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Joseph A. Mindell - One of the best experts on this subject based on the ideXlab platform.
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solvent accessibility changes in a na dependent c4 Dicarboxylate Transporter suggest differential substrate effects in a multistep mechanism
Journal of Biological Chemistry, 2020Co-Authors: Connor D. D. Sampson, Joseph A. Mindell, Matthew J. Stewart, Christopher MulliganAbstract:The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS Transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the Transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.
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Solvent accessibility changes in a Na+-dependent C4-Dicarboxylate Transporter suggest differential substrate effects in a multistep mechanism.
The Journal of biological chemistry, 2020Co-Authors: Connor D. D. Sampson, Joseph A. Mindell, Matthew J. Stewart, Christopher MulliganAbstract:The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS Transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the Transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.
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The bacterial Dicarboxylate Transporter VcINDY uses a two-domain elevator-type mechanism
Nature Structural & Molecular Biology, 2016Co-Authors: Christopher Mulligan, Gabriel A. Fitzgerald, Cristina Fenollar-ferrer, Ariela Vergara-jaque, Desirée Kaufmann, Yan Li, Lucy R Forrest, Joseph A. MindellAbstract:‘Repeat swap’ modeling of the outward-facing conformation and biochemical analyses show that the bacterial VcINDY symporter uses an elevator-type mechanism for substrate transport across the cell membrane. Secondary Transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known Transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-Transporter homolog Glt_Ph translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na^+/succinate Transporter VcINDY, from Vibrio cholerae . Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.
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The bacterial Dicarboxylate Transporter VcINDY uses a two-domain elevator-type mechanism.
Nature structural & molecular biology, 2016Co-Authors: Christopher Mulligan, Gabriel A. Fitzgerald, Cristina Fenollar-ferrer, Ariela Vergara-jaque, Desirée Kaufmann, Lucy R Forrest, Joseph A. MindellAbstract:Secondary Transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known Transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-Transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na(+)/succinate Transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 A and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.
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Assessing Protomer Independence of the Dimeric C4-Dicarboxylate Transporter, Vcindy
Biophysical Journal, 2015Co-Authors: Christopher Mulligan, Gabriel A. Fitzgerald, Joseph A. MindellAbstract:The divalent anion:sodium symporter (DASS) family includes characterized representatives from bacteria and humans (SLC13 family). DASS family members have been implicated in key physiological roles, including fatty acid synthesis and the transport of Krebs cycle intermediates and sulfate across the cytoplasmic membrane. Disruption of genes encoding DASS family members in mice and flies reveal roles in energy homeostasis, affecting lifespan determination, insulin resistance and obesity in these organisms.VcINDY, from the bacterium Vibrio cholerae is the only DASS family member for which there is high resolution structural information available and functional characterization reveals VcINDY to share key functional characteristics with mammalian DASS family members. VcINDY is a functional dimer exhibiting a large buried surface area at the dimer interface; a feature likely shared by all DASS family members. Characterization of other bacterial members of the family suggests cooperativity between the protomers. This apparent inter-protomer communication is likely transmitted via substrate-induced conformational changes at the dimer interface. If true, this has important mechanistic implications for the entire family.To investigate whether transport by VcINDY is a cooperative process we have introduced cysteine residues at the dimer interface that, under oxidizing conditions or in the presence of crosslinking agents, will staple regions of the interface together, limiting protein movement. Using this procedure we have demonstrated that pinning the interface together in several regions does not abrogate transport activity, indicating that no obligatory conformational changes occur across the dimer interface during transport. Although further confirmatory work is required, these data suggest that the VcINDY protomers function independently of one another during the transport cycle.
Christopher Mulligan - One of the best experts on this subject based on the ideXlab platform.
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solvent accessibility changes in a na dependent c4 Dicarboxylate Transporter suggest differential substrate effects in a multistep mechanism
Journal of Biological Chemistry, 2020Co-Authors: Connor D. D. Sampson, Joseph A. Mindell, Matthew J. Stewart, Christopher MulliganAbstract:The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS Transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the Transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.
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Solvent accessibility changes in a Na+-dependent C4-Dicarboxylate Transporter suggest differential substrate effects in a multistep mechanism.
The Journal of biological chemistry, 2020Co-Authors: Connor D. D. Sampson, Joseph A. Mindell, Matthew J. Stewart, Christopher MulliganAbstract:The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS Transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the Transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.
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The bacterial Dicarboxylate Transporter VcINDY uses a two-domain elevator-type mechanism
Nature Structural & Molecular Biology, 2016Co-Authors: Christopher Mulligan, Gabriel A. Fitzgerald, Cristina Fenollar-ferrer, Ariela Vergara-jaque, Desirée Kaufmann, Yan Li, Lucy R Forrest, Joseph A. MindellAbstract:‘Repeat swap’ modeling of the outward-facing conformation and biochemical analyses show that the bacterial VcINDY symporter uses an elevator-type mechanism for substrate transport across the cell membrane. Secondary Transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known Transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-Transporter homolog Glt_Ph translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na^+/succinate Transporter VcINDY, from Vibrio cholerae . Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.
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The bacterial Dicarboxylate Transporter VcINDY uses a two-domain elevator-type mechanism.
Nature structural & molecular biology, 2016Co-Authors: Christopher Mulligan, Gabriel A. Fitzgerald, Cristina Fenollar-ferrer, Ariela Vergara-jaque, Desirée Kaufmann, Lucy R Forrest, Joseph A. MindellAbstract:Secondary Transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known Transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-Transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na(+)/succinate Transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 A and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.
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Assessing Protomer Independence of the Dimeric C4-Dicarboxylate Transporter, Vcindy
Biophysical Journal, 2015Co-Authors: Christopher Mulligan, Gabriel A. Fitzgerald, Joseph A. MindellAbstract:The divalent anion:sodium symporter (DASS) family includes characterized representatives from bacteria and humans (SLC13 family). DASS family members have been implicated in key physiological roles, including fatty acid synthesis and the transport of Krebs cycle intermediates and sulfate across the cytoplasmic membrane. Disruption of genes encoding DASS family members in mice and flies reveal roles in energy homeostasis, affecting lifespan determination, insulin resistance and obesity in these organisms.VcINDY, from the bacterium Vibrio cholerae is the only DASS family member for which there is high resolution structural information available and functional characterization reveals VcINDY to share key functional characteristics with mammalian DASS family members. VcINDY is a functional dimer exhibiting a large buried surface area at the dimer interface; a feature likely shared by all DASS family members. Characterization of other bacterial members of the family suggests cooperativity between the protomers. This apparent inter-protomer communication is likely transmitted via substrate-induced conformational changes at the dimer interface. If true, this has important mechanistic implications for the entire family.To investigate whether transport by VcINDY is a cooperative process we have introduced cysteine residues at the dimer interface that, under oxidizing conditions or in the presence of crosslinking agents, will staple regions of the interface together, limiting protein movement. Using this procedure we have demonstrated that pinning the interface together in several regions does not abrogate transport activity, indicating that no obligatory conformational changes occur across the dimer interface during transport. Although further confirmatory work is required, these data suggest that the VcINDY protomers function independently of one another during the transport cycle.
Gerhard Burckhardt - One of the best experts on this subject based on the ideXlab platform.
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Interaction of Excitatory Amino Acid Transporters 1 - 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate.
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2017Co-Authors: Birgitta C. Burckhardt, Gerhard BurckhardtAbstract:BACKGROUND/AIMS Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid Transporters (EAATs). METHODS Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent Dicarboxylate Transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related Dicarboxylates were assayed. RESULTS L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all Dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. CONCLUSION EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and Dicarboxylates, EAAT3 and NaDC3 can complement each other.
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Interaction of Excitatory Amino Acid Transporters 1 – 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate
Karger Publishers, 2017Co-Authors: Birgitta c. Burckhardt, Gerhard BurckhardtAbstract:Background/Aims: Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid Transporters (EAATs). Methods: Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent Dicarboxylate Transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related Dicarboxylates were assayed. Results: L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all Dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. Conclusion: EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and Dicarboxylates, EAAT3 and NaDC3 can complement each other
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Glutathione Is a Low-Affinity Substrate of the Human Sodium-Dependent Dicarboxylate Transporter
Nephron. Physiology, 2013Co-Authors: Lena Schorbach, Gerhard Burckhardt, Wolfgang Krick, Birgitta C. BurckhardtAbstract:Background/Aims: During a single pass through the kidneys, more than 80% of glutathione (GSH) is excreted, indicating not only glomerular filtration, but also tubular secretion. The first step in tubular secretion is the uptake of a substance across the basolateral membrane of proximal tubule cells by sodium-dependent and -independent Transporters. Due to the Dicarboxylate-like structure, we postulated that GSH uptake across the basolateral membrane is mediated by the sodium-dependent Dicarboxylate Transporter 3 (NaDC3). Methods: Tracer uptake and electrophysiologic measurements using a two-electrode voltage clamp device were performed in Xenopus laevis oocytes expressing the human (h)NaDC3. Results: Uptake of succinate, the reference substrate of hNaDC3, was inhibited by GSH in a dose-dependent manner with an IC50 of 1.88 mM. GSH evoked potential-dependent inward currents, which were abolished under sodium-free conditions. At -60 mV, GSH currents showed saturation kinetics with a KM of 1.65 mM. Conclusion: hNaDC3 present at the basolateral membrane of proximal tubule cells mediates sodium-dependent GSH uptake. The kinetic data show that NaDC3 is a low-affinity GSH Transporter.
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Interactions of benzylpenicillin and non-steroidal anti-inflammatory drugs with the sodium-dependent Dicarboxylate Transporter NaDC-3.
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2004Co-Authors: Birgitta C. Burckhardt, Gerhard Burckhardt, Julia Lorenz, Jurgen SteffgenAbstract:Sodium-dependent Dicarboxylate Transporters located in the basolateral membrane (NaDC-3) of renal proximal tubule cells maintain the driving force for exchange of organic anions and drugs against alpha-ketoglutarate via organic anion Transporters OAT1 and OAT3. So far, information on direct interaction of drugs with the cloned NaDC-3 was missing. Here we tested the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) and benzylpenicillin with NaDC-3 cloned from winter flounder (fNaDC-3) and human (hNaDC-3) kidneys. Flufenamate and benzylpenicillin inhibited [14C]succinate uptake in oocytes expressing fNaDC-3. Flufenamate elicited Na(+)-dependent currents in oocytes expressing fNaDC-3 with a reversal potential around -60 mV. Raising extracellular K+ concentration depolarized fNaDC3-expressing oocytes more in the presence of flufenamate than in its absence, an effect not seen with water-injected control oocytes. These findings suggest that flufenamate via interaction with fNaDC-3 increased the K+ conductance. Acetylsalicylate, indomethacin, and salicylate showed small potential-dependent inward currents in fNaDC-3 but not in hNaDC-3 expressing oocytes. Benzylpenicillin induced voltage-dependent inward currents which were Na(+)-dependent in oocytes expressing fNaDC-3. The currents were, however, much smaller than those induced by succinate, reflecting probably a low fit of the monovalent benzylpenicillin to the Dicarboxylate binding site. The data show hitherto unknown effects of monovalent anionic drugs on a Transporter for divalent di- and tricarboxylates.
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the renal na dependent Dicarboxylate Transporter nadc 3 translocates dimethyl and disulfhydryl compounds and contributes to renal heavy metal detoxification
Journal of The American Society of Nephrology, 2002Co-Authors: Birgitta C. Burckhardt, Britta Drinkuth, Christine Menzel, Angela Konig, Jurgen Steffgen, Stephen H Wright, Gerhard BurckhardtAbstract:ABSTRACT. The active transport of Krebs cycle intermediates, such as succinate, α-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 μM 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 −90 and −10 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.
Ana M Pajor - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of a na coupled Dicarboxylate Transporter from bacillus licheniformis
Biochimica et Biophysica Acta, 2009Co-Authors: Melodie A. Strickler, Jason A. Hall, Olga Gaiko, Ana M PajorAbstract:Abstract 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 Km of ∼ 6 μM. SdcL may also transport aspartate, α-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na+ and Dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K0.5 for Na+ of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.
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Functional characterization of a Na(+)-coupled Dicarboxylate Transporter from Bacillus licheniformis.
Biochimica et biophysica acta, 2009Co-Authors: Melodie A. Strickler, Jason A. Hall, Olga Gaiko, Ana M PajorAbstract:Abstract 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 Km of ∼ 6 μM. SdcL may also transport aspartate, α-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na+ and Dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K0.5 for Na+ of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.
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Functional Characterization of a Na+-Coupled Dicarboxylate Carrier Protein from Staphylococcus aureus
Journal of bacteriology, 2005Co-Authors: Jason A. Hall, Ana M PajorAbstract:We have cloned and functionally characterized a Na+-coupled Dicarboxylate Transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na+ symporter (DASS) family and shares significant sequence homology with the mammalian Na+/Dicarboxylate coTransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the Dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na+-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na+ (K0.5 of 2.7 mM) binding before Dicarboxylate (Km of 4.5 μM). Because this Transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.
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Primary Structure and Functional Characteristics of a Mammalian Sodium-coupled High Affinity Dicarboxylate Transporter
The Journal of biological chemistry, 1999Co-Authors: Ramesh Kekuda, Ana M Pajor, Wei Huang, Frederick H. Leibach, Haiping Wang, Lawrence D. Devoe, Puttur D. Prasad, Vadivel GanapathyAbstract:Abstract We have cloned a Na+-dependent, high affinity Dicarboxylate Transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na+-dependent, low affinity Dicarboxylate Transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na+-dependent transport of succinate with aK t of 2 μm. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na+concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of Dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li+ inhibits NaDC3 in the presence of Na+. Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8.0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6.5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na+-dependent, high affinity Dicarboxylate Transporter.
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sodium coupled Transporters for krebs cycle intermediates
Annual Review of Physiology, 1999Co-Authors: Ana M PajorAbstract:▪ Abstract Krebs cycle intermediates such as succinate, citrate, and α-ketoglutarate are transferred across plasma membranes of cells by secondary active Transporters that couple the downhill movement of sodium to the concentrative uptake of substrate. Several Transporters have been identified in isolated membrane vesicles and cells based on their functional properties, suggesting the existence of at least three or more Na+/Dicarboxylate coTransporter proteins in a given species. Recently, several cDNAs, called NaDC-1, coding for the low-affinity Na+/Dicarboxylate coTransporters have been isolated from rabbit, human, and rat kidney. The Na+/Dicarboxylate coTransporters are part of a distinct gene family that includes the renal and intestinal Na+/sulfate coTransporters. Other members of this family include a Na+- and Li+-dependent Dicarboxylate Transporter from Xenopus intestine and a putative Na+/Dicarboxylate coTransporter from rat intestine. The current model of secondary structure in NaDC-1 contains 11...
Birgitta C. Burckhardt - One of the best experts on this subject based on the ideXlab platform.
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Interaction of Excitatory Amino Acid Transporters 1 - 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate.
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2017Co-Authors: Birgitta C. Burckhardt, Gerhard BurckhardtAbstract:BACKGROUND/AIMS Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid Transporters (EAATs). METHODS Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent Dicarboxylate Transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related Dicarboxylates were assayed. RESULTS L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all Dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. CONCLUSION EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and Dicarboxylates, EAAT3 and NaDC3 can complement each other.
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Glutathione Is a Low-Affinity Substrate of the Human Sodium-Dependent Dicarboxylate Transporter
Nephron. Physiology, 2013Co-Authors: Lena Schorbach, Gerhard Burckhardt, Wolfgang Krick, Birgitta C. BurckhardtAbstract:Background/Aims: During a single pass through the kidneys, more than 80% of glutathione (GSH) is excreted, indicating not only glomerular filtration, but also tubular secretion. The first step in tubular secretion is the uptake of a substance across the basolateral membrane of proximal tubule cells by sodium-dependent and -independent Transporters. Due to the Dicarboxylate-like structure, we postulated that GSH uptake across the basolateral membrane is mediated by the sodium-dependent Dicarboxylate Transporter 3 (NaDC3). Methods: Tracer uptake and electrophysiologic measurements using a two-electrode voltage clamp device were performed in Xenopus laevis oocytes expressing the human (h)NaDC3. Results: Uptake of succinate, the reference substrate of hNaDC3, was inhibited by GSH in a dose-dependent manner with an IC50 of 1.88 mM. GSH evoked potential-dependent inward currents, which were abolished under sodium-free conditions. At -60 mV, GSH currents showed saturation kinetics with a KM of 1.65 mM. Conclusion: hNaDC3 present at the basolateral membrane of proximal tubule cells mediates sodium-dependent GSH uptake. The kinetic data show that NaDC3 is a low-affinity GSH Transporter.
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3-Hydroxyglutaric acid is transported via the sodium-dependent Dicarboxylate Transporter NaDC3
Journal of Molecular Medicine, 2007Co-Authors: Franziska Stellmer, Birgitta C. Burckhardt, Britta Keyser, Hermann Koepsell, Thomas Streichert, Markus Glatzel, Sabrina Jabs, Joachim Thiem, Wilhelm Herdering, David M. KoellerAbstract:Patients with glutaryl-CoA dehydrogenase (GCDH) deficiency accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3OH-GA) in their blood and urine. To identify the Transporter mediating the translocation of 3OH-GA through membranes, kidney tissue of Gcdh −/− mice have been investigated because of its central role in urinary excretion of this metabolite. Using microarray analyses of kidney-expressed genes in Gcdh −/− mice, several differentially expressed genes encoding Transporter proteins were identified. Real-time polymerase chain reaction analysis confirmed the upregulation of the sodium-dependent Dicarboxylate coTransporter 3 (NaDC3) and the organic cation Transporter 2 (OCT2). Expression analysis of NaDC3 in Xenopus laevis oocytes by the two-electrode-voltage-clamp technique demonstrated the sodium-dependent translocation of 3OH-GA with a K _M value of 0.95 mM. Furthermore, tracer flux measurements in Chinese hamster ovary cells overexpressing OCT2 showed that 3OH-GA inhibited significantly the uptake of methyl-4-phenylpyridinium, whereas 3OH-GA is not transported by OCT2. The data demonstrate for the first time the membrane translocation of 3OH-GA mediated by NaDC3 and the cis -inhibitory effect on OCT2-mediated transport of cations.
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Interactions of benzylpenicillin and non-steroidal anti-inflammatory drugs with the sodium-dependent Dicarboxylate Transporter NaDC-3.
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2004Co-Authors: Birgitta C. Burckhardt, Gerhard Burckhardt, Julia Lorenz, Jurgen SteffgenAbstract:Sodium-dependent Dicarboxylate Transporters located in the basolateral membrane (NaDC-3) of renal proximal tubule cells maintain the driving force for exchange of organic anions and drugs against alpha-ketoglutarate via organic anion Transporters OAT1 and OAT3. So far, information on direct interaction of drugs with the cloned NaDC-3 was missing. Here we tested the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) and benzylpenicillin with NaDC-3 cloned from winter flounder (fNaDC-3) and human (hNaDC-3) kidneys. Flufenamate and benzylpenicillin inhibited [14C]succinate uptake in oocytes expressing fNaDC-3. Flufenamate elicited Na(+)-dependent currents in oocytes expressing fNaDC-3 with a reversal potential around -60 mV. Raising extracellular K+ concentration depolarized fNaDC3-expressing oocytes more in the presence of flufenamate than in its absence, an effect not seen with water-injected control oocytes. These findings suggest that flufenamate via interaction with fNaDC-3 increased the K+ conductance. Acetylsalicylate, indomethacin, and salicylate showed small potential-dependent inward currents in fNaDC-3 but not in hNaDC-3 expressing oocytes. Benzylpenicillin induced voltage-dependent inward currents which were Na(+)-dependent in oocytes expressing fNaDC-3. The currents were, however, much smaller than those induced by succinate, reflecting probably a low fit of the monovalent benzylpenicillin to the Dicarboxylate binding site. The data show hitherto unknown effects of monovalent anionic drugs on a Transporter for divalent di- and tricarboxylates.
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the renal na dependent Dicarboxylate Transporter nadc 3 translocates dimethyl and disulfhydryl compounds and contributes to renal heavy metal detoxification
Journal of The American Society of Nephrology, 2002Co-Authors: Birgitta C. Burckhardt, Britta Drinkuth, Christine Menzel, Angela Konig, Jurgen Steffgen, Stephen H Wright, Gerhard BurckhardtAbstract:ABSTRACT. The active transport of Krebs cycle intermediates, such as succinate, α-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 μM 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 −90 and −10 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.