RAPID COMMUNICATION
Functional characterization of NBC4: a new
electrogenic sodium-bicarbonate cotransporter
Pejvak
Sassani1,
Alexander
Pushkin1,
Eitan
Gross2,3,4,
Alla
Gomer4,
Natalia
Abuladze1,
Ramanath
Dukkipati1,
Gerardo
Carpenito1, and
Ira
Kurtz1
1 Division of Nephrology, Center for Health Sciences, UCLA
School of Medicine, Los Angeles, California, 90095-1698;
Departments of 2 Urology and 3 Physiology and
Biophysics, Case Western Reserve University and 4 Department
of Veterans Affairs Medical Center, Cleveland, Ohio 44106
 |
ABSTRACT |
Sodium-bicarbonate cotransporters
are homologous membrane proteins mediating the electrogenic or
electroneutral transport of sodium and bicarbonate. Of the functionally
characterized sodium-bicarbonate cotransporters (NBC), NBC1
proteins are known to be electrogenic. Here we report the cloning and
functional characterization of NBC4c, a new splice variant of the NBC4
gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein
variants and 40% identical to electroneutral NBC3. When expressed in
mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate
cotransport. The transport of sodium and bicarbonate is chloride
independent and is completely inhibited by DIDS. NBC4c transcripts were
detected in several tissues including brain, heart, kidney, testis,
pancreas, muscle, and peripheral blood leukocytes. The data indicate
that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.
transport; acid-base; testes; kidney
 |
INTRODUCTION |
SODIUM-BICARBONATE
COTRANSPORT (NBC) proteins contribute to intracellular pH
(pHi) regulation and transepithelial HCO
transport in several tissues (1, 2, 6, 8-10, 20-26,
29) by mediating electrogenic or electroneutral
Na+-HCO
cotransport in the absence of Cl
. NBC transporters are homologous proteins belonging to
the bicarbonate transporter superfamily (BTS), which also includes the
Cl
/HCO
exchanger proteins AE1-AE4
(5, 31) and Na+-driven
Cl
/HCO
exchangers (12, 27,
32). NBC1 is the only currently known gene in mammals
encoding electrogenic Na+-HCO
cotransport proteins (3). In humans, NBC1
encodes two electrogenic Na+-HCO
cotransport variants, kNBC1 and pNBC1, which differ in their
NH2 terminus (3). kNBC1 mediates the majority
of basolateral HCO
efflux in the renal proximal
tubule (2, 9, 26, 29). pNBC1 is highly expressed in
pancreas and various other tissues (1, 8, 14, 20, 21). An
electroneutral NBC, NBC3 in humans (rat ortholog NBCn1) mediates
Cl
-independent and stilbene-insensitive
Na+-HCO
cotransport in various tissues (10, 20, 22, 25).
We now report the cloning and functional characterization of NBC4c, a
new splice variant of the NBC4 gene (23, 24). The results
indicate that NBC4c is an electrogenic
Na+-HCO
cotransporter. There is
currently little information regarding the basis for the ion
specificity and transport stoichiometry of members of the BTS. The
finding that NBC1 and NBC4c proteins are functionally similar will
provide a basis for additional studies addressing the structural
requirements for electrogenic Na+-HCO
cotransport.
 |
METHODS |
Cloning and sequencing of NBC4c.
An arrayed human cDNA library (Origene Technologies, Rockville, MD) was
screened by a PCR-based approach according to the manufacturer's
instructions. PCR primers with sequence common to the previously
reported NBC4 splice variants (23, 24) were used to
identify new NBC4 clones: sense, 5'-CAGACCAGCCACAGCAGGAACTG-3' (535); antisense, 5'-GTGCTGCTGGATTTGGACAGTGG-3'
(867). The primer positions refer to NBC4c.
Vector-derived 5' and 3' primers were used to identify clones with the
longest inserts. Positive clones were verified by sequencing. An ~6.3
kb-clone was obtained from human testes that contained the coding
region of a new splice variant of NBC4, which was named NBC4c. The
predicted amino acid sequence of NBC4c is shown in Fig.
1. The NBC4c nucleotide sequence has been
submitted to GenBank/EMBL Data Bank with the accession number AF293337.
The 5' end of the coding sequence for this clone was confirmed by 5'
rapid amplification of cDNA ends (RACE) PCR amplification.
Furthermore, to confirm that the NBC4c amino acid sequence was derived
from a bona fide transcript, we amplified the entire open reading frame
of human NBC4c by RT-PCR with Marathon Ready cDNA prepared from
human testes (Clontech, Palo Alto, CA) as a template. Nucleotide
sequences were determined bidirectionally by automated sequencing (ABI
310, Perkin-Elmer, Foster City, CA) using Taq polymerase
(Ampli-Taq FS, Perkin-Elmer). Sequence assembly and analysis were
carried out with Geneworks software (Oxford, UK). The alignment of
human NBC4c with human NBC1 and NBC3 is shown in Fig. 1.

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Fig. 1.
Multiple protein sequence alignment of human
sodium-bicarbonate cotransport (NBC) proteins NBC4, NBC1, and NBC3. The
amino acid sequence of the NBC4c splice variant and the pNBC1 variant
of NBC1 are depicted.
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RT-PCR amplification of NBC4c.
In initial experiments using Northern blot analysis of human tissues
and various probes specific to the 3'-untranslated region (UTR) of
NBC4c, we were unable to detect specific labeling of NBC4c because of
either low abundance or mRNA instability. Therefore, a PCR approach was
used in which primers specific for NBC4c were used for amplification of
cDNA obtained from various tissues. The following primers used in the
PCR reactions were specific for the 3'-UTR of NBC4c and did not have
any sequence homology to other NBC transporters: sense,
5'-CACCTTGCACTTCAAAATATCCTGTCCAG-3' (3750-3778); antisense,
5'-GTTCAAACTTTTCATATAACCCTTAGGAAATTG-3' (4402-4434). Controls were
negative, and all PCR products were confirmed by sequencing.
Functional characterization of NBC4c.
HEK293-T cells were grown on fibronectin-coated coverslips and were
transiently transfected by the calcium phosphate precipitation method
with a pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) containing the
coding region for NBC4c. Mock-transfected cells were transfected with
the vector alone. The plasmids were purified with the Endofree plasmid
purification kit (Qiagen, Valencia, CA) before their use. Functional
studies were performed 24 h after transfection. pHi was monitored with the fluorescent probe
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
and a microfluorometer coupled to the microscope (19). Data was obtained from ~20 cells per coverslip. Calibration of intracellular BCECF was performed at the end of every experiment by
monitoring the 500- to 440-nm fluorescence excitation ratio at various
values of pHi in the presence of high-K+
nigericin standards (30). The following experimental
protocols were performed. 1) For Na+
addition/removal in Cl
-containing HCO
-buffered solutions, the cells were initially bathed for 25 min in a
Na+-free Cl
-containing HEPES-buffered
solution (solution A, Table
1). The cells were acutely acidified to
~pHi 6.5 by exposure to HCO
-buffered Na+-free Cl
-containing solution
(solution B, Table 1). The cells were then exposed to a
HCO
-buffered Na+- and
Cl
-containing solution (solution C, Table 1)
followed by subsequent Na+ removal (solution B).
Additional experiments were performed in cells bathed in DIDS (1 mM) or
ethylisopropylamiloride (EIPA; 50 µM). 2) For
Na+ addition/removal in Na+-free and
Cl
-free HCO
-buffered solutions, the cells were bathed in a Na+-containing Cl
-free
HEPES-buffered solution (solution D, Table 1) for 1 h
before experimentation. The cells were then exposed to a
Na+- and Cl
-free HEPES-buffered solution
(solution E, Table 1) for 25 min. The cells were acutely
acidified to ~pHi 6.5 by exposure to a HCO
-buffered Na+- and
Cl-
free solution (solution F, Table 1). A
Na+-containing Cl
-free solution was then
added (solution G, Table 1), followed by the removal of
Na+ (solution F). 3) For
Na+ removal/addition in Cl
-containing
HEPES-buffered solutions, the cells were initially bathed in a
Na+-free Cl
-containing HEPES-buffered
solution (solution A) for 25 min. The cells were acutely
acidified to ~pHi 6.5 by a brief exposure to and
subsequent removal of a solution containing 30 mM NH4Cl
(replacing 30 mM tetramethylammonium chloride; solution H,
Table 1). A HEPES-buffered Na+- and
Cl
-containing solution was then added (solution
I, Table 1), followed by the subsequent removal of Na+
(solution A).
Electrogenicity of NBC4c.
The methodology to measure the current through electrogenic
Na+-HCO
cotransporters in mammalian cell monolayers with an Ussing chamber was described previously (13, 14). HEK293-T cells were not suitable for these measurements because they do not form a high-resistance monolayer. The mPCT renal
cell line (13), which lacks endogenous electrogenic
Na+-HCO
cotransport function and forms a
high-resistance confluent monolayer (transepithelial resistance
1,000
cm2) when grown on filter inserts, was used to
characterize the electrogenicity of NBC4. The cells were grown
on filter inserts in mRTE medium (1:1 mixture of DMEM and Ham's F-12
medium and the following additives: 10 ng/ml epidermal growth factor, 5 µg/ml insulin, 5 µg/ml transferrin, 4 µg/ml dexamethasone, 10 U/ml interferon
, 2 mM glutamine, and 5% fetal bovine
serum). The cells were then transiently transfected with a
pcDNA3.1 plasmid (Invitrogen) containing the coding region for NBC4c
with Effectene (Qiagen) per the manufacturer's protocol. Control
(mock-transfected cells) were transfected with the vector alone. All
plasmids were purified with the Endofree plasmid purification kit
(Qiagen) before use. Confluent cells grown on permeable filter supports
(0.4 µm; Millipore, Bedford, MA) were mounted vertically in a
thermostated Ussing chamber equipped with gas inlets for CO2 bubbling. Functional studies were performed 48 h
after transfection. The cells were permeabilized apically with 10 µM
amphotericin B to remove the electrical resistance of the apical
membrane (4, 7, 16-18). The application of a
Na+ gradient across the epithelial cell monolayer in cells
expressing NBC4c in the presence of HCO
generates two currents with opposite signs. The absolute sign of each current in
the Ussing chamber depends on which compartment, basolateral or apical,
is connected to the ground electrode. In the experiments shown in Fig.
5, the apical chamber was connected to ground. In this case, the
short-circuit current generated by the flux of Na+ through
the paracellular shunt pathway (INa) has a
negative sign. In NBC4c-expressing cells, but not in mock-transfected
cells, an additional positive short-circuit current generated by the flux of Na+ and HCO
through NBC4c is
also present (INaHCO3). The
resultant net baseline short-circuit current in NBC4c expressing cells
is the vectorial sum of the two short-circuit currents:
INaHCO3 + INa. The
net baseline short-circuit current before the addition of DIDS depends
on both the magnitude of the paracellular pathway Na+
conductance and NBC4c conductance. The net baseline short-circuit current is not identical in each monolayer because of slight
differences in the paracellular Na+ conductance between
monolayers and the level of expression of NBC4c. We therefore offset
the net baseline short-circuit current to zero. When DIDS is added to
the basolateral solution of NBC4c-expressing cells in the presence of
both a Na+ gradient and HCO
, the net
short-circuit current becomes negative because the positive
short-circuit current generated by the flux of Na+ and
HCO
through NBC4c is blocked by basolateral DIDS,
leaving only the negative short-circuit current generated by the flux
of Na+ through the paracellular shunt pathway
(INa). The DIDS-sensitive short-circuit current,
i.e., the net current through NBC4c, is thus obtained by subtracting
the short-circuit current measured in the presence of DIDS from that
measured in the absence of DIDS
|
(1)
|
Only transfected cells for which the DIDS-sensitive current was
at least 10-fold larger then that of the corresponding mock-transfected cells were used; 30% of cell monolayers met this criterion. The stoichiometry of the cotransporter was determined from its reversal potential (Erev) and Eq. 2 as described
previously (13, 14, 16)
|
(2)
|
where n is the number of bicarbonate anions
cotransported with each sodium cation, the subscripts i and o represent
intra- and extracellular concentrations, respectively, of the indicated ion, R is the gas constant, T is temperature, and
F is the Faraday constant. For a symmetrical
HCO
concentration, the ratio
[HCO
]
/[HCO
]
equals 1 and Erev depends logarithmically only
on the magnitude and direction of the Na+ concentration
gradient. Erev of the cotransporter was measured for several different Na+ concentration gradients while
keeping HCO
concentrations symmetrical across the
basolateral membrane. Initially, a fivefold Na+
concentration gradient was applied across the monolayer by perfusing the basolateral compartment with a Cl
-free
HCO
-buffered solution containing 50 mM
Na+ (solution J, Table 1) and the apical
compartment with a Cl
-free
HCO
-buffered solution containing 10 mM
Na+ (solution K, Table 1). In separate
experiments, a twofold Na+ concentration gradient was used
by replacing solution J in the basolateral compartment with
a solution containing 20 mM Na+ (solution L,
Table 1). All solutions were Cl
free. To measure
Erev of the cotransporter, current-voltage
relationships were collected with an epithelial voltage-clamp amplifier
(EC825; Warner Inst., Hamden, CT). The data were digitized at 100 kHz by an analog/digital converter (PowerLab/400, ADInstruments, Castle Hill, Australia) for further analysis. Data were filtered at 0.5 Hz.
Current-voltage relationships were measured by stepping the voltage
command from
100 mV to +100 mV with a 10-mV step, using the
stimulator utility of the Chart program (ADInstruments).
Statistics.
Results are reported as means ± SE. Unpaired Student's
t-test and linear regression analyses were used as required.
Dunnett's t-test was used when more than one experimental
group was compared with a control group.
 |
RESULTS |
Sequence alignment and tissue distribution.
The amino acid sequence of the NBC4c clone used for functional analysis
in this study and its alignment with human NBC1 and NBC3 are shown in
Fig. 1. NBC4c is 56% identical to electrogenic NBC1 protein variants
and 40% identical to electroneutral NBC3 at the amino acid level.
Therefore, NBC4c has a higher homology with electrogenic than with
electroneutral Na+-HCO
cotransporters.
NBC4c transcripts are found in several tissues (Fig.
2).

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Fig. 2.
Expression of NBC4c mRNA in various tissues. Total RNA
from various human tissues was reverse transcribed using avian
myeloblastosis virus reverse transcriptase (RT). NBC4c was amplified in
25 cycles with the specific primers described in
METHODS.
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|
Functional characterization.
In Na+- and Cl
-containing
HCO
-buffered solutions, resting pHi was
6.88 ± 0.06 (n = 6) in mock-transfected control
HEK293-T cells and 6.87 ± 0.04 (n = 7) in
NBC4c-transfected cells [P = not significant (NS)].
The cells were initially bathed in Na+-free HEPES-buffered
solutions (pH 7.4). When the cells were acutely acidified by the
addition of Na+-free
CO2/HCO
-buffered solutions (pH 7.4),
pHi failed to recover in the absence of external
Na+ (Fig. 3). After the
addition of Na+ (140 mM), pHi recovered slowly
at a rate of 0.22 ± 0.03 pH units/min (n = 5) in
mock-transfected HEK293-T cells (Fig. 3A). In
NBC4c-transfected cells, pHi recovered at a significantly
faster rate of 1.00 ± 0.08 pH units/min (n = 8;
P < 0.001; Fig. 3B). Further experiments were done to determine the Cl
dependence of the
Na+-induced H+/base flux in NBC4c-transfected
cells. As shown in a typical experiment in Fig. 3C, the
Na+-induced pHi recovery was Cl
independent. To determine the HCO
dependence of
NBC4c-mediated transport, separate studies were done in the nominal
absence of HCO
in HEPES-buffered solutions utilizing
the NH4+ prepulse technique to acidify the
cells acutely. After the addition of Na+ (140 mM) in
mock-transfected HEK293-T cells, pHi recovered slowly at a
rate of 0.17 ± 0.02 pH units/min (n = 5). In
NBC4c-transfected cells (Fig. 3D), pHi recovered
at a rate that was not significantly different from that in
mock-transfected cells [0.19 ± 0.02 pH units/min
(n = 6); P = NS]. These results
indicate that NBC4c has an absolute requirement for Na+ and
HCO
. In separate experiments, the effect of DIDS
(Fig. 3E) and EIPA (Fig. 3F) on NBC4c-mediated transport was studied. In NBC4c-transfected cells, in
HCO
-buffered solutions DIDS (1 mM) significantly
inhibited the Na+-induced HCO
-dependent
pHi transients [0.13 ± 0.03 pH units/min
(n = 5); P < 0.001]. In
HCO
-buffered solutions, EIPA (50 µM) decreased the
rate of the Na+-dependent pHi recovery in
mock-transfected cells from 0.22 ± 0.03 (n = 5)
to 0.06 ± 0.01 (n = 7) pH units/min
(P < 0.01). In NBC4c-transfected cells, the magnitude
of the inhibition was similar in that EIPA (50 µM) decreased the rate
of pHi recovery from 1.00 ± 0.08 (n = 8) to 0.75 ± 0.06 (n = 10) pH units/min
(P < 0.02). Figure 4
depicts the calculated Na+-dependent equivalent base fluxes
in mock-transfected and NBC4c-transfected cells.

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Fig. 3.
Functional characterization of NBC4c. HEK293-T cells were bathed
initially in a Na+-free, HEPES-buffered solution, pH 7.4. After the addition of Na+-free
CO2/HCO -buffered solutions (pH 7.4),
Na+ addition/removal experiments were performed.
A: mock-transfected cells (Cl -containing
solutions). B: NBC4c-transfected cells
(Cl -containing solutions). C:
NBC4c-transfected cells (Cl -free solutions).
D: NBC4c-transfected cells acidified by NH4Cl
(30 mM) removal in HEPES-buffered solutions. E:
NBC4c-transfected cells: Na+ addition/removal in
CO2/HCO -buffered solutions in the
presence of DIDS (1 mM). F: NBC4c-transfected cells.
Na+ addition/removal in
CO2/HCO -buffered solutions in the
presence of ethylisopropylamiloride (EIPA; 50 µM).
pHi, intracellular pH.
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Fig. 4.
Na+-dependent equivalent base flux (EBF) in
mock-transfected and NBC4c-transfected HEK293-T cells.
Na+-dependent EBF was calculated as EBF = dpHi/dt × in each protocol, where
dpHi/dt represents the initial rate of change in
pHi measured in the first 10 s after Na+
addition and signifies the intrinsic buffer capacity
( i) in HEPES-containing solutions and the total buffer
capacity ( T) when HCO -buffered
solutions were used. Both i and T were
measured with the NH4Cl technique (28) over
the pHi range 6.35-6.55 in which
dpHi/dt was calculated. In mock-tranfected
cells, T was 20.7 ± 0.33 mM/pH unit
(n = 3), which was not significantly different than the
T of 21.1 ± 1.45 mM/pH unit (n = 3) in NBC4c-transfected cells. Similarly, the i of
14.5 ± 0.23 mM/pH unit (n = 3) in mock-tranfected
cells was not significantly different from the i of
14.9 ± 1.02 mM/pH unit (n = 3) in
NBC4c-transfected cells. In HCO -buffered solutions,
the Na+-dependent EBF in NBC4c-transfected cells
(n = 8) was significantly greater than in
mock-transfected cells (n = 5) (P < 0.001). In HEPES-containing solutions, the Na+-dependent
EBF of NBC4c-transfected cells (n = 6) was
significantly decreased compared with the EBF in
HCO -buffered solutions (P < 0.001).
Similarly, in NBC4c-transfected cells (n = 5) DIDS (1 mM) significantly decreased the Na+-dependent EBF
(P < 0.001).
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Electrogenicity of NBC4c.
To determine whether NBC4c is electrogenic, we measured the
stilbene-inhibitable Na+- and
HCO
-dependent short-circuit current in mPCT cells
tranfected with the transporter. The short-circuit current generated by
application of a Na+ concentration gradient across apically
permeabilized mPCT cells transfected with NBC4c was measured in an
Ussing chamber. In the presence of HCO
(25 mM), a
fivefold Na+ concentration gradient generated an average
DIDS-sensitive short-circuit current of 3.4 ± 0.6 µA/cm2 (n = 6; Fig.
5A) compared with 0.4 ± 0.2 µA/cm2 (n = 6) for mock-transfected
monolayers (not shown). In the nominal absence of
CO2/HCO
, application of a fivefold
Na+ gradient across monolayers of NBC4c-transfected cells
generated an average DIDS-sensitive short-circuit current of 0.2 ± 0.1 µA/cm2 (n = 6; Fig.
5B). These results suggested that the electrogenic flux of
Na+ through NBC4c is coupled to that of
HCO
and that the coupling ratio is at least 2 HCO
per 1 Na+. To determine the coupling
ratio (i.e, stoichiometry), we measured Erev in
NBC4c-transfected cells from which the stoichiometry (n) was
calculated as described in METHODS (see Eq. 2).
Current-voltage relationships of NBC4c were measured at two different
Na+ concentration gradients as shown in Fig.
6. The Erev data
indicate that the HCO
:Na+ stoichiometry
of NBC4c was ~3:1 (Table 2).

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Fig. 5.
Electrogenicity of NBC4c. In
these experiments, a 5-fold Na+ gradient was applied across
apically permeabilized mPCT cells transfected with NBC4c by
perfusing the apical compartment with a solution containing 10 mM
Na+ and the basolateral compartment with a solution
containing 50 mM Na+, in the presence or absence of
CO2/HCO . The DIDS-sensitive
short-circuit current, i.e., the net current through NBC4c, is
calculated by subtracting the short-circuit current measured in the
presence of DIDS from that measured in the absence of DIDS (see
Eq. 1 in METHODS). The baseline short-circuit
current was offset to zero in A and B. DIDS (1 mM) altered the short-circuit current in NBC4c-transfected cells only
in the presence (A) but not in the absence (B) of
HCO (25 mM). In A, the positive
short-circuit current generated by the flux of Na+ and
HCO through NBC4c is blocked by basolateral DIDS,
leaving a negative short-circuit current generated by the flux of
Na+ through the paracellular shunt pathway.
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Fig. 6.
Current-voltage (I-V) relations for
NBC4c expressed in mPCT cells. I is the DIDS-sensitive
current. The potential of the basolateral compartment is taken as zero.
The ratio of apical (AP) to basolateral (BL) Na+
concentrations (AP/BL) was as follows: 10/50 ( ); 10/20
( ). Also shown is the I-V
relation of mock-transfected cells at a 10/50 gradient
( ). The reversal potentials for the different gradients
were evaluated graphically from the intersection of the lines with the
x-axis and are tabulated in Table 2.
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 |
DISCUSSION |
In the present study, we have determined the transport properties
and electrogenicity of a new member of the NBC family, NBC4. We have
shown that the NBC4c splice variant is an electrogenic Na+-HCO
cotransporter. The transport of
Na+ and HCO
is Cl
independent and is completely stilbene sensitive. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes, indicating a
potentially widespread and functionally important role for this transporter in mediating electrogenic
Na+-HCO
cotransport.
Before this study, products of NBC1 were the only proteins
known to mediate electrogenic Na+-HCO
cotransport (3). Comparison of the amino acid sequence
of NBC4c with other members of the BTS demonstrates that
NBC4c has the highest similarity to the NBC1 electrogenic
Na+-HCO
cotransporter proteins. However, a detailed comparison of the amino acid sequence of NBC1 and NBC4c does
not reveal obvious structural motifs unique to these proteins that may
be responsible for conferring electrogenic
Na+-HCO
cotransport. These findings
suggest that the three-dimensional structure of these transporters
and/or regulatory factors may determine the electrogenic nature and
transport stoichiometry of NBC proteins. Indeed, we have recently shown that the phosphorylation of Ser984 in kNBC1 shifts the
HCO
:Na+ coupling ratio from 3:1 to 2:1
(15). Whether the HCO
:Na+
coupling ratio of NBC4c and other NBC proteins can be similarly regulated is currently unknown. There is presently a paucity of information regarding the basis for the ion specificity and transport stoichiometry of members of the BTS. The finding that NBC1 and NBC4c
proteins are functionally similar provides an additional basis for
addressing the minimum structural requirements, which are necessary for
a protein to mediate electrogenic
Na+-HCO
cotransport.
Although NBC1 and NBC4c proteins function as electrogenic
Na+-HCO
cotransporters, our results
demonstrate that NBC4c-mediated transport is strictly
HCO
dependent. NBC4c had an absolute requirement for
HCO
because in its absence, the magnitude of the
Na+-dependent H+/base fluxes in
NBC4c-transfected and control mock-transfected cells were identical and
there was no detectable stilbene-inhibitable current through the
transporter. When expressed in mammalian cells, kNBC1 has been reported
to function in the absence of HCO
(6),
although not all studies concur with this finding (13). Whereas NBC4c and NBC1 proteins may differ in their
HCO
dependence, they share the property of being
stilbene inhibitable. In addition to these electrogenic NBC proteins,
other known members of the BTS are stilbene sensitive with the
exception of NBC3 and AE4 (22, 31). The putative stilbene
binding motif KLFD (amino acids 742-745) in NBC3 and KMLN (amino
acids 518-521) in AE4 may alter their affinity for stilbenes and
account for their DIDS insensitivity (22, 31). A
comparison of the amino acid sequence of NBC4 with NBC1 and other
members of the BTS reveals that the putative stilbene binding motif
[K(M/L)(X)K] is lacking in NBC4, which has the sequence KMIG (amino
acids 655-658). Therefore, replacement of the highly conserved
lysine at position 658 by glycine does not appear to appreciably alter
the binding of negatively charged stilbene disulfonates.
Our finding that NBC4c transcripts are expressed in several tissues
including brain, heart, kidney, testis, pancreas, muscle, and
peripheral blood leukocytes suggests that the transporter may have a
housekeeping function in regulating pHi. pNBC1 transcripts have also been detected in many organs, indicating the ubiquitous expression of this transporter (1). The second known NBC1
protein variant, kNBC1, is more restricted in its tissue expression and has thus far been detected in kidney and eye (8, 29).
Additional studies of the cellular/subcellular localization in various
tissues, transport kinetics, and regulation of these electrogenic
Na+-HCO
cotransporters are required to address the question as to why in the mammalian genome there are two
distinct genes that encode electrogenic
Na+-HCO
cotransporters. Examples of
other proteins in the BTS in which functionally similar transporters are encoded by separate genes are the AE1-4
Na+-independent Cl
-HCO
exchangers (5, 31).
The functional characterization of NBC4 is of additional
importance given that the disease gene in Alstrom syndrome, a rare multisystemic autosomal recessive disorder characterized by infantile cardiomyopathy, hepatic dysfunction, progressive sensorineural hearing
loss, retinopathy, truncal obesity, asthma, diabetes mellitus, and
hypogonadism, has been mapped to a 6.1-cM region of chromosome 2p13 containing the coding region of NBC4 (11).
Given that NBC4 is therefore a candidate gene for this syndrome,
further studies are ongoing in patients with this syndrome to determine
whether mutations in the coding region of NBC4 exist.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-46976, DK-58563, and DK-07789,
the Iris and B. Gerald Cantor Foundation, the Max Factor Family
Foundation, the Richard and Hinda Rosenthal Foundation, the Fredericka
Taubitz Foundation, a Cystic Fibrosis Foundation grant Gross01G0, and
American Heart Association Grant 9706507. A. N. Abuladze is
supported by National Kidney Foundation of Southern California Training
Grant J891002.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: I. Kurtz, UCLA Division of Nephrology, 10833 Le Conte Ave., Rm. 7-155 Factor Bldg., Los Angeles, CA 90095-1689 (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/ajpcell.00409.2001
Received 20 August 2001; accepted in final form 25 October 2001.
 |
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