Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney

Ana M. Pajor and Nina N. Sun

Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations.1 Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na+-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is ~24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant (Km) for succinate was 0.35 mM, and the Km for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at -50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.

citrate; succinate; dicarboxylate transport; sodium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE NA+-dicarboxylate cotransporter of the apical membrane of the renal proximal tubule, NaDC-1, reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the tubular filtrate (19). This transporter has a broad substrate specificity for di- and tricarboxylates, but tricarboxylate substrates, such as citrate, are carried in protonated form. NaDC-1 is electrogenic, coupling three sodium ions with each divalent anion substrate molecule, with the net transfer of one positive charge (20). The Na+-dicarboxylate cotransporters belong to a gene family that includes the Na+-sulfate cotransporters, NaSi-1 and SUT-1 (6, 15). Three orthologs of NaDC-1 have been isolated from rabbit (rb), human (h), and rat (r) (17, 18, 28). This gene family also contains the Xenopus laevis (X. laevis) intestinal NaDC-2, which uses both sodium and lithium to drive dicarboxylate transport (2), and the high-affinity transporters, NaDC-3, found on the basolateral membrane of kidney proximal tubule and brush-border membrane of placenta (3, 11, 30).

One of the physiological functions of NaDC-1 is to regulate the concentration of urinary citrate, an endogenous inhibitor of calcium stone formation (8, 23). Low urinary citrate concentrations are associated with an increased risk of kidney stone formation (23). However, there is very little information on the relationship between NaDC-1 activity and the mechanisms that produce hypocitraturia. For example, it is not known whether NaDC-1 contributes to hypocitraturia directly, although there are reports of kidney stone patients with idiopathic hypocitraturia (24, 16). Alternately, the role of NaDC-1 in producing hypocitraturia could be secondary to another problem, such as metabolic acidosis. The transport of citrate by NaDC-1 is stimulated by acidic pH (21, 34). One mechanism for this transport activation is an increase in the concentration of the preferred substrate, citrate2-, as the pH is decreased. However, chronic metabolic acidosis also stimulates citrate uptake by inducing transporter activity (9), which is the result of an increase in NaDC-1 mRNA and protein (1). Other chronic conditions, such as K+ deficiency and starvation, also stimulate citrate transport (1, 29, 33).

As a first step in studying the regulatory mechanisms in NaDC-1 and preparatory to the production of transgenic or "knockout" mice, the gene structure and functional properties of the Na+-dicarboxylate cotransporter in mouse were studied. We find that the gene structure in mice is remarkably similar to that in humans and that the NaDC-1 found in mouse kidney has functional similarities to the other NaDC-1 orthologs. However, there are some species differences in substrate selectivity and in interaction with lithium, suggesting that mouse Na+-dicarboxylate cotransporter (mNaDC-1) may also be useful for studying structure-function relationships in this family of transporters.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

mNaDC-1 cloning. The cDNA coding for the mNaDC-1 was amplified from reverse-transcribed mouse kidney mRNA by PCR. The primers were designed based on sequence alignments of the rabbit Na+-dicarboxylate cotransporter (rbNaDC-1) with the mouse genomic sequence of chromosome 11 (GenBank AC002324) and were designed to amplify the coding region and part of the 3' untranslated region of mNaDC-1. The sense primer included the start codon (in bold): 5'-GACAGGCTGGTCCTCACCATGGC-3' and the antisense primer was: 5'-CCTGAACACCGGGAAACACACCC-3'. The PCR was done using the Advantage cDNA PCR kit (Clontech) containing a proofreading DNA polymerase, Klentaq, to minimize mutations. The PCR product was cloned into the pCRII vector using the TopoTA cloning kit (In Vitrogen). The pCRII vector contains EcoR I sites on both sides of the insertion site of the PCR product. This allowed the excision of the insert from the pCRII vector using EcoR I and subcloning into the EcoR I sites of a construct of pSPORT I containing the 5' untranslated region (UTR) and part of the 3' UTR and poly(A+) tail of rbNaDC-1, as described previously (18). Both strands of the mNaDC-1 cDNA were sequenced at the Sealy Center for Molecular Science (University of Texas Medical Branch, Galveston, TX). The sequence was assembled and analyzed, and the gene was mapped, using programs from the Genetics Computer Group package. Sequence comparisons were run using the BLAST server at the National Center for Biotechnology Information. The secondary structure model was prepared based on Kyte-Doolittle hydropathy analysis of the sequence (13).

X. laevis oocytes and transport measurements. Stage V and VI oocytes from X. laevis were dissected and injected as described previously (17). Transport of [3H]succinate (DuPont-NEN) and [14C]citrate (Moravek) was measured between 4 and 7 days after injection, also as described (17). The sodium transport buffer contained (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, buffered to pH 7.5 with Tris base. For choline or lithium transport buffers the NaCl was replaced by 100 mM choline Cl or LiCl, respectively. In the transport assays, the oocytes were rinsed briefly with choline buffer to remove sodium and serum. Transport was initiated by replacement of the choline rinse with 0.4 ml of the appropriate transport buffer as described in the figure legends. Transport was stopped by the addition of 4 ml ice-cold choline buffer followed by removal of extracellular radioactivity with three additional washes in cold choline buffer. Individual oocytes were transferred to scintillation vials and dissolved in 0.25 ml 10% SDS. Scintillation cocktail was added, and radioactivity was counted. Counts in control uninjected oocytes were subtracted from the counts in cRNA-injected oocytes. Data are presented as means ± SE, except for kinetic constants for which the error represents the error of the fit. Kinetic constants were calculated by nonlinear regression to the Michaelis-Menten and Hill equations, using SigmaPlot 5.0 software (Jandel Scientific).

Electrophysiology experiments. Measurements of substrate-induced inward currents in X. laevis oocytes expressing mNaDC-1 were made using the two-electrode voltage clamp technique, as described (20). The pulse protocol was controlled using pClamp6 software (Axon instruments) and consisted of 100-ms voltage steps from a holding potential of -50 mV between +50 and -150 mV in 20-mV decrements. Substrate-dependent currents were determined from the difference between currents measured in sodium buffer with and without substrate.

Northern blot. A mouse multiple-tissue Northern blot, purchased from Origene Technologies, contained 2 µg of poly(A+) RNA in each lane. The blot was probed with the full-length mNaDC-1 insert (EcoR I fragment) at high stringency in 50% formamide at 42°C (17). High-stringency washes in 0.1× SSC, 0.1% SDS at 55°C were also done as described previously (17).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of mNaDC-1. A routine search of the GenBank database revealed a genomic sequence of a portion of mouse chromosome 11 (GenBank accession no. AC002324) that is very similar in sequence to other NaDC-1-type Na+-dicarboxylate cotransporters (17, 18). Gene-specific primers were designed based on the sequence alignment with rabbit NaDC-1. PCR amplification of mouse kidney cDNA using the gene-specific primers produced a cDNA of 2,200 bases containing a single open reading frame. The mNaDC-1 cDNA sequence codes for a protein of 586 amino acids (Fig. 1). There are two consensus sequences for N-glycosylation, Asn570 and Asn580, both located at the COOH terminus, a glycosylation pattern that is similar to other members of this gene family (19). The sequence also contains two potential protein kinase C phosphorylation sites (Ser211 and Ser435), of which only Ser435 is predicted to be located in an intracellular loop. The alignment of the amino acid sequence of mNaDC-1 with other NaDC-1 orthologs is also shown in Fig. 1. The closest relative of mNaDC-1 is the rat NaDC-1, (rNaDC-1), which is 92% identical in sequence. The mNaDC-1 is 77% identical to human Na+-dicarboxylate cotransporter (hNaDC-1) and 75% identical to rbNaDC-1. The mNaDC-1 is ~47% identical to the NaDC-3 orthologs (from rat and flounder) and 45% identical to the Na+-sulfate cotransporter, NaSi-1 (4, 15, 28, 30).


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Fig. 1.   Alignment of Na+-dicarboxylate cotransporter type 1 (NaDC-1) amino acid sequences from mouse (GenBank accession no. AF201903), rat (AF058714), human (U26209), and rabbit (U12186). The alignment was made by using the Genetics Computer Group software package (GCG) program Pileup. The amino acids that are identical in all of the NaDC-1 orthologs are shown in the bold consensus line (Cons). The locations of the 11 predicted transmembrane domains are indicated by lines above the sequences.

Mouse Slc13a2 gene. The human gene coding for hNaDC-1 has been assigned the name SLC13A2, which represents the solute carrier family 13, member 2 (member 1 of this family is NaSi-1). Therefore, using the nomenclature for mouse genes, the mNaDC-1 gene is called Slc13a2. The intron-exon organization of the Slc13a2 gene, shown in Table 1, was determined by sequence alignment between the mNaDC-1 cDNA and the sequence of mouse chromosome 11 (GenBank AC002324). The total length of the gene is ~24.9 kb, and it is divided into 12 exons (Fig. 2). The sequence of the mNaDC-1 cDNA and the exons in the gene are identical. The gene sequence also contains a TATA box located 93 bases upstream of the start codon, suggesting that the translation start site is likely to be between 53-68 bases upstream of the start codon.

                              
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Table 1.   Exon/intron organization of the mouse Slc13a2 gene



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Fig. 2.   Top: secondary structure model of mouse Na+-dicarboxylate cotransporter (mNaDC-1) with 11 transmembrane domains and two N-glycosylation sites at the COOH terminus (Y). The amino acids encoded by each of the 12 exons in the Slc12a2 gene are shown as alternate shaded and nonshaded circles. Bottom: intron-exon organization of the Slc13a2 gene. The gene coding for mNaDC-1 contains 12 exons and a total size of ~24.9 kb. The sequence of the entire Slc13a2 gene is found in GenBank AC002324.

The predicted secondary structure model of mNaDC-1 (Fig. 2) contains 11 transmembrane domains and an extracellular COOH terminus, similar to the structure of the other NaDC-1s (19). The exons in the Slc13a2 gene code for different transmembrane domains, and the boundaries often occur near the interface between the membrane- and aqueous-exposed portions of the protein (Fig. 2). The intron-exon boundaries in the mouse gene are identical to those of the corresponding human gene, SLC13A2 [the human gene was mapped by comparing the hNaDC-1 sequence with the sequence of chromosome 17 (GenBank AC005726), results not shown]. The human gene is found on chromosome 17 p11.1-q11.1 (14, 18).

Functional characterization of mNaDC-1: substrates. X. laevis oocytes injected with cRNA coding for mNaDC-1 had sodium-dependent dicarboxylate transport activity, whereas control oocytes do not express dicarboxylate or tricarboxylate transporters (17, 20). As shown in Fig. 3, the Michaelis-Menten constant (Km) for succinate in mNaDC-1 was 318 µM, and in a second experiment the Km was 373 µM. The Km for citrate was 732 µM (Fig. 3), and in three experiments the mean Km for citrate was 612 ± 132 µM (means ± SE). The mNaDC-1 substrate affinities are more similar to those of the rbNaDC-1 (17), despite being more closely related in sequence to rNaDC-1, which has a Km for succinate of ~25 µM (4, 27). Also, there was no evidence of substrate inhibition by high concentrations of citrate or succinate in mNaDC-1, as reported for the rNaDC-1 (SDCT1) (4). As shown previously for other NaDC-1 orthologs, the transport of succinate by mNaDC-1 was unaffected by changes in pH whereas the transport of citrate was markedly stimulated at acidic pH (Fig. 4). This result supports the hypothesis that tricarboxylates are preferentially carried in protonated form and also shows that mNaDC-1 is functionally related to other NaDC-1s. The response to pH in NaDC-2 and the NaDC-3s are different from those of the NaDC-1 orthologs (2, 3, 11).


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Fig. 3.   Substrate kinetics in oocytes expressing mNaDC-1. Five-minute uptakes were measured in sodium-containing buffers. A: succinate transport as a function of increasing concentrations of succinate in the medium. The Michaelis-Menten constant (Km) for succinate is 318 ± 37 µM (the error represents the SE of the regression), and the maximum velocity (Vmax) is 18 ± 1 nmol  ·  oocyte-1  ·  h-1. B: kinetics of citrate transport. The Km for citrate is 732 ± 217 µM, and the Vmax is 19 ± 2 nmol  ·  oocyte-1  ·  h-1.



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Fig. 4.   Effect of pH on transport of succinate and citrate by mNaDC-1. Transport of 100 µM [3H]succinate or [14C]citrate was measured in mNaDC-1-injected Xenopus laevis oocytes for 15 min. Transport solutions contained 100 mM sodium buffered to pH 7.5 or 5.5. Data shown are means ± SE; n = 5 oocytes.

The substrate specificity of mNaDC-1 was determined by inhibition of succinate transport and by measurement of substrate-induced currents. The most potent inhibitor of succinate transport by mNaDC-1 was 2,2-dimethylsuccinate (DMS) (Fig. 5), a substrate previously thought to be specific for the high-affinity transporter, NaDC-3, found on the renal basolateral membrane (32). Other substrates that produced >50% inhibition of transport include fumarate, succinate, glutarate, tricarballylate, and alpha -ketoglutarate (Fig. 5). The substrate selectivity of mNaDC-1 was further tested by measurement of substrate-induced currents using the two-electrode voltage clamp technique. As is the case for other members of this family, transport of succinate by mNaDC-1 is electrogenic, producing inward currents in the presence of 1 mM substrate and 100 mM sodium. Figure 6A shows the voltage-dependence of succinate-induced currents in mNaDC-1. The currents produced by test substrates at -50 mV expressed as a percentage of the succinate-induced current are shown in Fig. 6B. As predicted by the transport inhibition profile, large currents were produced by dicarboxylates (fumarate, alpha -ketoglutarate, and dimethylsuccinate) and tricarboxylates (citrate and tricarballylate). Smaller currents, 10-30% of the succinate-induced currents, were seen in the presence of the acidic amino acids, aspartate and glutamate, and no currents were produced by the monocarboxylate, pyruvate.


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Fig. 5.   Substrate specificity of mNaDC-1. Transport of 10 µM [3H]succinate was measured for 15 min in the presence or absence of 1 mM test inhibitors. The transport in the presence of inhibitor is expressed as a percentage of control measured in the absence of inhibitor. DMS, 2,2-dimethylsuccinate; alpha -KG, alpha -ketoglutarate. Data shown are means ± SE; n = 3 experiments. There was significant inhibition (P < 0.05) by all substrates except for aspartate and pyruvate.



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Fig. 6.   A: voltage dependence of succinate-induced currents in an oocyte expressing mNaDC-1. The pulse protocol was as described in METHODS. Vm, membrane voltage. B: substrate-dependent inward currents (I) measured at -50 mV expressed as a percentage of the currents measured with succinate (Isuccinate). Data shown are means ± SE; n = 3 experiments.

Functional characterization: cations. The mouse NaDC-1 is a sodium-dependent transporter. Replacement of sodium with choline resulted in complete abolishment of transport (Fig. 7 and 8). Increasing the concentrations of sodium in the transport buffers resulted in a sigmoid activation of succinate transport (Fig. 7). The half-saturation constant for sodium (KNa) was 9.5 mM, and the apparent Hill coefficient, nH, was 1.48 (Fig. 7). In a second experiment (not shown) the apparent KNa was 11 mM and nH was 1.87. 


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Fig. 7.   Sodium activation of succinate transport in oocytes expressing mNaDC-1. The transport of 100 µM [3H]succinate was measured for 5 min in buffers containing 0-100 mM Na+ (Na+ replaced by choline). The data were fit by the Hill equation. The half-saturation constant for sodium (KNa): 9.5 ± 1.5 mM, Vmax: 1,359 ± 83 pmol · oocyte-1 · h-1, Hill coefficient (nH) = 1.48 ± 0.35 (± SE of regression). Each data point represents the means ± SE of 5 oocytes.



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Fig. 8.   Sodium activation of succinate-induced currents in an oocyte expressing mNaDC-1 measured with the 2-electrode voltage clamp method. A: succinate-dependent inward currents as a function of sodium concentration. The succinate concentration was 100 µM. The curves at four of the voltages tested are shown. B: the effect of voltage on Imax (top), the half-saturation constant for sodium (K0.5Na; middle), and apparent nH (bottom), determined from nonlinear regression to the Hill equation. The error bars represent errors of the fit.

The sodium-activation experiment was also done using the two-electrode voltage clamp technique (Fig. 8). The substrate-dependent currents in oocytes expressing mNaDC-1 were measured at different sodium concentrations. Similar to the radiotracer uptake experiment, there was a sigmoidal relationship between sodium concentration and substrate-dependent inward currents in mNaDC-1 (Fig. 8A). The half-saturation constant for sodium (K0.5sodium) was 28 mM at -50 mV [in 3 experiments, K0.5sodium at -50 mV was 22.5 ± 3.1 mM, means ± SE]. The K0.5sodium decreased as the membrane voltage became more negative (Fig. 8B), indicating that sodium binding is a voltage-sensitive step. The maximum current observed at saturating substrate concentrations, (Imax), became larger with more negative membrane potentials. The nH under voltage clamp conditions was ~2 (between 1.9-2.1) at all voltages tested (Fig. 8B). In three experiments, the nH at -50 mV was 2.1 ± 0.04.

In the rbNaDC-1, lithium competes with sodium with high affinity at one of the three sodium binding sites, resulting in transport inhibition with an apparent inhibition constant of 2 mM (20). However, in the absence of sodium, lithium can drive transport of succinate in rbNaDC-1, although the succinate Km measured in lithium is very high, around 3 mM (20). Therefore, we examined the effects of lithium in mNaDC-1. As shown in Fig. 9A, lithium can partially substitute for sodium, producing ~30% of the transport rate seen in sodium. The inhibition by lithium was lower than that seen in the rbNaDC-1, however. In 5 mM lithium the inhibition was only ~20% (Fig. 9B).


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Fig. 9.   Cation dependence of mNaDC-1. A: transport of 100 µM [3H]succinate was measured in transport buffers containing 100 mM concentrations of sodium, lithium, or choline (as chloride salts). B: transport of 100 µM [3H]succinate was measured in transport buffers containing either 95 mM Na+:5 mM choline or 95 mM Na+:5 mM Li+. Fifteen-minute uptakes were measured. Data shown are means ± SE of 5 oocytes.

The cation selectivity of currents induced by mNaDC-1 is shown in Fig. 10. There are no inward currents in the presence of choline. Interestingly, the substrate-dependent currents in lithium are ~8% of the currents measured in sodium, which is lower than the uptake measurements in lithium (Fig. 9A). In three experiments, the currents measured in lithium at -50 mV and 1 mM succinate were 10 ± 1.5% (means ± SE) of the currents measured in sodium.


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Fig. 10.   Cation specificity of mNaDC-1 measured using the 2-electrode voltage clamp technique. Steady-state substrate-dependent currents measured in 100 mM sodium, lithium, or choline are plotted as a function of membrane potential. The succinate concentration was 1 mM.

Northern blot. The tissue distribution of mNaDC-1 mRNA is shown in Fig. 11. The predominant hybridization signal was seen at 2.4 kb in kidney and small intestine although there was a less abundant message of ~5 kb. There was no hybridization signal with brain, heart, stomach and skeletal muscle, similar to the tissue distributions of other NaDC-1 orthologs (19).


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Fig. 11.   Multiple tissue Northern blot of mouse poly(A+) RNA probed at high stringency with mNaDC-1 cDNA. The major hybridization signal was at 2.4 kb in kidney and small intestine. Sk muscle, skeletal muscle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mouse low-affinity Na+-dicarboxylate cotransporter, called mNaDC-1, is found in the kidney and small intestine. The Slc13a2 gene, coding for mouse NaDC-1, is located on chromosome 11, and has been mapped to ~10 kb upstream of the nude gene, whn (25). Slc13a2 is divided into 12 exons, each of which codes for approximately one transmembrane domain of mNaDC-1, which resembles the gene structure of other membrane transporters, such as the Na+-glucose cotransporter, hSGLT1 (31). Remarkably, the gene structure of Slc13a2 is identical to the structure of the human gene, SLC13A2, which is found on chromosome 17 (14, 18).

The functional properties of mNaDC-1 resemble those of other NaDC-1 orthologs, although there are some interesting species differences. Transport by mNaDC-1 is sodium dependent, electrogenic, and the preferred substrates appear to be divalent anions, as seen in the other members of this family (19). The mNaDC-1 transporter has a broad substrate specificity for a range of di- and tricarboxylates, including fumarate, succinate, and citrate. Interestingly, mNaDC-1 handles DMS as a substrate. Although DMS was previously thought to be specific for the high-affinity Na+-dicarboxylate cotransporter (NaDC-3) found on the basolateral membrane in kidney proximal tubule (32), some of the low-affinity (NaDC-1) transporters, such as rNaDC-1, also carry DMS (20).

The coupling coefficient in mNaDC-1 is likely to be 3 Na+:1 divalent anion substrate, similar to the other NaDC transporters (19). Inward currents were measured in oocytes expressing mNaDC-1 in the presence of substrate and sodium, suggesting that there is a net movement of positive charge into the cells. Citrate transport was stimulated by acidic pH, which supports the idea that citrate is carried in divalent form. There was no effect of pH on succinate transport, suggesting that both succinate2- and succinate1- may be substrates of the transporter, although succinate exists predominantly as a divalent anion at physiological pH. Finally, the apparent nH of 1.5-2.1 in mNaDC-1 is also consistent with three sodium binding sites. The nH indicates the minimal number of sodium binding sites, and is affected by the strength of interaction or cooperativity between the sites (26).

The m- and rNaDC-1 orthologs are ~92% identical in amino acid sequence. Despite the sequence similarity, some of their functional properties are very different. For example, the apparent Km for succinate in mNaDC-1 is around 0.35 mM, similar to the Km of the rb- and hNaDC-1 (21). Similarly, previous studies using rabbit brush-border membrane vesicles showed that the Km for succinate is around 0.5 mM (12, 35). In contrast, rNaDC-1 has a higher apparent substrate affinity, with a Km for succinate of 25 µM for the cloned transporter expressed in X. laevis oocytes (27, 4) and a Km of ~150 µM in rat renal brush-border membrane vesicles (5, 29).

There are also differences between m- and rNaDC-1 in their sensitivity to inhibition by lithium. The mNaDC-1 is relatively insensitive to inhibition by lithium, with only ~20% inhibition in the presence of 5 mM lithium. Furthermore, lithium can partially substitute for sodium in mNaDC-1. In contrast, the rNaDC-1 is sensitive to inhibition by lithium, with an IC50 of ~3 mM, and lithium cannot substitute for sodium (4). Because lithium competes with sodium at one of the three cation binding sites, the identification of residues contributing to lithium binding or inhibition may provide information on differences in the structures of cation binding sites. Chimeras made between h- and rbNaDC-1 transporters show that transmembrane domain 11 and the COOH-terminal tail contain the residues that determine their differences in lithium sensitivity (10). The rat and mouse sequences only differ by 8%, and in exon 12, which codes for the last transmembrane domain, there are only 4 amino acid differences.

One of the physiological functions of NaDC-1 is to reabsorb filtered citrate from the proximal tubule lumen, thus helping to regulate the concentration of urinary citrate. Citrate forms soluble complexes with calcium, which prevents the precipitation of calcium in the form of calcium oxalate or calcium phosphate stones. Urinary citrate concentrations are low in approximately one-half of patients with renal stones (23). However, there is still very little information on the relationship between NaDC-1 and hypocitraturia. To produce hypocitraturia, the activity of NaDC-1 has to increase, which could be a consequence of several events. For example, in many kidney stone patients, there are disturbances in acid-base balance. Chronic metabolic acidosis leads to an increased Vmax for citrate, mediated through increased NaDC-1 mRNA and protein concentrations (1). However, there are also reports of kidney stone patients with idiopathic hypocitraturia or low urinary citrate in the absence of other disorders (16, 24). One possible mechanism that could produce low urinary citrate would be an increase in NaDC-1 activity by direct effects on the transporter properties. In humans the fractional excretion of citrate is quite high, ~30%, compared with ~2% in other species (8). This difference may be due to species differences in citrate Km that are approximately tenfold between hNaDC-1 and rNaDC-1 (21). Our mutagenesis studies have shown that a single mutation in NaDC-1 could potentially produce a large change in Km (7). An alternate mechanism of activation of NaDC-1 could involve second messenger systems, producing an increase in turnover number. There is some evidence that NaDC-1 activity can be modulated by increasing the activity of protein kinases. Treatment of oocytes with phorbol esters results in a decreased activity by NaDC-1, of which ~30% can be accounted for by endocytosis of the transporter from the plasma membrane, suggesting that the remaining decrease in activity is due to an effect on the transporter itself (22).

In conclusion, we have presented the sequence, chromosomal organziation, and tissue distribution of mNaDC-1. The mNaDC-1 is a low-affinity Na+-dicarboxylate cotransporter with broad substrate selectivity, found in kidney and intestine. This report is the first characterization of the NaDC-1 from mouse and provides fundamental information about the transporter as a basis for production of transgenic or knockout animals. Furthermore, the functional differences between the mouse and other species identified in this study should also be useful for future structure-function studies.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants (DK-46269 and DK-02429).


    FOOTNOTES

Address for reprint requests and other correspondence: A. M. Pajor, Univ. of Texas Medical Branch, Dept. of Physiology and Biophysics, Galveston, TX 77555 (E-mail: ampajor{at}utmb.edu).

1  The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL data bank with accession number AF201903.

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. §1734 solely to indicate this fact.

Received 10 December 1999; accepted in final form 13 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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