From the Departments of Biochemistry and Molecular
Biology and ¶ Obstetrics and Gynecology, Medical College of
Georgia, Augusta, Georgia 30912 and the § Department of
Physiology and Biophysics, University of Texas Medical Branch,
Galveston, Texas 77555
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ABSTRACT |
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We have cloned a
Na+-dependent, high affinity
dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits
48% identity in amino acid sequence with rat NaDC1, a
Na+-dependent, low affinity dicarboxylate
transporter. NaDC3-specific mRNA is detectable in kidney, brain,
liver, and placenta. When expressed in mammalian cells, NaDC3 mediates
Na+-dependent transport of succinate with a
Kt of 2 µM. The transport function of
NaDC3 shows a sigmoidal relationship with regard to Na+
concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number
of dicarboxylates including dimethylsuccinate as substrates and
excludes monocarboxylates. Li+ inhibits NaDC3 in the
presence of Na+. Transport of succinate by NaDC3 is
markedly influenced by pH, the transport function gradually decreasing
when pH is acidified from 8.0 to 5.5. In contrast, the influence of pH
on NaDC3-mediated transport of citrate is biphasic in which a pH change
from 8.0 to 6.5 stimulates the transport and any further
acidification inhibits the transport. In addition, the potency of
citrate to compete with NaDC3-mediated transport of succinate increases
25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate.
This represents the first report on the cloning and functional characterization of a mammalian Na+-dependent,
high affinity dicarboxylate transporter.
Placenta plays an obligatory role in providing the developing
fetus with essential nutrients and metabolic fuels (1, 2). This
function is facilitated by the presence of specific transport mechanisms in the maternal facing brush border membrane and the fetal
facing basal membrane of the placental syncytiotrophoblast. Several
years ago, studies from our laboratory demonstrated the existence of a
Na+-coupled transport system for dicarboxylates in brush
border membrane vesicles prepared from human term placenta (3). This
transport system accepts succinate and other Krebs cycle intermediates
as substrates. The functional characteristics of the placental
sodium/dicarboxylate transporter are distinct from those of the
sodium/dicarboxylate transporter described in brush border membrane
vesicles from mammalian kidney and intestine. The primary distinction
is in substrate affinity. The transporter in the placental brush border
membrane recognizes succinate with high affinity (Kt
~5 µM). In contrast, the transporter in the renal and
intestinal brush border membranes exhibits relatively much lower
affinity (Kt ~100-500 µM) for
succinate (4-8). However, a high affinity sodium/dicarboxylate transporter, similar to the one expressed in the placental brush border
membrane, is present in the renal basolateral membrane (9, 10) and in
the liver canalicular membrane (11, 12).
There have been several reports on the cloning and functional
characterization of the low affinity sodium/dicarboxylate transporter from mammalian kidney (13-16) and intestine (17). This transporter has
been designated as either NaDC1 (Na+/dicarboxylate
cotransporter 1) or SDCT1 (sodium/dicarboxylate transporter 1). We
refer to this transporter as NaDC1 throughout this paper. The amino
acid sequence of NaDC1 exhibits 78% identity between rabbit and human
homologs (13, 14). The rat NaDC1 shows ~70% identity with rabbit and
human NaDC1 (15-17). Rabbit NaDC1 and human NaDC1 interact with
succinate with a Kt value of around 400 µM (13, 14). Interestingly, rat NaDC1 exhibits a
Kt value of 25-30 µM for succinate
(15, 16), and the observed significant divergence in the amino acid sequence of rat NaDC1 in comparison with rabbit NaDC1 and human NaDC1
is likely to be related to the difference in the substrate affinity
between rat NaDC1 and rabbit/human NaDC1. Recently a nonmammalian low
affinity sodium/dicarboxylate transporter has been cloned from the
intestine of Xenopus laevis (18). Cation specificity and
tissue distribution of this transporter clearly differentiate it from
mammalian NaDC1. Based on these differences, the X. laevis
sodium/dicarboxylate transporter has been designated NaDC2. The
Kt value for succinate for the X. laevis
transporter is in the range of 300-700 µM, depending on
whether Na+ or Li+ is used as the cotransported
cation. The amphibian NaDC2 may be a species-specific isoform of the
mammalian NaDC1.
There is no information available in the literature on the molecular
nature of the high affinity sodium/dicarboxylate transporter. We report
here on the cloning and functional characterization of a high affinity
sodium/dicarboxylate transporter NaDC3 from rat placenta. This
transporter, when heterologously expressed in mammalian cells, mediates
the transport of succinate with a Kt value of 2 µM. The rat NaDC3 is 48% identical to rat NaDC1 in amino
acid sequence. Northern blot analysis indicates that NaDC3 transcript
is also present in the brain, in addition to placenta, kidney, and
liver, the tissues in which the existence of a high affinity
sodium/dicarboxylate transporter has been demonstrated by functional
studies. This represents the first report on the molecular nature of a
mammalian high affinity sodium/dicarboxylate transporter.
Materials--
SuperScript Plasmid System for cDNA cloning
and Lipofectin were purchased from Life Technologies, Inc. Restriction
enzymes were obtained from New England Biolabs. NitroPure transfer
membranes were purchased from Micron Separations, Inc. The human
retinal pigmental epithelial
(HRPE)1 cell line was kindly
provided by M. A. Del Monte (Department of Ophthalmology, W. K. Kellogg Eye Center, Ann Arbor, MI). The cells were routinely
cultured in Dulbecco's modified Eagle's medium/F-12 medium
supplemented with 10% fetal bovine serum and 100 µg/ml of penicillin
and 100 units/ml of streptomycin. [2,3- 3H]Succinic acid
(specific radioactivity, 37.5 Ci/mmol) was purchased from Moravek
Biochemicals (Brea, CA), and [14C]citrate (specific
radioactivity, 65 mCi/mmol) was purchased from American Radiolabeled
Chemicals (St. Louis, MO). [ Screening of the cDNA Library--
This was done by colony
screening of the plasmid cDNA library grown on NitroPure transfer
membranes as described by Vogeli and Kaytes (19). The cDNA probe
used for screening was 1.8 kilobase pairs long and consisted of almost
the entire coding region and some of the 3'-untranslated region
(nucleotides 105-1953 of human NaDC1) (14). The probe was labeled with
[ DNA Sequencing--
Sequencing by the dideoxynucleotide chain
termination method was performed by Taq DyeDeoxy terminator
cycle sequencing with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA Sequencer. Data base searches were done using the
commercially available program GenBankTM (22).
Northern Blot Analysis--
Tissue distribution of the
NaDC3-specific transcripts was determined by Northern analysis. A
commercially available membrane blot containing size-fractionated
mRNA (2 µg each) from brain, heart, kidney, stomach, small
intestine, and skeletal muscle of rat was used for this purpose. A
second blot containing mRNA from rat placenta and liver was
prepared in our laboratory. A BamHI-EcoRI fragment (~2.0 kilobase pairs) consisting of most of the open reading
frame of the rNaDC3 cDNA was used as the probe.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and
Restriction Analysis--
To detect the presence of NaDC3 transcripts
in rat placenta and in rat liver, RT-PCR was done with
poly(A)+ RNA isolated from these tissues. The primers used
were 5'-GGCTTCCACCGCAATGAT-3' (upstream primer) and
5'-TGAAGGCGTAGGAACAGC-3' (downstream primer). These primers
corresponded to nucleotide positions 465-482 and 1571-1588,
respectively, in the rNaDC3 cDNA. The expected size of the PCR
product is 1124 bp. This sequence in rNaDC3 cDNA possesses a single
restriction site each for DraIII and NcoI.
Restriction analysis of the RT-PCR products was done by subjecting the
purified RT-PCR products (Qiaex II, Qiagen, Chatsworth, CA) to
digestion with DraIII or NcoI and analyzing the
size of the resulting fragments. The sizes of the expected fragments
are 368 and 756 bp in the case of DraIII digestion and
251 and 873 bp in the case of NcoI digestion.
Functional Expression of the cDNA--
The cDNA was
functionally expressed in HRPE cells by vaccinia virus expression
system (23) as described previously (20, 21). HRPE cells were used
instead of HeLa cells because they had lesser endogenous succinate
transport activity (data not shown). Transport measurements were made
at room temperature using [3H]succinate or
[14C]citrate. In most experiments, the incubation time
for transport measurements was 1 min to represent initial uptake rates.
Since the uptake rates for [14C]citrate were much less
than the uptake rates for [3H]succinate, a 10-min
incubation was used instead of a 1-min incubation to measure
[14C]citrate uptake. Transport was terminated by
aspiration of the uptake buffer followed by two rapid washes with 2 ml
of ice-cold transport buffer. Following this, the cells were
solubilized with 0.5 ml of 1% SDS in 0.2 N NaOH and
transferred to vials for quantitation of the radioactivity associated
with the cells. The transport buffer was 25 mM Hepes/Tris
(pH 7.5), supplemented with 140 mM NaCl, 5.4 mM
KCl, 1.8 mM CaCl2, 0.8 mM
MgSO4, and 5 mM glucose in most of the
measurements. In experiments dealing with the anion dependence of
succinate transport, the composition of the transport buffer was
modified by substituting potassium gluconate for KCl and calcium
gluconate for CaCl2. When the influence of Na+
on succinate transport was being investigated, the buffers containing 140 mM NaCl or 140 mM
N-methyl-D-glucamine (NMDG) chloride were mixed
to give uptake buffers of desired Na+ composition. When the
influence of pH on transport was investigated, transport buffers of
different pH values were prepared by varying the concentration of Tris,
Hepes, and Mes.
Data Analysis--
Uptake measurements were made in duplicate
and each experiment was repeated two or three times with separate
transfections. Results are given as means ± S.D. of these
replicate values. Kinetic analyses were carried out by nonlinear as
well as linear regression methods using the commercially available
computer programs Fig.P, version 6.0 (Cambridge, UK) or Sigma Plot
(Chicago, IL).
Structural Features of the NaDC3 cDNA--
The cloned cDNA
is 3239 bp long and has an open reading frame of 1803 bp including the
termination codon. The open reading frame is flanked by a 30-bp-long
5'-noncoding sequence and 1406-bp-long 3'-noncoding sequence. The
putative initiation codon is preceded by a Kozak consensus sequence
(GCGCGG) (24). At the 3'-end, the cDNA has two polyadenylation
signals in tandem (AATAAATAAA) preceding the poly(A) tail. The open
reading frame encodes a protein of 600 amino acids (Fig.
1). The protein has an estimated core molecular mass of 66.1 kDa and a pI of 8.09. Hydropathy analysis of the
primary amino acid sequence using the Kyte-Doolittle method (25) with a
window size of 20 amino acids shows that the protein is highly
hydrophobic with 12 putative transmembrane domains (Fig. 2). Two potential sites
(NX(T/S)) for N-linked glycosylation are present
at positions 584 and 594. When rat NaDC3 is modeled to accommodate
these N-glycosylation sites toward the extracytoplasmic side, both the N-terminal and the C-terminal ends of the protein are
directed toward the extracellular side of the cell membrane. This is,
however, only a hypothetical model, and further experimentation is
needed to confirm the exact number of transmembrane domains and the
orientation of the N and C termini and the loops between the
transmembrane domains. A comparison of the amino acid sequence of rat
NaDC3 with the protein sequences in the SwissProt sequence data base
revealed that NaDC3 bears significant homology to the known
sodium-dependent dicarboxylate and
sodium-dependent sulfate transporters (13-17, 26). The
closest relative, rat NaDC1, has a sequence similarity of 71% and
identity of 48% at the level of amino acid sequence (Fig. 1).
Tissue Distribution of NaDC3 Transcripts--
Poly(A)+
RNA isolated from several tissues of rat was analyzed by Northern blot
hybridization for the presence of mRNA transcripts of NaDC3 (Fig.
3). In the Northern blot obtained
commercially, transcripts were detected in brain and kidney (Fig.
3A). mRNA from heart, stomach, small intestine, and
skeletal muscle did not show any hybridization signal. The size of the
primary transcript was 3.3 kilobases. An additional minor hybridizing
transcript of 5.4 kilobases was also seen in both the positive tissues.
In the second blot with mRNA from placenta and liver, although
NaDC3-specific transcripts of 3.3 kilobases were detected in both
placenta and liver, the hybridization signal obtained from placental
mRNA was very weak. The hybridization signal in the mRNA from
liver was comparable with that obtained in the mRNA from brain
(data not shown). Since the cDNA was isolated from a rat placental
cDNA library, RT-PCR was performed on mRNA isolated from rat
placental tissue to conclusively prove the presence of NaDC3-specific
mRNA in the placenta. Poly(A)+ RNA isolated from liver
was used as a positive control, and reaction in which reverse
transcriptase was omitted was used as negative control. As seen in Fig.
3B, an RT-PCR product of the expected size (1124 bp) was
obtained with mRNA isolated from the rat placenta as well as liver.
The identity of the RT-PCR product was confirmed by restriction
analysis using the enzymes DraIII and NcoI as
detailed under "Experimental Procedures." The restriction fragments
obtained from the RT-PCR products are of the expected size (Fig.
3C), confirming that NaDC3 is expressed in rat placenta.
Functional Characterization--
The functional expression of the
NaDC3 cDNA was done in HRPE cells by transient transfection
followed by vaccinia virus-induced expression of the cDNA. The
function was monitored by the transport of radiolabeled succinate. Fig.
4 shows the time course of the uptake of
succinate by HRPE cells transfected with vector alone or NaDC3
cDNA. Na+-dependent uptake of succinate in
control cells transfected with empty vector showed negligible
transport, indicating that HRPE cells themselves do not have
significant levels of NaDC3-like transport activity. Under these
conditions, the uptake of succinate in NaDC3 cDNA-transfected
cells was 75-100-fold higher. The uptake of succinate in the absence
of extracellular Na+ in HRPE cells expressing NaDC3 was
similar to the levels of Na+-dependent uptake
measured in cells transfected with empty vector. These results clearly
demonstrate that NaDC3 is a sodium-dependent succinate
transporter. Since the NaDC3-mediated
Na+-dependent uptake of succinate linearly
increased at least up to 3 min, subsequent initial transport rate
measurements were made using a 1-min incubation period.
The ionic dependence of the cDNA-stimulated succinate uptake was
investigated by measuring succinate transport in the presence of
various inorganic salts (Table I).
Control uptake was measured in the presence of NaCl. Replacement of
Na+ with equimolar concentrations of cations such as
K+, choline, or NMDG almost completely (~99%) inhibited
the succinate uptake, indicating that Na+ is essential for
the transport function. Replacement of Na+ with
Li+ decreased the uptake markedly, but there was, however,
significant residual uptake of succinate, indicating that
Li+ can substitute for Na+ to a small extent.
When Cl
Substrate saturation kinetics of the cloned NaDC3 were analyzed in
cDNA-transfected HRPE cells (Fig. 5).
The initial uptake rate of succinate was measured at varying
concentrations of succinate in the uptake buffer (0.5-15
µM). The concentration of
[3H]succinate was kept constant at 50 nM. The
data obtained were analyzed first by nonlinear regression analysis
(Fig. 5) and confirmed by linear regression (Fig. 5, inset).
The cDNA-induced transport of succinate was saturable. The
experimental values were found to fit best to a transport model
consisting of a single transport system. The values for the kinetic
constants, Kt (Michaelis-Menten constant) and
Vmax (maximal velocity), for the uptake of
succinate in cDNA-transfected cells are 2.0 ± 0.1 µM and 115 ± 6 pmol/106 cells/min,
respectively.
The effect of Na+ on the uptake of succinate was
investigated by measuring the uptake of succinate in HRPE cells
transfected with NaDC3 cDNA in the presence of varying
concentrations of extracellular Na+. The concentration of
NaCl in the extracellular medium was varied from 0 to 75 mM. The relationship between the uptake rate and the
Na+ concentration was sigmoidal (Fig.
6), suggesting the involvement of more
than one Na+ per succinate molecule transported. The data
were fit to the Hill equation, and the Hill co-efficient, which is the
number of Na+ ions interacting with the carrier, was
calculated. The value, determined from the slope of the Hill plot (Fig.
6, inset) was 2.7 ± 0.2. This indicates that for every
succinate molecule transported, three Na+ ions are
cotransported. Since succinate is a divalent anion at physiological pH,
one extra positive charge enters the cell with every succinate
molecule, thereby rendering the transport process rheogenic. The
rheogenic nature of the transport process is supported by the findings
that K+-induced depolarization of the cells inhibit the
transport function of NaDC3. Transport of succinate (20 nM)
in cells exposed to a physiological concentration of K+
(5.6 mM) was 6.5 ± 0.4 pmol/106
cells/min, whereas this transport was reduced 32% to 4.4 ± 0.2 pmol/106 cells/min in cells exposed to 56 mM
K+. Thus, both the Na+ gradient as well as the
difference in the membrane potential across the cell membrane energize
the transport process.
The substrate specificity of the transporter was evaluated by assessing
the ability of various unlabeled mono-, di-, and tricarboxylic compounds to inhibit the transport of labeled succinate in
cDNA-transfected HRPE cells (Table
II). Monocarboxylates lactate and
pyruvate showed very little inhibition of [3H]succinate
uptake, indicating that monocarboxylates are not substrates. The two-
and three-carbon chain dicarboxylates oxalate and malonate also were
poor inhibitors, suggesting that the length of the carbon chain plays a
crucial role in substrate recognition. Dicarboxylates of four- and
five-carbon chain length (e.g. succinate, fumarate, and
glutarate) markedly inhibited the uptake of radiolabeled succinate, indicating that these are the most ideal substrates recognized by the
transporter. The transporter is also able to distinguish between
trans and cis isomers of unsaturated
dicarboxylates. Fumarate (a trans isomer) was able to
inhibit totally (~99%) the uptake of [3H]succinate,
whereas the corresponding cis isomer, maleate, caused significantly much lesser inhibition (~25%). Malate and
When we examined the effect of Li+ on
[3H]succinate uptake in the absence of Na+,
we observed significant residual uptake of succinate, which indicated
that in the absence of Na+, Li+ was able to
substitute for Na+ to a small extent. But several studies
with native renal brush border membrane vesicles as well as with cloned
NaDC1 have shown that Li+ can inhibit the activity of the
low affinity sodium/dicarboxylate transporter in the presence of
Na+. Therefore, we studied the effect of Li+ on
the activity of NaDC3 in the presence of Na+ to determine
if the high affinity sodium/dicarboxylate transporter also interacts
with Li+ in a similar manner. Potent inhibition was seen at
very low concentrations of Li+, which seemed to plateau off
at around 2.5 mM (data not shown). At this concentration,
Li+ was able to inhibit the transport activity of NaDC3 by
~60%. Further increase in the concentration of Li+ up to
40 mM did not increase the inhibition significantly. Thus, Li+ is a potent inhibitor of NaDC3-mediated
Na+-dependent succinate uptake. Interestingly,
the inhibition is only partial.
Citrate exists predominantly as a tricarboxylate anion at pH 7.5, but
the concentration of the dicarboxylate anion species increases
considerably when the pH is changed to 5.5. In contrast, succinate
exists predominantly as a dicarboxylate anion at pH 7.5 as well as at
pH 5.5. Therefore, we investigated the influence of pH on the
interaction of succinate and citrate with NaDC3 by assessing the
ability of these compounds in unlabeled form to inhibit NaDC3-mediated
[3H]succinate uptake at pH 7.5 and at pH 5.5 (Fig.
7). These experiments revealed that the
uptake of NaDC3-mediated [3H]succinate uptake was
markedly influenced by pH, the uptake rate at pH 7.5 being about
3-4-fold higher than at pH 5.5. However, when compared with the
corresponding control uptake, unlabeled succinate was able to inhibit
[3H]succinate uptake at both pH values. There was a
2-fold difference in the inhibitory potency between pH 5.5 and 7.5. The
IC50 value (concentration of unlabeled compound to inhibit
the uptake of radiolabeled succinate by 50%) for succinate was
1.5 ± 0.1 µM at pH 7.5. This value decreased to
0.7 ± 0.2 µM at pH 5.5. The maximal velocities for
rNaDC3-mediated succinate uptake, calculated from these experiments,
were 75 ± 2 pmol/106 cells/min at pH 7.5 and 19 ± 4 pmol/106 cells/min at pH 5.5, respectively. When
citrate was used as the inhibitor, pH was found to have a much greater
effect on the inhibitory potency. The IC50 value for the
inhibition of [3H]succinate uptake by citrate was
2.1 ± 0.2 mM at pH 7.5. This value decreased 25-fold
to 0.08 ± 0.01 at pH 5.5. These data show that the transport
function of NaDC3 is significantly inhibited by acid pH and that
citrate is preferentially recognized by NaDC3 as a substrate in the
divalent form.
The influence of pH on the NaDC3-mediated uptake of succinate and
citrate is compared in Fig. 8. The uptake
of succinate was markedly inhibited when the pH was changed from 8.0 to
5.5 (Fig. 8A). The uptake at pH 8.0 was almost 5-fold
greater than the uptake at pH 5.5. The influence of pH on citrate
uptake was very different from the influence on succinate uptake (Fig.
8B). The process of citrate uptake displayed a distinct pH
optimum at about pH 7.0. Decreasing the pH from 8.0 to 6.5-7.0
stimulated citrate uptake, but further decrease below pH 6.5 inhibited
citrate uptake. The uptake of succinate and citrate in cells
transfected with vector alone was much less compared with uptake in
cells transfected with NaDC3 cDNA, and this endogenous uptake was
not influenced by pH. These data show that the function of NaDC3 is
inhibited by acidic pH, NaDC3 accepts preferentially dicarboxylates as
substrates, and the biphasic pH influence on citrate uptake is due to
the opposing effects of acidic pH on the function of NaDC3 and on the
concentration of the dianionic species of citrate.
This report describes the cloning and functional characterization
of a mammalian high affinity Na+-dependent
dicarboxylate transporter (NaDC3). The NaDC3 was cloned from a rat
placental cDNA library. This is the first high affinity dicarboxylate transporter to be characterized at the molecular level.
The functional characteristics of rat NaDC3 were investigated by
heterologously expressing the NaDC3 cDNA in HRPE cells. Rat NaDC3
transports succinate, and its function is obligatorily dependent on the
presence of Na+. Li+ is able to substitute for
Na+ to a very small but significant extent. The
Na+:succinate stoichiometry is 3:1. The
Kt for succinate is 2 µM. The
transporter recognizes various dicarboxylates consisting of four- or
five-carbon chain length. Dimethylsuccinate and
dimercaptosuccinate are also recognized as substrates by NaDC3.
There are marked differences in substrate affinity and substrate
specificity between NaDC3 and NaDC1. The Kt for succinate is 500-800 µM in the case of rabbit and human
NaDC1 when evaluated by tracer uptake in X. laevis oocytes
(13, 14). The rat NaDC1 exhibits a much lower Kt for
succinate, in the range of 25-30 µM, when analyzed by
electrophysiological approaches in X. laevis oocytes (15,
16). These differences in Kt values among NaDC1s
from various animal species may not be entirely due to the differences
in the experimental approaches employed in the determination of the
Kt values. A recent study (28) has shown that rabbit
NaDC1 has a Kt of 180 µM in X. laevis oocytes when analyzed electrophysiologically, in contrast
to the Kt value of 25-30 µM for rat
NaDC1 obtained with similar experimental approaches (15, 16).
Therefore, the possibility of actual differences in the affinity of
NaDC1 among various animal species cannot be ruled out. The
Kt for the transport of succinate by rat NaDC3 is 2 µM, a value 10- to 15-fold less than the corresponding
value for rat NaDC1. The difference is even greater when compared with
Kt values reported for rabbit and human NaDC1.
Another significant difference between NaDC3 and NaDC1 is in substrate
specificity. NaDC1s from various animal species exhibit similar
substrate specificity. This includes preferential recognition of
dicarboxylates with four- or five-carbon chain length, trans
isomer selectivity for unsaturated dicarboxylates, and lack of
interaction with monocarboxylates (13-16). NaDC3 also exhibits all of
these characteristics with respect to substrate specificity. However,
NaDC3 and NaDC1 markedly differ in the case of interaction with
dimethylsuccinate. Rabbit NaDC1 does not recognize dimethylsuccinate as
a substrate (13). In contrast, rat NaDC3 interacts very well with this
substituted succinate derivative. These differences in the affinity for
succinate and in the interaction with dimethylsuccinate between NaDC1
and NaDC3 constitute essential criteria for the identification of NaDC3
as the high affinity dicarboxylate transporter. Mammalian kidney
expresses a low affinity dicarboxylate transporter as well as a high
affinity dicarboxylate transporter, the former in the brush border
membrane and the latter in the basolateral membrane (4-6, 9, 10). Only
the high affinity transporter in the basolateral membrane interacts
with dimethylsuccinate, and therefore this succinate derivative is
considered as the test substrate for the high affinity transporter
(29). Since NaDC3 has a Kt of 2 µM for
succinate and also interacts with dimethylsuccinate, it is clearly
evident that NaDC3 represents the high affinity dicarboxylate transporter.
The high affinity dicarboxylate transporter has so far been shown to be
expressed by functional studies in three tissues, namely kidney, liver,
and placenta (3, 9-12). The present study provides evidence for the
expression of NaDC3 in these three tissues. A surprising finding,
however, is the evidence for the presence of NaDC3 mRNA transcripts
in brain. Dicarboxylate transport function has not been described in
this tissue.
With respect to interaction with Li+, NaDC3 behaves similar
to NaDC1. In the presence of Na+, the transport function of
NaDC3 is inhibited by Li+. The inhibition is maximal (60%)
at 1 mM Li+. Interestingly, the inhibition does
not go beyond 60% even when the concentration of Li+ is
increased to as high as 40 mM. In the case of NaDC1, there appears to be a species-dependent variation in sensitivity
to Li+ (30). The rabbit NaDC1 is inhibited 60% at 2.5 mM Li+, whereas the human NaDC1 is inhibited
only 20% even at 10 mM Li+. These results show
that rat NaDC3 and rabbit NaDC1 interact with Li+ with high
affinity. Furthermore, the present study shows that Li+ can
substitute for Na+, although to a small extent, in
supporting succinate transport by NaDC3. Similar results have been
obtained for rabbit NaDC1 (28). Thus, Li+ is a stimulator
as well as an inhibitor for NaDC3 and NaDC1. This cation stimulates the
activity of these transporters in the absence of Na+ but
inhibits the activity for these transporters in the presence of
Na+. Pajor et al. (28) have recently analyzed
the interaction of Na+ and Li+ with rabbit
NaDC1 in great detail. This analysis has led to the following
conclusions: (a) although Na+ is the preferred
cation for NaDC1, Li+ can support transport; (b)
one of the three cation binding sites in NaDC1 exhibits a higher
affinity for Li+ than for Na+, and
(c) the binding of Li+ to this site results in
inhibition of Na+-dependent transport function
of NaDC1. Our current data with NaDC3 suggest that NaDC3 behaves
essentially in a similar manner with regard to interaction with
Na+ and Li+.
NaDC3 and NaDC1 differ markedly in pH sensitivity. With succinate as
the substrate, rat NaDC1 shows no change in transport function between
pH 7.5 and 5.5 (15). Rabbit NaDC1 also behaves similarly in the pH
range of 7.5-5.5 with glutarate as the substrate (30). Transport of
glutarate by human NaDC1, on the other hand, is stimulated when pH is
changed from 7.5 to 5.5 (30). The influence of pH on the transport of
succinate by rat NaDC3 is dramatically different. The transport
function of NaDC3 is markedly inhibited by acidic pH. The pH
sensitivity profile of NaDC3 in the present study is similar to that of
succinate transport via the high affinity dicarboxylate transporter in
rat renal basolateral membrane vesicles (9).
The results of the influence of pH on the interaction of NaDC3 with
citrate are interesting. Citrate exists predominantly as a trivalent
anion at pH 7.5, but the concentration of the divalent anionic species
of citrate increases to significant levels when the pH is changed from
7.5 to 5.5. Our results with NaDC3 show that citrate is 25-fold more
potent in competing with the transport of succinate at pH 5.5 than 7.5. This suggests that the divalent anionic species of citrate rather than
the trivalent anionic species is preferred as the substrate by NaDC3.
This suggestion is supported by the differential influence of pH on the
NaDC3-mediated transport of succinate and citrate. While the transport
of succinate is inhibited steadily by a decrease in pH from 8.0 to 5.5, the transport of citrate actually increases when the pH is changed from
8.0 to 6.5. However, when the pH is made more acidic by decreasing the
pH below 6.5, the transport of citrate is inhibited. These observations
can be explained based on the opposing effects of acidic pH on the
transport activity of NaDC3 and the concentration of the transportable
ionic species of citrate. A gradual decrease in pH from 8.0 to 5.5 steadily decreases the transport function of NaDC3 but steadily
increases the concentration of the transportable divalent anionic
species of citrate. These two opposing effects of pH on the transport
function of NaDC3 and on the concentration of the transportable citrate
species are responsible for the biphasic influence of pH on
NaDC3-mediated citrate transport. These results are significant because
Chen et al. (15) have recently suggested that the high
affinity dicarboxylate transporter present in the renal basolateral
membrane is capable of transporting the divalent as well as trivalent
forms of citrate. This suggestion was primarily based on the findings
by Wright and Wunz (10) that citrate transport in renal basolateral
membrane vesicles is not affected markedly by pH. However, if the
inhibitory influence of acidic pH on the carrier function is taken into
account, it is obvious that the actual stimulatory effect of acidic pH
on citrate transport is significantly underestimated under these
conditions. We therefore conclude that there is no difference between
the low affinity and high affinity dicarboxylate transporters in terms
of preferential recognition of the divalent citrate over trivalent
citrate as the substrate.
Functional studies have demonstrated that the low affinity and high
affinity dicarboxylate transporters are differentially localized in the
kidney tubular cells (4-6, 9, 10). The low affinity transporter is
expressed in the brush border membrane, whereas the high affinity
transporter is expressed in the basolateral membrane.
Immunohistochemical studies using antibodies raised against the cloned
rat NaDC1 has recently established the brush border membrane
localization of the low affinity dicarboxylate transporter in kidney
(16). Functional studies have also demonstrated that the low affinity
transporter is expressed in the brush border membrane of intestinal
epithelial cells (7, 8) and the high affinity transporter is expressed
in the canalicular membrane of liver cells (11, 12). In the placenta,
the high affinity dicarboxylate transporter is present in the brush
border membrane (3). This membrane is in direct contact with maternal
blood, and therefore the transporter may mediate the entry of succinate and other Krebs cycle intermediates from maternal blood into the placental syncytiotrophoblast. We speculate that monocarboxylates such
as lactate and pyruvate and dicarboxylates such as succinate,
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]dCTP and Ready-to-go
oligolabeling kit were purchased from Amersham Pharmacia Biotech. Rat
multiple tissue Northern blot was purchased from OriGene Technologies
Inc. (Rockville, MD). All compounds used in the inhibition studies were
procured from Sigma.
-32P]dCTP using the ready-to-go oligolabeling kit and
used to screen a rat placental cDNA library (20, 21) under low
stringency conditions.
RESULTS
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Fig. 1.
Comparison of amino acid sequences of rat
NaDC3 and rat NaDC1.
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Fig. 2.
Hydropathy plot of rNaDC3 using the
Kyte-Doolittle method with a window size of 20 amino acids.
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Fig. 3.
Tissue distribution of NaDC3 mRNA
transcripts in rat. A, a commercially available
Northern blot containing 2 µg/lane of poly(A)+ RNA
isolated from brain, heart, kidney, stomach, small intestine, and
skeletal muscle (lanes 1-6, respectively) was
used for this purpose. The blot was probed with rNaDC3 cDNA, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.
The sizes of RNA hybridizing bands were determined using RNA standards
run in parallel in an adjacent lane. B, the expression of
NaDC3 mRNA transcripts in rat placenta (lanes
1 and 2) and rat liver (lanes
3 and 4) as analyzed by RT-PCR. Lanes
1 and 3 are minus RT controls. C,
restriction analysis of the RT-PCR products obtained from rat placenta
(lanes 1, 3, and 5) and rat
liver (lanes 2, 4, and 6).
Lanes 1 and 2, undigested;
lanes 3 and 4,
DraIII-digested; lanes 5 and
6, NcoI-digested.
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Fig. 4.
Time course of succinate uptake in
rNaDC3-transfected HRPE cells. Uptake of
[3H]succinate (20 nM) was measured in the
absence ( ) and presence (
) of Na+ at pH 7.5 in HRPE
cells transfected with rNaDC3 cDNA. In Na+-free buffer,
the NaCl in the uptake buffer was replaced with NMDG chloride. The
endogenous Na+-dependent transport of succinate
in HRPE cells was determined by measuring succinate uptake in parallel
in cells transfected with vector alone (
).
in the uptake buffer was replaced with
F
, I
, SCN
, or
NO3
, the initial uptake rate of
[3H]succinate was inhibited only marginally, suggesting
the noninvolvement of the anion in the transport process. This anion
independence is typical of transporters belonging to the
sodium-dependent glucose transporter family (27).
Influence of monovalent cations and anions on the uptake of
succinate in rNaDC3-expressing HRPE cells
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Fig. 5.
Kinetic analysis of rNaDC3 cDNA-induced
succinate uptake in HRPE cells. Uptake of succinate was measured
in cDNA-transfected cells with 1 min incubation in NaCl-containing
buffer at pH 7.5 over a succinate concentration range of 0.5-15
µM. The concentration of labeled succinate was kept
constant at 50 nM. Since the endogenous succinate transport
activity was negligible, it was not considered in the analysis of the
data. Inset, Eadie-Hofstee plot.
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Fig. 6.
Effect of Na+ on the uptake of
succinate in HRPE cells expressing the cloned rNaDC3 cDNA.
Uptake of 0.25 µM succinate (0.05 µM
radiolabeled succinate plus 0.2 µM unlabeled succinate)
was studied in HRPE cells transiently expressing the cloned rNaDC3
cDNA with a 1-min incubation in the presence of increasing
concentrations of Na+ (0-75 mM) and a fixed
concentration of Cl (140 mM) in the
extracellular medium. The osmolality of the medium was kept constant by
replacing Na+ with appropriate concentrations of NMDG.
Inset, Hill plot of the same data. v, uptake rate
in pmol/106 cells/min; Vm, the maximal
uptake rate calculated from the experimental data using the Hill
equation.
-ketoglutarate also inhibited the uptake of radiolabeled succinate
very potently, indicating that the presence of a hydroxyl group or a
keto group on the
-carbon atom does not interfere with the ability
to interact with the transporter. Similarly, dimethylsuccinate and
dimercaptosuccinate were also potent inhibitors of succinate uptake,
indicating that substitution at the second and third carbon atoms in
the succinate molecule with methyl or thiol groups does not abolish the
interaction with the transporter. Dipicolinate, on the other hand
exhibited a very small but significant ability to inhibit succinate
uptake. Citrate, which is predominantly trivalent at pH 7.5, also
inhibited succinate uptake by 60%. While the amino acid aspartate
inhibited succinate uptake significantly (~60%), glutamate had no
effect.
Substrate specificity of NaDC3
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Fig. 7.
Effect of pH on the inhibition of
[3H]succinate uptake by unlabeled succinate and citrate
in HRPE cells expressing rNaDC3 cDNA. HRPE cells were
transfected with rNaDC3 cDNA. Uptake of 20 nM
[3H]succinate was measured at pH 5.5 ( ) and 7.5 (
)
in the presence of Na+ with a 1-min incubation in the
absence and presence of varying concentrations of unlabeled succinate
(A) and citrate (B). The uptake measured in the
absence of inhibitors at each pH was taken as the corresponding control
uptake (100%). The control uptake was 0.23 ± 0.02 pmol/106 cells/min at pH 5.5 and 0.78 ± 0.03 pmol/106 cells/min at pH 7.5.
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Fig. 8.
Influence of pH on rNaDC3-induced uptake of
succinate (A) and citrate (B) in HRPE
cells. Uptake of [3H]succinate (20 nM)
and [14C]citrate (100 µM) was measured in
HRPE cells transfected with either rNaDC3 cDNA ( ) or vector
alone (
). The uptake was carried out for 1 min (succinate) or 10 min
(citrate) in Na+-containing buffers of varying pH.
DISCUSSION
-ketoglutarate, malate, and fumarate may be actively transported from mother to fetus across the placenta to serve as metabolic fuels
for fetal utilization. Our earlier studies have demonstrated the
existence of a H+-dependent monocarboxylate
transporter in the placental brush border membrane (31). The exit
mechanisms for these monocarboxylates and dicarboxylates in the
placental basal membrane remain to be investigated.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HD 33347 and DA 10045.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF081825.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Medical College of Georgia,
Augusta, GA 30912-2100. Tel.: 706-721-7652; Fax: 706-721-6608; E-mail: vganapat{at}mail.mcg.edu.
The abbreviations used are: HRPE, human retinal pigmental epithelial; rNaDC3, rat NaDC3; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); NMDG, N-methyl-D-glucamine; Mes, 4-morpholineethanesulfonic acid.
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