined crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 A resolution.
J Mol Biol
160:
475-498,
1982[ISI][Medline].
98.
Rogalski, AA,
Steck TL,
and
Waseem A.
Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell.
J Biol Chem
264:
6438-6446,
1989[Abstract/Free Full Text].
99.
Romero, MF,
Hediger MA,
Boulpaep EL,
and
Boron WF.
Expression cloning and characterization of a renal electrogenic Na/HCO3 cotransporter.
Nature
387:
409-413,
1997[ISI][Medline].
100.
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].
101.
Ruhlmann, A,
Kukla D,
Schwager P,
Bartels K,
and
Huber R.
Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region.
J Mol Biol
77:
417-436,
1973[ISI][Medline].
102.
Ruiz, OS,
and
Arruda JAL
Regulation of the renal Na-HCO3 cotransporter by cAMP and Ca-dependent protein kinases.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F560-F565,
1992[Abstract/Free Full Text].
103.
Salhany, JM.
Allosteric effects in stilbenedisulfonate binding to band 3 protein (AE1).
Cell Mol Biol
42:
1065-1096,
1996[ISI].
104.
Sassani, P,
Pushkin A,
Gross E,
Gomer A,
Abuladze N,
Dukkipati R,
Carpenito G,
and
Kurtz I.
Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter.
Am J Physiol Cell Physiol
282:
C408-C416,
2002[Abstract/Free Full Text].
105.
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-F38,
1999[Abstract/Free Full Text].
106.
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].
107.
Seki G, Coppola S, and Fromter E. 1993. The Na-HCO3
cotransporer operates with a coupling ratio of 2 HCO3 to 1 Na in isolated rabbit renal proximal tubule. Pflügers Arch
425: 409-416, 1993.
108.
Sekler, I,
Lo RS,
Mastrocola T,
and
Kopito RR.
Sulfate transport mediated by the mammalian anion exchangers in reconstituted proteoliposomes.
J Biol Chem
270:
11251-11256,
1995[Abstract/Free Full Text].
109.
Sewell, WA,
and
Young JA.
Secretion of electrolytes by the pancreas of the anaesthetized rat.
J Physiol
252:
379-396,
1975[Abstract].
110.
Shumaker, H,
Amlal H,
Frizzell R,
Ulrich CD., 2nd,
and
Soleimani M.
CFTR drives Na+- nHCO
cotransport in pancreatic duct cells: a basis for defective HCO
secretion in CF.
Am J Physiol Cell Physiol
276:
C16-C25,
1999[Abstract/Free Full Text].
111.
Smith, JJ,
and
Welsh MJ.
cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia.
J Clin Invest
89:
1148-1153,
1992[ISI][Medline].
112.
Soleimani, M,
and
Aronson PS.
Ionic mechanism of Na+:HCO
cotransport in renal basolateral membrane vesicles.
J Biol Chem
264:
18302-18308,
1989[Abstract/Free Full Text].
113.
Soleimani, M,
Grassl SM,
and
Aronson PS.
Stoichiometry of Na-HCO3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex.
J Clin Invest
79:
1276-1280,
1987[ISI][Medline].
114.
Sterling, D,
Reithmeier RA,
and
Casey JR.
A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers.
J Biol Chem
276:
47886-47894,
2001[Abstract/Free Full Text].
115.
Sun, XC,
Bonanno JA,
Jelamskii S,
and
Xie Q.
Expression and localization of Na+-HCO
cotransporter in bovine corneal endothelium.
Am J Physiol Cell Physiol
279:
C1648-C1655,
2000[Abstract/Free Full Text].
116.
Tsuganezawa, H,
Kobayashi K,
Iyori M,
Araki T,
Koizumi A,
Watanabe S,
Kaneko A,
Fukao T,
Monkawa T,
Yoshida T,
Kim DK,
Kanai Y,
Endou H,
Hayashi M,
and
Saruta T.
A new member of the HCO
transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney.
J Biol Chem
276:
8180-8189,
2001[Abstract/Free Full Text].
117.
Van't Hof, W,
Malik A,
Vijayakumar S,
Qiao J,
van Adelsberg J,
and
Al-Awqati Q.
The effect of apical and basolateral lipids on the function of the band 3 anion exchange protein.
J Cell Biol
139:
941-949,
1997[Abstract/Free Full Text].
118.
Villanger, O,
Veel T,
and
Raeder MG.
Secretin causes H+/HCO
secretion from pig pancreatic ductules by vacuolar-type H+-adenosine triphosphatase.
Gastroenterology
108:
850-859,
1995[ISI][Medline].
119.
Vince, JW,
Carlsson U,
and
Reithmeier RA.
Localization of the Cl
/HCO
anion exchanger binding site to the amino-terminal region of carbonic anhydrase II.
Biochemistry.
39:
13344-13349,
2000[ISI][Medline].
120.
Vince, JW,
and
Reithmeier RA.
Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1
/HCO
exchanger.
J Biol Chem
273:
28430-284377,
1998[Abstract/Free Full Text].
121.
Vince, JW,
and
Reithmeier RA.
Identification of the carbonic anhydrase II binding site in the Cl
/HCO
anion exchanger AE1.
Biochemistry
39:
5527-5533,
2000[ISI][Medline].
122.
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].
123.
Weinman, EJ,
Evangelista CM,
Steplock D,
Liu MZ,
Shenolikar S,
and
Bernardo A.
Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO
co-transporter in BSC-1 cells.
J Biol Chem
276:
42339-42346,
2001[Abstract/Free Full Text].
124.
Wolosin, JM,
Alvarez LJ,
and
Candia OA.
HCO
transport in the toad lens epithelium is mediated by an electronegative Na+-dependent symport.
Am J Physiol Cell Physiol
258:
C855-C861,
1990[Abstract/Free Full Text].
125.
Yoshitomi, K,
Burckhardt BC,
and
Fromter E.
Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule.
Pflügers Arch
405:
360-366,
1985[ISI][Medline].
126.
Young, JA,
Cook DI,
ew van Lennep,
and
Roberts M.
Secretion by the major salivary glands.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, vol. 1, p. 773-815.
127.
Zhao, H,
Star RA,
and
Muallem S.
Membrane localization of H+ and HCO
transporters in the rat pancreatic duct.
J Gen Physiol
104:
57-85,
1994[Abstract].
128.
Zhao, H,
Wiederkehr MR,
Fan L,
Collazo RL,
Crowder LA,
and
Moe OW.
Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase A and NHE-3 phosphoserines 552 and 605.
J Biol Chem
274:
3978-3987,
1999[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 283(5):F876-F887