Structure, function, and genomic organization of human  Na<sup>+</sup>-dependent high-affinity dicarboxylate transporter

Wang, Haiping; Fei, You Jun; Kekuda, Ramesh; Yang‐Feng, Teresa L.; Devoe, Lawrence D.; Leibach, Frederick H.; Prasad, Puttur D.; Ganapathy, Vadivel

doi:10.1152/ajpcell.2000.278.5.c1019

Cited by 121 publications

(130 citation statements)

References 29 publications

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“…This cell line has low background dicarboxylate transport activity (20), and it is useful for plate transport assays because the cells are strongly attached to the plastic culture dishes.…”

Section: Transport Activity and Expression Of Cysteine-substitutedmentioning

confidence: 99%

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Pajor

Randolph

2005

Journal of Biological Chemistry

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The Na ؉ /dicarboxylate co-transporter, NaDC-1, from the kidney and small intestine, transports three sodium ions together with one divalent anion substrate, such as succinate 2؊ . The Na ϩ /dicarboxylate co-transporter, NaDC-1, is found on the apical membrane of the epithelial cells of the renal proximal tubule and the small intestine (1). NaDC-1 carries a broad range of divalent anion substrates including dicarboxylates, such as succinate and ␣-ketoglutarate, and protonated tricarboxylates, such as citrate 2Ϫ . NaDC-1 is involved in regulating concentrations of citric acid cycle intermediates in kidney cells and in the urine. Consequently, NaDC-1 activity affects the development of kidney stones (2) and regulation of blood pressure (3). NaDC-1 has also been implicated in life span determination; the Drosophila homolog, Indy, is a longevity gene also expressed in the gastrointestinal tract (4). NaDC-1 belongs to the SCL13 gene family that includes sodium-coupled transporters for sulfate and di-and tricarboxylates in vertebrates (5) and the sodium-independent malate transporter from plant vacuoles (6).The identification of the substrate and cation binding sites in NaDC-1 and the transmembrane helices (TMs) 1 involved in forming the permeation pathway remains an area of active investigation. Our previous studies have shown that the carboxyl-terminal half of NaDC-1, TM7-TM11, contains residues that determine substrate specificity (7). Chimera studies suggest that TM10 and the adjacent loops contain amino acids that determine differences in K m for citrate and sodium (8). Recently, we have found that TM9 is involved in the conformational changes seen during the transport cycle and likely forms part of the permeation pathway through the transporter (9). The extracellular end of TM9 in NaDC-1 contains four conformationally sensitive residues, Ser-479, Ala-480, Ala-481, and Thr-482 (9). Cysteine substitutions of these amino acids resulted in proteins that were sensitive to inhibition by the membrane-impermeant methanethiosulfonate reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET). In the present study, the cysteine scan was continued through the extracellular loop between TM9 and TM10 (extracellular loop 5) and in TM10, from Thr-483 to Val-528, in order to identify conformationally sensitive residues. As described previously, the membrane-impermeant reagent MTSET was used to react with accessible thiolate anions (10), and the functional consequences of the labeling were measured.The major finding of this study is that MTSET-accessible residues are found in the loop between TM9 and TM10 and not in the transmembrane region. Eleven of the cysteine-substituted mutants were sensitive to MTSET, with either increased or decreased activity after chemical labeling. The most sensitive cysteine mutants were T483C, T484C, L485C, L487C, I489C, and M493C, all of which exhibited conformational differences in their sensitivity to MTSET labeling as well as substrate protection. All six cysteine-substituted mutants wer...

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“…This cell line has low background dicarboxylate transport activity (20), and it is useful for plate transport assays because the cells are strongly attached to the plastic culture dishes.…”

Section: Transport Activity and Expression Of Cysteine-substitutedmentioning

confidence: 99%

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Pajor

Randolph

2005

Journal of Biological Chemistry

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“…This experiment utilized the Fetchex program within the pCLAMP 6.0 software package. We have used this method previously in our laboratory to determine the substrate/charge transfer ratio for the H ϩ -coupled peptide transporter PEPT2 (32) and Na ϩ -coupled dicarboxylate transporter NaDC3 (33). region.…”

Section: Materials-[mentioning

confidence: 99%

Primary Structure, Genomic Organization, and Functional and Electrogenic Characteristics of Human System N 1, a Na+- and H+-coupled Glutamine Transporter

Fei¹,

Sugawara²,

Nakanishi³

et al. 2000

Journal of Biological Chemistry

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We have cloned the human Na ؉ -and H ؉ -coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li ؉ activation kinetics showed a Li ؉ :glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine-or asparagine-induced currents, was saturable, Na ؉ -dependent, Li ؉ -tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na ؉ ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na ؉ and one amino acid substrate per transport cycle coupled to the efflux of one H ؉ , rendering the transport process electrogenic.

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“…Within the cloned NaDC-1 orthologs, DMS analogs left either radiolabeled succinate uptake unaffected (41,45,46) or induced currents comparable in magnitude to succinate under two-electrode voltage clamp conditions (44,47,48). In contrast, NaDC-3 orthologs were generally sensitive to DMS analogs (12)(13)(14)(15)(16). Thus, 2,3-DMS can be used as a discriminator between luminal and basolateral Na ϩ -dependent dicarboxylate transporters.…”

Section: Discussionmentioning

confidence: 98%

“…The high-affinity Na ϩ -dependent dicarboxylate transporter with a K 0.5 Ͻ 0.2 mM for succinate is located at the basolateral membrane of renal proximal tubular cells, sinusoidal membrane of hepatocytes, brain synaptosomes, and the maternal-facing brush border membrane of the placental syncytiotrophoblast. This transporter, designated NaDC-3, has also been cloned from different species and functionally characterized (12)(13)(14)(15)(16). We have recently reported the cloning of fNaDC-3 from winter flounder kidney (16).…”

Section: Discussionmentioning

confidence: 99%

“…By immunohistochemical studies, rabbit and rat NaDC-1 were located to the luminal membrane of proximal tubule cells. NaDC-3 (or SDCT2) cloned from human (12), rat (13,14), mouse (15), and flounder (16) showed a comparatively high affinity for succinate in radiotracer uptake experiments (K m : 20 Ϯ 1 M for human, 15 Ϯ 1.3 M for rat, and 30.4 Ϯ 13.5 M for flounder NaDC-3). In the flounder kidney, NaDC-3 is located at the basolateral membrane of proximal tubule cells (Steffgen et al, unpublished results).…”

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confidence: 99%

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The Renal Na+-Dependent Dicarboxylate Transporter, NaDC-3, Translocates Dimethyl- and Disulfhydryl-Compounds and Contributes to Renal Heavy Metal Detoxification

Burckhardt

Drinkuth

Menzel

et al. 2002

Journal of the American Society of Nephrology

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Abstract. The active transport of Krebs cycle intermediates, such as succinate, ␣-ketoglutarate, and citrate, is mediated by sodium-coupled transporters found in the luminal (NaDC-1) and basolateral plasma membranes (NaDC-3) of proximal tubule cells. This study used the two-electrode voltage clamp technique to examine steady-state currents associated with the influx of three sodium ions and one divalent dicarboxylate into oocytes expressing the sodium-dicarboxylate transporter from winter flounder kidney, fNaDC-3. The substrate concentration, where half-maximal current was observed (K 0.5 ), was 30 M for succinate. Besides 2,2-dimethylsuccinate, fNaDC-3 also accepted 2,3-dimethylsuccinate and the oral lead-chelating agent, meso-2,3-dimercaptosuccinate (DMSA or Succimer).Whereas the K 0.5 for succinate and 2,2-dimethylsuccinate was independent of membrane voltage within Ϫ90 and Ϫ10 mV, K 0.5 for 2,3-dimethylsuccinate and 2,3-dimercaptosuccinate increased with decreasing voltage, indicating a critical role of the position of the methyl-or sulfhydryl-group in voltage-sensitive affinity. In addition to meso-2,3-dimercaptosuccinate, fNaDC-3 translocated dimercaptopropane-1-sulfonate (DMPS or Dimaval), an oral chelator for the treatment of mercury intoxication. The chelates formed by HgCl 2 and DMSA or DMPS and by Pb(NO 3 ) 2 and DMSA, however, were not translocated by fNaDC-3. The data suggest that NaDC-3 is an essential component in the delivery of uncomplexed antidotes for renal heavy metal detoxification.Di-and tricarboxylates, such as succinate, ␣-ketoglutarate, and citrate, are actively taken up by renal proximal tubule cells both from the peritubular capillaries and the glomerular filtrate. These compounds serve as metabolic fuels and as substrates for gluconeogenesis (1,2,3). In addition, intracellular ␣-ketoglutarate drives the uptake of organic anions by an organic anion/ dicarboxylate exchanger, OAT1, located in the basolateral membrane (4,5). This exchange process constitutes the first step in the proximal tubular excretion of a large number of organic anions, including widely used drugs such as ␤-lactam antibiotics, antiviral drugs, diuretics, and nonsteroidal antiinflammatory drugs (6 -9).The transport of di-and tricarboxylates across the luminal and the basolateral cell membrane of proximal tubule cells is mediated by two distinct Na ϩ -dependent dicarboxylate transporters, NaDC-1 and NaDC-3 [reviewed in references 10 and 11]. Both translocate three sodium ions and a di-or tricarboxylate in its divalent form. NaDC-1 has been cloned from rabbit, rat, mouse, and human. Except for rat NaDC-1 (or SDCT1), these transporters exhibit a comparatively low affinity for succinate (K m above 0.2 mM). By immunohistochemical studies, rabbit and rat NaDC-1 were located to the luminal membrane of proximal tubule cells. NaDC-3 (or SDCT2) cloned from human (12), rat (13,14), mouse (15), and flounder (16) showed a comparatively high affinity for succinate in radiotracer uptake experiments (K m : 20 Ϯ 1 M for human, 15 Ϯ 1...

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Structure, function, and genomic organization of human Na⁺-dependent high-affinity dicarboxylate transporter

Cited by 121 publications

References 29 publications

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Primary Structure, Genomic Organization, and Functional and Electrogenic Characteristics of Human System N 1, a Na+- and H+-coupled Glutamine Transporter

The Renal Na+-Dependent Dicarboxylate Transporter, NaDC-3, Translocates Dimethyl- and Disulfhydryl-Compounds and Contributes to Renal Heavy Metal Detoxification

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Structure, function, and genomic organization of human Na+-dependent high-affinity dicarboxylate transporter

Cited by 121 publications

References 29 publications

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Primary Structure, Genomic Organization, and Functional and Electrogenic Characteristics of Human System N 1, a Na+- and H+-coupled Glutamine Transporter

The Renal Na+-Dependent Dicarboxylate Transporter, NaDC-3, Translocates Dimethyl- and Disulfhydryl-Compounds and Contributes to Renal Heavy Metal Detoxification

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Structure, function, and genomic organization of human Na⁺-dependent high-affinity dicarboxylate transporter