NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions.

Citrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons.

Mapping Functionally Important Residues in the Na/Dicarboxylate Cotransporter, NaDC1.

Transporters from the SLC13 family couple the transport of two to four Na ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites.

Molecular Basis for Inhibition of the Na+/Citrate Transporter NaCT (SLC13A5) by Dicarboxylate Inhibitors.

The Na/citrate transporter, NaCT (SLC13A5), is a therapeutic target for metabolic diseases. Citrate is an important signaling molecule that regulates the activity of lipid- and glucose-metabolizing enzymes in cells. Previous studies identified two compounds, PF-06649298 (compound 2: ) and PF-06678419 (compound 4: ), that inhibit human NaCT with high affinity, and one of the compounds demonstrated specificity relative to other SLC13 family members.

Mutations in the Na(+)/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay.

Mutations in the SLC13A5 gene that codes for the Na(+)/citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In the present study we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and treatment strategies.

Structure-Based Identification of Inhibitors for the SLC13 Family of Na(+)/Dicarboxylate Cotransporters.

In mammals, citric acid cycle intermediates play a key role in regulating various metabolic processes, such as fatty acid synthesis and glycolysis. Members of the sodium-dependent SLC13 transporter family mediate the transport of di- and tricarboxylates into cells. SLC13 family members have been implicated in lifespan extension and resistance to high-fat diets; thus, they are emerging drug targets for aging and metabolic disorders.

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

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.

Localization of the calcium-regulated citrate transport process in proximal tubule cells.

Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport.

Sodium-coupled dicarboxylate and citrate transporters from the SLC13 family.

The SLC13 family in humans and other mammals consists of sodium-coupled transporters for anionic substrates: three transporters for dicarboxylates/citrate and two transporters for sulfate. This review will focus on the di- and tricarboxylate transporters: NaDC1 (SLC13A2), NaDC3 (SLC13A3), and NaCT (SLC13A5). The substrates of these transporters are metabolic intermediates of the citric acid cycle, including citrate, succinate, and α-ketoglutarate, which can exert signaling effects through specific receptors or can affect metabolic enzymes directly.

Functional characterization of SdcF from Bacillus licheniformis, a homolog of the SLC13 Na⁺/dicarboxylate transporters.

The SdcF transporter from Bacillus licheniformis (gene BL02343) is a member of the divalent anion sodium symporter (DASS)/SLC13 family that includes Na⁺/dicarboxylate transporters from bacteria to humans. SdcF was functionally expressed in Escherichia coli (BL21) and assayed in right side out membrane vesicles. ScdF catalyzed the sodium-coupled transport of succinate and α-ketoglutarate.

Nonsteroidal anti-inflammatory drugs and other anthranilic acids inhibit the Na(+)/dicarboxylate symporter from Staphylococcus aureus.

The Na(+)/dicarboxylate symporter from Staphylococcus aureus, named SdcS, is a member of the divalent anion sodium symporter (DASS) family that also includes the mammalian SLC13 Na(+)/dicarboxylate cotransporters, NaDC1 and NaCT. The mammalian members of the family are sensitive to inhibition by anthranilic acid derivatives such as N-(p-amylcinnamoyl)anthranilic acid (ACA), which act as slow inhibitors. This study shows that SdcS is inhibited by ACA as well as the fenamate nonsteroidal anti-inflammatory drugs, flufenamate and niflumate.

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function.

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport.

Role of isoleucine-554 in lithium binding by the Na+/dicarboxylate cotransporter NaDC1.

Sodium-coupled transport of citric acid cycle intermediates, such as succinate and citrate, is mediated by the NaDC1 transporter located on the apical membrane of kidney proximal tubule and small intestine cells. Our previous study showed that transmembrane helix (TM) 11 of NaDC1 is important for sodium and lithium binding, as well as for determining citrate affinity [Kahn and Pajor (1999) Biochemistry 38, 6151]. In the present study, 21 amino acids in TM11 and the extracellular loop of NaDC1 were mutated one at a time to cysteine.

Single nucleotide polymorphisms in the human Na+-dicarboxylate cotransporter affect transport activity and protein expression.

The sodium-coupled transport of citric acid cycle intermediates in the intestine and kidney is mediated by the Na(+)-dicarboxylate cotransporter, NaDC1. In the kidney, NaDC1 plays an important role in regulating succinate and citrate concentrations in the urine, which may have physiological consequences including the development of kidney stones. In the present study, the impact of nonsynonymous single nucleotide polymorphisms (SNPs) on NaDC1 expression and function was characterized using the COS-7 cell heterologous expression system.

Functional characterization of a Na(+)-coupled dicarboxylate transporter from Bacillus licheniformis.

The Na(+)-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na(+) symporter (DASS) family that includes the bacterial Na(+)/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na(+)/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na(+)-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K(m) of approximately 6 microM. SdcL may also transport aspartate, alpha-ketoglutarate and oxaloacetate with low affinity.

Identification of conformationally sensitive amino acids in the Na(+)/dicarboxylate symporter (SdcS).

The Na(+)/dicarboxylate symporter (SdcS) from Staphylococcus aureus is a homologue of the mammalian Na(+)/dicarboxylate cotransporters (NaDC1) from the solute carrier 13 (SLC13) family. This study examined succinate transport by SdcS heterologously expressed in Escherichia coli, using right-side-out (RSO) and inside-out (ISO) membrane vesicles. The K(m) values for succinate in RSO and ISO vesicles were similar, approximately 30 microM.

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2.

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time.

Threonine-509 is a determinant of apparent affinity for both substrate and cations in the human Na+/dicarboxylate cotransporter.

The Na+/dicarboxylate cotransporter (NaDC1) is involved in the absorption of citric acid cycle intermediates from the lumen of the renal proximal tubule and small intestine. The NaDC1 orthologues from human (h) and rabbit (rb) exhibit differences in citrate and cation transport properties. The citrate Km and sodium KNa values are much larger in human than rabbit NaDC1.

Inhibitor binding in the human renal low- and high-affinity Na+/glucose cotransporters.

The kidney contains two Na(+)/glucose cotransporters, called SGLT2 and SGLT1, arranged in series along the length of the proximal tubule. The low-affinity transporter, SGLT2, is responsible for the reabsorption of most of the glucose in the kidney. There is recent interest in SGLT2 as a target for the treatment of type II diabetes using selective inhibitors based on the structure of the phenylglucoside, phlorizin (phloretin-2'-beta-glucoside).

Inhibition of the Na+/dicarboxylate cotransporter by anthranilic acid derivatives.

The Na(+)/dicarboxylate cotransporter NaDC1 absorbs citric acid cycle intermediates from the lumen of the small intestine and kidney proximal tubule. No effective inhibitor has been identified yet, although previous studies showed that the nonsteroidal anti-inflammatory drug, flufenamate, inhibits the human (h) NaDC1 with an IC(50) value of 2 mM. In the present study, we have tested compounds related in structure to flufenamate, all anthranilic acid derivatives, as potential inhibitors of hNaDC1.

Sodium-dependent extracellular accessibility of Lys-84 in the sodium/dicarboxylate cotransporter.

The Na(+)/dicarboxylate cotransporter transports Na(+) with citric acid cycle intermediates such as succinate and citrate. The present study focuses on transmembrane helix 3, which is highly conserved among the members of the SLC13 family. Fifteen amino acids in the extracellular half of transmembrane helix (amino acids 98-112) as well as Lys-84, previously shown to affect substrate affinity, were mutated individually to cysteine and expressed in the human retinal pigment epithelial cell line.

Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus.

In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J.

Ala-504 is a determinant of substrate binding affinity in the mouse Na(+)/dicarboxylate cotransporter.

The Na(+)/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp.

Role of conserved prolines in the structure and function of the Na+/dicarboxylate cotransporter 1, NaDC1.

The Na+/dicarboxylate cotransporter 1 (NaDC1) is a low-affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TMs) 7 and 10. These transmembrane domains are of particular importance because they may be involved in determining the substrate or cation-binding affinity in NaDC1.

Transmembrane helices 3 and 4 are involved in substrate recognition by the Na+/dicarboxylate cotransporter, NaDC1.

The Na(+)/dicarboxylate cotransporters (NaDC1) from mouse (m) and rabbit (rb) differ in their ability to handle glutarate. Substrate-dependent inward currents, measured using two-electrode voltage clamp, were similar for glutarate and succinate in Xenopus oocytes expressing mNaDC1. In contrast, currents evoked by glutarate in rbNaDC1 were only about 5% of the succinate-dependent currents.