Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates
Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates
复制标题
DOI：
10.1042/bj20031261
复制
发表时间：
2004-03-15
影响因子：
4.1
通讯作者：
Ganapathy, V
中科院分区：
文献类型：
--
作者：
Inoue, K;Fei, YJ;Ganapathy, V
在本研究中，我们报道了果蝇Indy的小鼠同源基因--小鼠Na+-耦合柠檬酸转运蛋白(NACT)的克隆和功能鉴定。小鼠NACT由572个氨基酸组成，在一级序列上与大鼠和人类NACT高度相似。编码转运蛋白的小鼠NACT基因大约是。全长23kb，由12个外显子组成。当在哺乳动物细胞中表达时，克隆的转运蛋白介导柠檬酸和琥珀酸的Na+偶联转运。竞争实验表明，小鼠NACT还识别其他三元酸循环中间体，如苹果酸、富马酸和2-氧代戊二酸作为优良的底物。在pH 7.5时，转运过程的米氏常数柠檬酸盐为38+5微米，琥珀酸盐为37+/-6微米。传输过程是电生的，对Na+表现出强制性的要求。Na~+活化动力学表明，多个Na~‘离子参与了活化过程。胞外pH对小鼠NACT的转运功能有不同的影响，这取决于转运的底物是柠檬酸还是琥珀酸。这些底物的米氏常数也受pH的显著影响。在非洲爪哇卵母细胞表达系统中用双微电极电压钳技术检测时，小鼠NACT介导的转运过程是电生的。柠檬酸盐的电荷底物比为I，琥珀酸根的电荷底物比为2。这些研究预测的最可能的运输机制包括柠檬酸作为三价阴离子和琥珀酸作为二价阴离子的运输，其固定的Na+/底物化学计量比为4：1。本研究为哺乳动物NACT的电生性质提供了第一个明确的证据。
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 muM for citrate and 37 +/- 6 muM 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 I 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.