(Received for publication, July 22, 1994; and in revised form, December 23, 1994)
From the
The cDNA coding for a rabbit renal
Na/dicarboxylate cotransporter, designated NaDC-1, was
isolated by functional expression in Xenopus oocytes. NaDC-1
cDNA is approximately 2.3 kilobases in length and codes for a protein
of 593 amino acids. NaDC-1 protein contains eight putative
transmembrane domains, and the sequence and secondary structure are
related to the renal Na
/sulfate transporter, NaSi-1.
Northern analysis shows that the NaDC-1 message is abundant in kidney
and small intestine, and related transporters may be found in liver,
lung, and adrenal. The transport of succinate by NaDC-1 was
sodium-dependent, sensitive to inhibition by lithium, and inhibited by
a range of di- and tricarboxylic acids. This transporter also carries
citrate, but it does not transport lactate. In kinetic experiments, the K
for succinate was around 0.4 mM and the V
was 15 nmol/oocyte/h, while the
Hill coefficient of Na
activation of succinate
transport was 1.9. The transport of succinate by NaDC-1 was insensitive
to changes in pH, whereas the transport of citrate increased with
decreasing pH, in parallel with the concentration of divalent citrate
in the medium. The results of the functional characterization indicate
that NaDC-1 likely corresponds to the renal brush-border
Na
/dicarboxylate cotransporter.
Sodium-coupled transport is a widely distributed mechanism for
the concentrative accumulation of solutes across biological membranes
and is found in organisms ranging from bacteria to humans(1) .
In recent years, molecular cloning techniques have revealed several
gene families of sodium-coupled transporters, including those related
to the Na/glucose or the
Na
/Cl
/
-aminobutyric acid
cotransporters (1) , each with distinct structural and
functional properties. Some of these families consist of proteins with
12 predicted transmembrane domains, while others, such as the
Na
/bile acid cotransporters(2) , appear to
have only 7. There is also diversity in reaction mechanisms. Although
some families of sodium cotransporters couple one Na
ion per substrate molecule, additional mechanisms are found,
including coupling of Cl
or K
as
well as Na
.
The low affinity
Na/dicarboxylate cotransporter of the mammalian kidney
has been functionally characterized in isolated brush-border membrane
vesicles (3) and intact proximal tubules(4) . The
reaction cycle of this transporter is thought to involve the ordered
binding of three Na
ions followed by a divalent anion
substrate(3) . Furthermore, this transporter has a unique
sodium binding site with a high affinity for lithium(5) . Two
different Na
/dicarboxylate cotransporters have been
identified on opposite membranes of the kidney proximal tubule: the low
affinity transporter, with K
for
succinate around 0.4 to 0.8 mM, is found on the apical
membrane(6, 7) , while the high affinity transporter,
with K
for succinate around 10
µM, is found on the basolateral
membrane(7, 8) . Molecular studies of these
transporters should help to identify residues or domains involved in
determining the differences in substrate affinity, as well as resolving
the mechanism of concentrative transport involving four charged
substrates.
While the functional characteristics of the mammalian
Na/dicarboxylate cotransporters have been studied in
detail, nothing was known about these transporters at the molecular
level. Previously, we showed that a renal Na
/succinate
cotransporter could be functionally expressed from a mRNA size fraction
of 1.5-3 kb (
)injected into Xenopus oocytes(9) . This study reports the isolation of the cDNA
coding for a renal Na
/dicarboxylate cotransporter,
NaDC-1, by expression cloning in Xenopus oocytes. Based on its
functional properties, it is likely that NaDC-1 corresponds to the low
affinity Na
/dicarboxylate cotransporter found on the
apical membrane of the renal proximal tubule. The initial findings of
this study have been published in abstract form(10) .
Figure 1: Succinate transport in Xenopus oocytes during stages in expression cloning of NaDC-1. Oocytes were injected with (left to right): (i) controls, (ii) total renal cortex mRNA, (iii) Fraction 7 mRNA (1.5-3 kb), the size fraction of renal mRNA that gave the highest succinate transport of the six size fractions tested, (iv) cRNA prepared from a pool of 500 clones in the first screen of the cDNA library, (v) cRNA prepared from a pool of 50 clones during the second round of library screening, (vi) cRNA prepared from a pool of 8 clones during the third round of library screening, (vi) clone 201 (NaDC-1) cRNA. Oocytes were injected with 50 nl of RNA, and transport of 250 µM succinate was measured 6 days later in sodium-containing buffer. Data shown are means ± S.E. (n = 5).
The cDNA library was screened by the process of sib selection(15) , during which the expression of sodium-dependent succinate transport was monitored in cRNA-injected Xenopus oocytes. The initial screen of the library involved 16 pools of about 500 colonies each. Only one group of oocytes, injected with Pool 10, exhibited greater succinate transport than controls. The signal was only 2-fold above background (Fig. 1, Screen 1). To continue selecting for the succinate transporter, Pool 10 was subdivided into pools of 48-50 colonies each, and one of these groups produced higher succinate transport than controls, with a signal about 3-fold above background (Fig. 1, Screen 2). This pool was further subdivided into groups of about 8 colonies, and the positive group among these induced succinate transport by approximately 70-fold above controls (Fig. 1, Screen 3). Finally, oocytes injected with cRNA from purified clone 201 exhibited succinate transport rates approximately 1000-fold above controls (Fig. 1). Bacteria containing clone 201 produced smaller colonies, which could account for the relatively low succinate transport during the initial library screens compared with the extremely high expression from the pure clone. The cDNA was called NaDC-1 or sodium dicarboxylate transporter 1.
Figure 2: Nucleotide (below) and deduced amino acid sequence (above) of NaDC-1 cDNA. The nucleotides are numbered in the left margin, and the amino acids are numbered in italics in the right margin.
Figure 3: Upper panel, hydropathy analysis of the deduced amino acid sequence of NaDC-1 by the method of Kyte and Doolittle (17) using a window of 9 amino acids. The eight potential transmembrane domains are indicated by numbers. Lower panel, predicted secondary structure model of NaDC-1 based on hydropathy analysis. There are eight putative transmembrane domains, the N and C termini are both inside the cell. The predicted N-glycosylation site is indicated by a Y.
The amino acid sequence of NaDC-1 is
related to the rat renal Na/sulfate transporter,
NaSi-1(18) . There is 43% sequence identity and 66% similarity
between the two proteins (GCG program, GAP) (Fig. 4). The
predicted secondary structure models of both proteins are similar.
Eight membrane-spanning regions have also been predicted for the
otherwise unrelated Na
/phosphate
transporters(19) . However, the relative sizes of the
connecting loops are not similar to those of NaDC-1 and NaSi-1. The 8
transmembrane domain model differs from the 7 (2) or 12 (1) transmembrane domain secondary structures predicted by
Kyte-Doolittle analysis for other sodium-coupled transporters. However,
all of these structural models remain to be tested.
Figure 4: Sequence alignments of NaDC-1. Upper panel, comparison of the deduced amino acid sequences of NaDC-1 (above) and the rat renal sodium/sulfate transporter, NaSi-1 (18) (below). The alignment was made using the Genetics Computer Group program, GAP. Lines denote identical amino acids, and colons show chemically similar amino acids. There is 43% identity and 66% similarity between the two sequences. Lower panel, comparison of amino acids 482-568 of NaDC-1 (above) with amino acids 71-157 of rat intestinal mucin (20) (below). The alignment was made using the Genetics Computer Group program, Bestfit. There is 86% identity and 94% similarity between these two sequence segments.
There are no
other mammalian proteins with significant overall sequence similarity
to NaDC-1, although there are hypothetical proteins based on gene
sequences in yeast and Caenorhabditis elegans (GenBank Z30974 and X77395) that show some sequence similarity. There is,
however, an 87-amino acid domain near the C terminus (amino acids
477-586) of NaDC-1 that is 86% identical with the C terminus of
rat intestinal mucin (20) (Fig. 4). The corresponding
region of NaSi-1 is 53% identical with rat mucin (data not shown).
Epithelial mucins are heavily glycosylated proteins containing numerous
sequence repeats, which serve as attachment sites for O-linked
oligosaccharides, and unique segments at the N and C
terminus(20) . It is the nonrepeating, unique domain of rat
mucin that is 86% identical with NaDC-1. The possible function of this
domain in either protein is unknown at the moment. The sequence of
NaDC-1 is not related to the bacterial Na
- or
H
-dependent citrate transporters(21) .
Figure 5: Northern blot of rabbit RNA probed at high stringency with NaDC-1 cDNA. The adrenal and testis samples were total RNA (25 µg each), and the other samples were mRNA (5 µg each). The upper panel shows an overnight exposure, the lower panel shows a 7-day exposure. The positions of size standards (2.4 and 4.4 kb) are indicated at the right.
Studies in isolated membrane vesicles indicate that the
Na/dicarboxylate transporters exhibit a broad
substrate specificity for di- and tricarboxylic acids, but exclude
monocarboxylic acids(7, 25) . Fig. 6shows the
uptake of three different substrates in control and NaDC-1-injected
oocytes. There was very little or no transport of succinate and citrate
in control, uninjected oocytes ( Fig. 6and results not shown).
Oocytes injected with NaDC-1 cRNA exhibited transport of both succinate
and citrate, although the rate of succinate transport was higher. There
was no expression of lactate transport in oocytes injected with NaDC-1 (Fig. 6). The inhibition of succinate transport by test
substrates is shown in Fig. 7. As seen in previous
studies(25) , transport was inhibited by di- and tricarboxylic
acids including succinate, malate, citrate, and fumarate, with the same
relative inhibitory effect. There was no inhibition of succinate
transport by a 1 mM concentration (100-fold excess) of
lactate, pyruvate, aspartate, or sulfate. Furthermore, a test inhibitor
of basolateral Na
/dicarboxylate transport,
dimethylsuccinate(25, 26) , did not inhibit succinate
transport (Fig. 7) by NaDC-1.
Figure 6:
Transport of 250 µM [C]succinate, -citrate, or -lactate in
control (uninjected) or NaDC-1-injected (25 ng) oocytes. Uptakes were
measured 6 days after injection in a sodium-containing buffer (100
mM NaCl) for 30-min time periods. Data shown are means
± S.E. (n = 5).
Figure 7:
Inhibition of succinate transport by test
inhibitors in oocytes injected with NaDC-1 cRNA. The initial rate (5
min) of 10 µM [C]succinate was
measured in the presence or absence of 1 mM test inhibitor. DMS = dimethylsuccinate. Data shown are means ±
S.E. (n = 5), * = p < 0.05 relative
to controls.
Since the greatest difference
between the two Na/dicarboxylate cotransporters is in
substrate affinity (7) , the kinetics of succinate transport by
NaDC-1 was examined. The concentration dependence of succinate
transport in oocytes injected with NaDC-1 cRNA is shown in Fig. 8. Preliminary experiments indicated that the time course
of uptake of 1 mM succinate is linear through 5 min (data not
shown). Therefore, 5-min uptakes were used to calculate the initial
rates of succinate uptake in kinetic experiments. The uptake was
saturable and could be described by the Michaelis-Menten equation. The
Woolf-Augustinsson-Hofstee plot of the data (Fig. 8) was linear,
which is consistent with a single carrier-mediated mechanism, and
verifies the lack of diffusive pathways for succinate in oocytes. The
apparent K
for succinate was 446 µM,
and the V
was 15 nmol/oocyte/h. This V
indicates extremely high expression of NaDC-1
in oocytes.
Figure 8:
Concentration dependence of succinate
transport in oocytes injected with NaDC-1 cRNA. Oocytes were injected
with 25 ng of NaDC-1 cRNA, and uptakes were measured 6 days later in
sodium-containing buffer. The data have been corrected for background
counts (control oocytes had only background counts, between 18 and 25
dpm, at all concentrations tested). Five-minute uptakes were measured.
The concentration of succinate ranged from 50 µM to 5
mM. The inset shows a Woolf-Augustinsson-Hofstee plot
of the same data. The K and V
values are 446 µM and 15
nmol/oocyte/h, respectively, and were calculated using linear
regression. Data shown are means ± S.E. (n =
5).
The transport of succinate by NaDC-1 was sodium-dependent. As shown in Fig. 9, when sodium was removed from the medium and replaced with choline, the uptake of succinate was reduced to control levels. Fig. 9also shows that there is a sigmoid relationship between the initial rate of succinate transport and the concentration of sodium in the transport buffer. The Hill coefficient of the data is 1.9, indicating that at least two sodium ions are involved in the transport of succinate.
Figure 9:
Sodium
activation of succinate uptake in oocytes injected with NaDC-1 cRNA.
Five-minute uptakes of 100 µM [C]succinate were measured in the presence
of increasing concentrations of sodium in the transport buffer. The
NaCl was replaced by choline Cl. The data have been corrected for
background counts in uninjected oocytes (see legend to Fig. 8).
Nonlinear regression analysis was used to determine the apparent Hill
coefficient (n
= 1.9) and K
(50 mM). Data shown are means ±
S.E. (n = 5).
Additional features
characteristic of renal Na/dicarboxylate cotransport
include sensitivity to inhibition by lithium(5) , which
competes with sodium at one of the sodium-binding sites, and
sensitivity of citrate transport to changes in pH(27) . Fig. 10shows that succinate transport by NaDC-1 is also
inhibited by lithium. While replacement of 25 mM sodium in the
transport buffer by choline decreases succinate transport very slightly
(similar to what was seen in Fig. 9), the presence of 25 mM lithium inhibits transport by more than 50%. The effect of pH on
the transport of succinate and citrate by NaDC-1 is shown in Fig. 11. The transport of succinate was not affected by changes
in pH between 5.5 and 8. However, there was a marked change in citrate
transport with pH, with much higher transport of citrate at low pH and
lower transport at high pH. The concentrations of the four different
species of citrate (citrate
,
citrate
, citrate
,
citrate
) at these pH values were calculated using the
Henderson-Hasselbach equation, as described in previous
studies(27, 28) . The decrease in citrate transport
with increasing pH followed the concentration of citrate
and citrate
in the transport buffer (results
not shown).
Figure 10:
Inhibition of succinate transport by
lithium in oocytes injected with NaDC-1 cRNA. Five-minute uptakes of
100 µM [C]succinate were measured
in 100 mM NaCl or in 75 mM NaCl plus 25 mM LiCl, or 75 mM NaCl plus 25 mM choline Cl. Data
shown are means ± S.E. (n =
5).
Figure 11:
pH
dependence of succinate and citrate transport in oocytes injected with
NaDC-1 cRNA. The uptakes have been corrected for transport in control
oocytes. The transport of succinate (left) and citrate (right) was measured in Na-containing
transport buffers adjusted to pH ranging from 5.5 to 8. Five-minute
uptakes were measured. Data shown are means ± S.E. (n = 5).
This study reports the expression cloning and initial
transport characterization of the rabbit renal
Na/dicarboxylate cotransporter, NaDC-1. Based on the
results of the transport experiments reported here, it appears that
NaDC-1 corresponds to the Na
/dicarboxylate
cotransporter previously characterized on the apical membrane of the
renal proximal tubule. The sequence of NaDC-1 indicates that it
represents a new family in the superfamily of transporters related to
the renal Na
/sulfate transporter, NaSi-1. NaDC-1 is
the first mammalian Na
/dicarboxylate cotransporter to
be cloned.
Injection of NaDC-1 cRNA into Xenopus oocytes
results in an increase in succinate transport up to several
thousandfold above background. The transport properties of NaDC-1
expressed in Xenopus oocytes are remarkably similar to those
of the Na/dicarboxylate cotransporter characterized in
brush-border membranes from the outer renal cortex (3) and in
isolated perfused early proximal tubules(4) . Previous studies
in rabbit renal brush-border membrane vesicles found a K
for succinate between 0.4 and 0.8
mM(3, 7) . The K
of 450
µM for succinate transport by NaDC-1 agrees very well with
that of the native transporter. By comparison, the rabbit renal
basolateral Na
/dicarboxylate cotransporter has a K
for succinate of 10 µM(7) ,
while the rat basolateral Na
/dicarboxylate transporter
expressed in Xenopus oocytes after injection of total renal
mRNA has a K
for succinate of 24
µM(29) .
The substrate specificity and pH
dependence of NaDC-1 are also similar to those of the renal
brush-border Na/dicarboxylate cotransporter. The
transporter characterized in brush-border membranes carries citrate,
while the basolateral transporter excludes citrate, although citrate
does inhibit the transport of succinate(7) . NaDC-1 transports
both succinate and citrate and was inhibited by a range of di- and
tricarboxylates, just as the native transporter. In addition, there was
no inhibition of succinate transport by dimethylsuccinate, a test
inhibitor of the basolateral transporter(26) . The pH
independence of succinate transport seen in NaDC-1 is another feature
of the brush-border transporter(7, 8, 27) .
In contrast, the basolateral Na
/dicarboxylate
transporter shows a pH optimum around pH 7-7.5, although the
magnitude of this effect varies between studies (7, 8) . The transport of citrate by NaDC-1 is
strongly affected by pH, in parallel with the concentration of divalent
and monovalent citrate in the transport buffer. This result agrees with
previous studies indicating that citrate is transported predominantly
in divalent form (27, 28) .
Analysis of the
secondary structure of NaDC-1 indicates a protein of eight
membrane-spanning domains, similar in sequence and predicted secondary
structure to the renal Na/sulfate transporter, NaSi-1.
This similarity in structure may be related to similarities in
transport mechanism. For example, both transporters couple multiple
sodium ions to the transport of each divalent anion substrate. The Hill
coefficient for NaDC-1 was 1.9, and for NaSi-1 1.8(18) ,
indicating a minimum of two highly cooperative sodium binding sites.
Recent studies, using a two-electrode voltage clamp, of oocytes
expressing NaSi-1 (30) have shown that NaSi-1 is electrogenic
and couples three sodium ions to the transport of each sulfate. It is
likely that NaDC-1 also has a sodium coupling coefficient of 3. The
substrates of both transporters are divalent anions, although there are
distinct differences in substrate specificity, and the transport of
succinate by NaDC-1 is not inhibited by sulfate. In addition, there may
be differences in the sodium ion binding sites between the two
proteins, since NaDC-1 transport is inhibited by lithium, which is
thought to bind to one of the sodium binding sites. Further studies
based on comparisons between the two sequences may reveal information
about the mechanism of transport in this superfamily.
The results of
the Northern analysis indicate that the same
Na/dicarboxylate cotransporter may be expressed in
both kidney and small intestine. The succinate transport properties of
jejunum (and colon) are similar to those of the renal brush-border (22) . Transport in rat jejunum was sodium-dependent, inhibited
by citrate, and had a K
for succinate of 670
µM(22) . In contrast, transport studies of liver
canalicular membrane vesicles have identified a high affinity (K
10 µM)
Na
/dicarboxylate transport
pathway(23, 24) , similar to the basolateral
transporter of the renal proximal tubule(7) . The weak signal
seen on the Northern blot in liver, adrenal, and lung could indicate
either a low abundance of NaDC-1 mRNA or a closely related message,
possibly coding for the high affinity Na
/dicarboxylate
cotransporter, in those organs.
In conclusion, the cDNA coding for a
renal sodium-dicarboxylate transporter has been cloned by functional
expression in Xenopus oocytes. The functional characteristics
of NaDC-1 expressed in Xenopus oocytes, including the K for succinate around 450 µM,
transport of citrate, and insensitivity of succinate transport to
extracellular pH, are very similar to the functional properties of the
Na
/dicarboxylate cotransporter found on the apical
membrane of the renal proximal tubule. NaDC-1 is a protein of 593 amino
acids, with eight putative transmembrane domains and a single predicted N-glycosylation site. The sequence and predicted secondary
structure of NaDC-1 are related to the Na
/sulfate
transporter, NaSi-1. NaDC-1 is found primarily in kidney and small
intestine, consistent with previous functional studies of
Na
/dicarboxylate cotransport. The molecular cloning of
NaDC-1 cDNA will provide valuable insights into the mechanism of
sodium/dicarboxylate transport and will help to elucidate the potential
role of NaDC-1 in the formation of kidney stones (31) and
secretion of organic anions(32) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U12120[GenBank].