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. 2014 Jun 13;289(24):16998-7008.
doi: 10.1074/jbc.M114.554790. Epub 2014 May 7.

Determinants of substrate and cation transport in the human Na+/dicarboxylate cotransporter NaDC3

Avner Schlessinger  1 , Nina N Sun  2 , Claire Colas  3 , Ana M Pajor  4
Affiliations

Determinants of substrate and cation transport in the human Na+/dicarboxylate cotransporter NaDC3

Avner Schlessinger et al. J Biol Chem. .

Abstract

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.

Keywords: Citrate; Homology Modeling; Membrane; Molecular Docking; SLC13 Family; Sodium Transport; Succinate; Tricarboxylic Acid Cycle (TCA Cycle) (Krebs Cycle).

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Figures

FIGURE 1.
FIGURE 1.
hNaDC3-vcINDY alignment. The alignment between the sequences of hNaDC3 (Genpept AAF73251, top sequence) and vcINDY (Genpept AAF95939, bottom sequence) was visualized with Jalview (40) using the “ClustalX” color scheme. Helical segments in the vcINDY structure are indicated with red rectangles. The helical secondary structure elements (SS) were defined on the basis of the Protein Data Bank (PDB) secondary structure assignment (i.e. using the Dictionary of Secondary Structure of Proteins (DSSP) program (41)), the literature (13), and visual inspection of the structure. The helix nomenclature is on the basis of that defined by Mancusso et al. (13). Residues that are important for ligand and Na+ binding on the basis of the homology model are highlighted with gray stars and purple circles, respectively. Positions of amino acids that have been mutated in this study are marked with gray horizontal bars. The NaDC3 regions that were excluded from modeling (e.g. residues 302–340) and the vcINDY disordered region (i.e. residues 243–252) are marked with slashes (/).
FIGURE 2.
FIGURE 2.
Predicted NaDC3 structure. Shown are a side view (A) and cytoplasmic view (B) of the NaDC3 monomer model. Transmembrane helices are displayed in a color spectrum, indicating succession from the N (N-term, blue) to the C (C-term, red) terminus. Atoms of the substrate succinate are shown as spheres where oxygen atoms are displayed in red and carbon and hydrogen atoms in gray. Na+ is depicted as a purple sphere.
FIGURE 3.
FIGURE 3.
Predicted NaDC3 substrate and Na+ binding mode. The NaDC3 model is visualized as gray ribbons with key residues shown as sticks. Succinate is shown as green sticks. Oxygen, nitrogen, and hydrogen atoms are depicted in red, blue, and white, respectively, and the Na+ ion is visualized as a purple sphere. A, key hydrogen bonds between succinate and NaDC3 involving residues Ser-143, Asn-144, Thr-253, and Ala-254, with the Na+ ion, are shown as dotted gray lines. B, hydrogen bonds between the Na+ ion and NaDC3 involving residues Ser-139, Asn-144, and Thr-253 and the substrate succinate.
FIGURE 4.
FIGURE 4.
Binding sites of NaDC3 and vcINDY. The final model of NaDC3 (gray ribbons) is superimposed on the x-ray structure of vcINDY (pink). Key residues are displayed as sticks, where oxygen and nitrogen atoms are colored in red and blue, respectively. The sodium ion Na1 is visualized as a purple sphere. The citrate coordinates from the vcINDY structure are depicted by yellow sticks, and succinate coordinates from the NaDC3 model are shown as green sticks. Hydrogen bonds between citrate and vcINDY are shown as dotted yellow lines and between succinate and the NaDC3 model as dotted gray lines.
FIGURE 5.
FIGURE 5.
Predicted binding modes of representative NaDC3 substrates. Shown are the predicted binding modes of known substrates derived from homology modeling (A) and molecular docking (B–F). The NaDC3 binding site is visualized as surface representation (gray), and known substrates are displayed as sticks. Oxygen, nitrogen, and hydrogen atoms are depicted in red, blue, and white, respectively. Hydrogen bonds between the substrates and NaDC3 (including Na+) are displayed as dotted yellow lines. The coordinates of the substrate citrate from the initial NaDC3 homology model are shown as cyan sticks (A) and thin lines (B–F). The predicted docking poses of selected known substrates, including succinate (B), α-ketoglutarate (C), citrate (D), glutarate (E), and malate (F), are displayed as green sticks.
FIGURE 6.
FIGURE 6.
Succinate transport activity in NaDC3 mutants. Transport of [14C]succinate (10 μm) was measured in Na+-containing buffer for 30 min. The transport activity for each mutant is expressed as a percentage of the activity of WT NaDC3 from the same transfection experiment. Error bars represent mean ± S.E. (n = 4–10 separate experiments, n = 20 for the WT). Mutants with no activity were tested twice at room temperature and once at 37 °C. *, p < 0.05 compared with WT NaDC3. The mutants with low activity were compared with the background. #, p < 0.05.
FIGURE 7.
FIGURE 7.
NaDC3 binding site mutants have altered substrate specificity. The WT and mutant NaDC3 transporters were expressed in COS-7 cells. [14C]succinate (10 μm) uptake was measured in the presence and absence of 1 mm test substrate. The uptake rates for the wild-type and mutant transporters are shown as a percentage of the [14C]succinate transport activity measured in the absence of non-radioactive test substrate. Error bars represent mean ± S.E. (n = 3 separate experiments; WT, n = 9). MeS, methylsuccinate; AKG, α-ketoglutarate. *, p < 0.05 compared with WT NaDC3.
FIGURE 8.
FIGURE 8.
A dual-label competitive transport assay shows alterations in succinate and glutarate handling in NaDC3 binding site mutants. A, competitive uptake of [3H]succinate (10 μm) and [14C]glutarate (20 μm) by WT and mutant NaDC3 expressed in COS-7 cells. The transport was assayed for 20 min. Error bars represent mean ± S.E. (n = 3 separate transfection experiments except for S143A and S483A, where n = 1). B, TSRs (succinate:glutarate) of NaDC3 mutants calculated from competitive uptake data shown in A. Error bars represent mean ± S.E. (n = 3 experiments (S143A and S483A, n = 1)). *, p < 0.05, significantly different from the WT. Because of low succinate transport activity in the T527N mutant, particularly in the presence of the competitive inhibitor glutarate, the assays were done at 37 °C rather than room temperature. WT-37 and T527N-37 refer to the wild-type and T527N mutants assayed at 37 °C.
FIGURE 9.
FIGURE 9.
Altered Na+ handling by NaDC3 binding site mutants. A, sodium affinity screen of [14C]succinate transport (10 μm) in buffer containing 25 mm Na+, expressed as a percentage of the uptake rate measured in 140 mm Na+. Data represent mean ± S.E. (n = 3 experiments). *, p < 0.05, significantly different from WT NaDC3. B, Na+-activation kinetics of succinate transport in WT and mutant NaDC3. Transport of 10 μm [14C]succinate was measured in Na+ concentrations up to 140 mm (NaCl was replaced by choline chloride). Time points were 10 min. Each data point shows the mean of duplicate measurements from a single experiment, and the error bars indicate the range. The KNa values were as follows: WT, 26 mm; S483A, 17 mm; T253S, 95 mm. The data for T485V could not be fitted accurately because the curve did not show saturation.
FIGURE 10.
FIGURE 10.
Estimated location of the second Na+ ion, Na2. The NaDC3 model is visualized as gray ribbons with key residues shown as sticks. The substrate malate, which is predicted to be in close proximity to the C-terminal SNT motif, is displayed as green sticks. Oxygen, nitrogen, and hydrogen atoms are depicted in red, blue, and white, respectively. The Na+ ion Na1 is shown as a purple sphere, and the estimated location of the second Na+ binding site, Na2, is shown as a filled circle with a dotted line (purple).
FIGURE 11.
FIGURE 11.
Lithium interactions in NaDC3 mutants. The transport of 10 μm [14C]succinate was measured in transport buffer containing 140 mm LiCl, 10 mm LiCl + 130 mm NaCl, or 10 mm LiCl + 130 mm choline chloride. All data were corrected for background transport in vector-transfected cells. The transport activity in the presence of lithium was expressed as a percentage of the transport activity in the presence of 140 mm NaCl (control). Note the break in scale for T527N, which was stimulated by the combination of 10 mm LiCl + 130 mm NaCl. Error bars represent mean ± S.E. (n = 3–5 separate experiments). *, p < 0.05 compared with WT NaDC3.
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References

    1. Gullans S. R., Kone B. C., Avison M. J., Giebisch G. (1988) Succinate alters respiration, membrane potential, and intracellular K+ in proximal tubule. Am. J. Physiol. 255, F1170-F1177 - PubMed
    1. Ruderman N. B., Saha A. K., Vavvas D., Witters L. A. (1999) Malonyl-CoA, fuel sensing, and insulin resistance. Am. J. Physiol. 276, E1–E18 - PubMed
    1. Stoppa G. R., Cesquini M., Roman E. A., Prada P. O., Torsoni A. S., Romanatto T., Saad M. J., Velloso L. A., Torsoni M. A. (2008) Intracerebroventricular injection of citrate inhibits hypothalamic AMPK and modulates feeding behavior and peripheral insulin signaling. J. Endocrinol. 198, 157–168 - PubMed
    1. Pajor A. M. (2014) Sodium-coupled dicarboxylate and citrate transporters from the SLC13 family. Pflugers Arch. 466, 119–130 - PubMed
    1. Rogina B., Reenan R. A., Nilsen S. P., Helfand S. L. (2000) Extended life-span conferred by cotransporter gene mutations in Drosophila. Science 290, 2137–2140 - PubMed

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