Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates.
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 mM for citrate and 37+/-6 mM 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.
Inoue
Katsuhisa
K
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
Fei
You-Jun
YJ
Zhuang
Lina
L
Gopal
Elangovan
E
Miyauchi
Seiji
S
Ganapathy
Vadivel
V
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D019343:Citric Acid / Q000378:metabolism; D002952:Citric Acid Cycle; D003001:Cloning, Molecular; D029382:Dicarboxylic Acid Transporters; D005091:Exons; D016678:Genome; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity; D019802:Succinic Acid / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
D029382:Dicarboxylic Acid Transporters; C487219:Slc13a5 protein, mouse; D027981:Symporters; D019343:Citric Acid; D012964:Sodium; D019802:Succinic Acid