©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence and Functional Characterization of a Renal Sodium/Dicarboxylate Cotransporter (*)

(Received for publication, July 22, 1994; and in revised form, December 23, 1994)

Ana M. Pajor

From the Department of Physiology, University of Arizona, College of Medicine, Tucson, Arizona 85724

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(max) 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.


INTRODUCTION

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 (^1)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) .


EXPERIMENTAL PROCEDURES

Xenopus Oocytes

Stage V and VI oocytes from Xenopus laevis (NASCO) were dissected and defolliculated as described previously(9, 11) . They were injected with 50 nl of RNA (0.5 µg/µl) 1 day following isolation. Uptakes were measured 6 to 7 days later. The oocytes were maintained at 18 °C in Barth's solution (9) containing 50 mg/ml gentamicin sulfate, 2.5 mM sodium pyruvate, and 5% heat-inactivated horse serum(12) .

Transport Measurements

For measurement of succinate transport, groups of 5 oocytes were washed briefly in choline buffer (13) to remove serum, and transport was started with 0.4 ml (or 0.75 ml for kinetic studies) of [^14C]succinate or [^14C]citrate in sodium buffer. Oocyte uptake buffers consisted of: 100 mM NaCl (sodium buffer) or choline Cl (choline buffer), 2 mM KCl, 1 mM CaCl(2), 1 mM MgCl(2), and 10 mM HEPES-Tris (pH 7.5). After the transport time period, uptakes were stopped with 4 times 4 ml washes of ice-cold choline buffer. Each oocyte was dissolved in 0.5 ml of 10% SDS, and the ^14C was assayed by scintillation counting.

RNA Preparation and Size Fractionation

Rabbit organs were dissected and quick frozen in liquid nitrogen. RNA was prepared by the method of Chomczynski and Sacchi(14) , and poly(A) RNA was selected by oligo(dT) chromatography(13) . Approximately 200 µg of renal cortex mRNA was denatured at 65 °C, quick-chilled on ice, and then loaded onto a linear 5-15% sucrose density gradient, prepared in 1 mM EDTA, 10 mM Tris (pH 7.4). The gradient was centrifuged for 15 h at 4 °C in a Beckman SW28 rotor at 27,000 rpm. 2-ml fractions were collected and mRNA in each fraction was ethanol-precipitated and resuspended in water. Samples were stored at -80 °C.

cDNA Library Construction and Screening

The size fraction of rabbit renal cortex mRNA that induced the highest uptake of succinate transport when injected into Xenopus oocytes (1.5-3 kb, Fig. 1) was used for library construction. A directional cDNA library was made in the vector pSPORT1 using the Superscript Plasmid System (Life Technologies, Inc.) according to the manufacturer's directions. Approximately 8000 recombinants were screened by sib selection(15) . Bacteria were grown on solid media at all times during library screening. Replicas of each master plate were made onto nitrocellulose (S& BA85), and the colonies were allowed to grow overnight. The nitrocellulose facilitates recovery of colonies when purifying plasmid DNA. The colonies from each plate were scraped from the nitrocellulose, pelleted by centrifugation, and the plasmid DNA was purified using Qiaprep spin columns (Qiagen) according to the manufacturer's directions. The plasmid DNA was linearized with NotI, and capped cRNA was synthesized using the mMessage mMachine-T7 in vitro transcription kit (Ambion).


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).



cDNA Sequencing and Analysis

Both strands of the cDNA were sequenced using the dideoxy chain termination method (CircumVent, New England Biolabs) with oligonucleotide primers. The sequence was assembled and analyzed with the Genetics Computer Group package. Sequence comparisons were run using the BLAST server at the National Center for Biotechnology Information.

Northern Blot

Northern blots were prepared as described previously(11, 13) . The blot was probed (50% formamide, 42 °C) and washed (0.1 times SSC, 0.1% SDS, 55 °C) at high stringency.


RESULTS

Expression Cloning in Xenopus Oocytes

As we showed previously (9) , Xenopus oocytes injected with rabbit renal mRNA exhibit increased transport of succinate compared with uninjected controls (Fig. 1). Total renal cortex mRNA was fractionated by sucrose density centrifugation, and each fraction was injected into oocytes for measurement of succinate transporter expression. Injection of one of the size fractions, Fraction 7 (1.5-3 kb), resulted in the highest expression of succinate transport, to approximately 15-fold above background (Fig. 1). This size fraction of mRNA was used to make a cDNA library in the plasmid pSPORT1.

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.

NaDC-1 Sequence

The nucleotide and predicted amino acid sequence of NaDC-1 are shown in Fig. 2. NaDC-1 cDNA is 2357 nucleotides long and codes for a protein of 593 amino acids with a predicted molecular mass of 65,577 Da. The cDNA contains a Kozak consensus sequence for translation initiation, CACCAUGG(16) , and two potential N-glycosylation sites (Asn and Asn). In vitro translation experiments show a primary translation product of NaDC-1 at 57 kDa and a shift in mass to 64 kDa in the presence of microsomes, indicating core glycosylation of only one of the two potential sites (not shown). Kyte-Doolittle hydropathy analysis (17) of the NaDC-1 protein reveals at least eight hydrophobic stretches long enough to span the membrane as alpha-helices (Fig. 3). A secondary structure model of NaDC-1, based on this analysis, is also shown in Fig. 3. The model contains eight putative transmembrane domains, a large extracellular loop between transmembrane domains 3 and 4 containing a single N-glycosylation site, and a large intracellular loop between transmembrane domains 6 and 7 (Fig. 3). This model does not contain any putative phosphorylation sites for protein kinase C or cAMP-dependent protein kinase.


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) .

Tissue Distribution of NaDC-1

Since Na/dicarboxylate cotransport has been reported in organs other than the kidney, including intestine (22) and liver(23, 24) , the tissue distribution of mRNA coding for NaDC-1 was examined (Fig. 5). A rabbit Northern blot containing mRNA from different organs was probed at high stringency with the full-length NaDC-1 cDNA. A single transcript of about 2.8 kb was detected in both kidney and jejunum after an overnight exposure of the blot. After 7 days of exposure, hybridization signals of the same size were also evident in liver, lung, and adrenal. No hybridization signals were observed in mRNA from rabbit testis, stomach, salivary gland, thymus, spleen, heart, or brain (Fig. 5). Preliminary studies confirm that the renal and intestinal mRNAs may be identical. The sequences of two overlapping polymerase chain reaction fragments amplified from jejunum cDNA were identical with the sequence of NaDC-1 and corresponded to amino acids 63-344 (not shown).


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.



Transport Characterization of NaDC-1

Xenopus oocytes were used as an expression system in which to characterize the functional properties of NaDC-1. The expression of succinate transport in oocytes injected with NaDC-1 cRNA was proportional to the amount of cRNA injected. Expression was linearly related to the amount of cRNA injected up to approximately 25 ng, above which there was maximal expression of succinate transport (data not shown). Likewise, the expression of succinate transport increased with time after injection. Succinate transport was not detectable until 3 days after injection and then increased linearly through 6-7 days (data not shown). In addition, the variability between oocytes decreased with time after injection. All uptake experiments reported here were measured 6 to 7 days after injection of 25 ng of cRNA per oocyte.

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 [^14C]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 [^14C]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(m) for succinate was 446 µM, and the V(max) was 15 nmol/oocyte/h. This V(max) 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(max) 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 [^14C]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^0) 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 [^14C]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).




DISCUSSION

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(m) for succinate between 0.4 and 0.8 mM(3, 7) . The K(m) 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(m) 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(m) 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(m) for succinate of 670 µM(22) . In contrast, transport studies of liver canalicular membrane vesicles have identified a high affinity (K(m) approx 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(m) 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) .


FOOTNOTES

*
This study was supported by National Institutes of Health Grant DK46269. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) U12120[GenBank].

(^1)
The abbreviation used is: kb, kilobase(s).


ACKNOWLEDGEMENTS

Thanks to Ning Sun for assistance with part of the sequence. Thanks also to Drs. E.M. Wright and J. R. Halpert for critical comments on this manuscript.


REFERENCES

  1. Wright, E. M., Hager, K. M., and Turk, E. (1992) Curr. Opin. Cell Biol. 4, 696-702 [Medline] [Order article via Infotrieve]
  2. Hagenbuch, B., Stieger, B., Foguet, M., Lubbert, H., and Meier, P. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10629-10633 [Abstract]
  3. Wright, S. H., Hirayama, B., Kaunitz, J. D., Kippen, I., and Wright, E. M. (1983) J. Biol. Chem. 258, 5456-5462 [Abstract/Free Full Text]
  4. Brennan, T. S., Klahr, S., and Hamm, L. L. (1986) Am. J. Physiol. 251, F683-F689
  5. Wright, E. M., Wright, S. H., Hirayama, B. A., and Kippen, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7514-7517 [Abstract]
  6. Wright, E. M. (1985) Annu. Rev. Physiol. 47, 127-141 [CrossRef][Medline] [Order article via Infotrieve]
  7. Wright, S. H., and Wunz, T. M. (1987) Am. J. Physiol. 253, F432-F439
  8. Burckhardt, G. (1984) Pfluegers Arch. 401, 254-261 [Medline] [Order article via Infotrieve]
  9. Coady, M. J., Pajor, A. M., Toloza, E. M., and Wright, E. M. (1990) Arch. Biochem. Biophys. 283, 130-134 [Medline] [Order article via Infotrieve]
  10. Pajor, A. M. (1994) J. Am. Soc. Nephrol. 5, 296 (abstr.)
  11. Pajor, A. M., and Wright, E. M. (1992) J. Biol. Chem. 267, 3557-3560 [Abstract/Free Full Text]
  12. Quick, M. W., Naeve, J., Davidson, N., and Lester, H. A. (1992) BioTechniques 13, 358-362
  13. Pajor, A. M., Hirayama, B. A., and Wright, E. M. (1992) Biochim. Biophys. Acta 1106, 216-220 [Medline] [Order article via Infotrieve]
  14. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  15. McCormick, M. (1987) Methods Enzymol. 151, 445-449 [Medline] [Order article via Infotrieve]
  16. Kozak, M. (1987) J. Mol. Biol. 196, 947-950 [Medline] [Order article via Infotrieve]
  17. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  18. Markovich, D., Forgo, J., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8073-8077 [Abstract/Free Full Text]
  19. Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983 [Abstract]
  20. Khatri, S. A., Forstner, G. G., and Forstner, J. F. (1993) Biochem. J. 294, 391-399 [Medline] [Order article via Infotrieve]
  21. van der Rest, M. E., Siewe, R. M., Abee, T., Schwarz, E., Oesterhelt, D., and Konings, W. N. (1992) J. Biol. Chem. 267, 8971-8976 [Abstract/Free Full Text]
  22. Wolffram, S., Badertscher, M., and Scharrer, E. (1994) Exp. Physiol. 79, 215-226 [Abstract]
  23. Moseley, R. H., Jarose, S., and Permoad, P. (1992) Am. J. Physiol. 263, G871-G879
  24. Zimmerli, B., O'Neill, B., and Meier, P. J. (1992) Pfluegers Arch. 421, 329-335 [Medline] [Order article via Infotrieve]
  25. Wright, S. H., Kippen, I., Klinenberg, J. R., and Wright, E. M. (1980) J. Membr. Biol. 57, 73-82 [Medline] [Order article via Infotrieve]
  26. Ullrich, K. J. (1994) Biochim. Biophys. Acta 1197, 45-62 [Medline] [Order article via Infotrieve]
  27. Wright, S. H., Kippen, I., and Wright, E. M. (1982) Biochim. Biophys. Acta 684, 287-290 [Medline] [Order article via Infotrieve]
  28. Brennan, S., Hering-Smith, K., and Hamm, L. L. (1988) Am. J. Physiol. 255, F301-F306
  29. Steffgen, J., Kienle, S., Scheyerl, F., and Franz, H. E. (1994) Biochem. J. 297, 35-39 [Medline] [Order article via Infotrieve]
  30. Busch, A. E., Waldegger, S., Herzer, T., Biber, J., Markovich, D., Murer, H., and Lang, F. (1994) J. Biol. Chem. 269, 12407-12409 [Abstract/Free Full Text]
  31. Hamm, L. L. (1990) Kidney Int. 38, 728-735 [Medline] [Order article via Infotrieve]
  32. Pritchard, J. B., and Miller, D. S. (1992) in The Kidney: Physiology and Pathophysiology (Seldin, D. W., and Giebisch, G., eds) pp. 2921-2945, Raven Press, New York

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.