In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis–Menten constant for the transport process is 38±5 µM for citrate and 37±6 µM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis–Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.
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March 2004
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Research Article|
March 15 2004
Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates
Katsuhisa INOUE;
Katsuhisa INOUE
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
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You-Jun FEI;
You-Jun FEI
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
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Lina ZHUANG;
Lina ZHUANG
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
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Elangovan GOPAL;
Elangovan GOPAL
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
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Seiji MIYAUCHI;
Seiji MIYAUCHI
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
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Vadivel GANAPATHY
Vadivel GANAPATHY
1
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
1To whom correspondence should be addressed (e-mail vganapat@mail.mcg.edu).
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Publisher: Portland Press Ltd
Received:
August 18 2003
Revision Received:
December 01 2003
Accepted:
December 04 2003
Accepted Manuscript online:
December 04 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 378 (3): 949–957.
Article history
Received:
August 18 2003
Revision Received:
December 01 2003
Accepted:
December 04 2003
Accepted Manuscript online:
December 04 2003
Citation
Katsuhisa INOUE, You-Jun FEI, Lina ZHUANG, Elangovan GOPAL, Seiji MIYAUCHI, Vadivel GANAPATHY; Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates. Biochem J 15 March 2004; 378 (3): 949–957. doi: https://doi.org/10.1042/bj20031261
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