Characterization of a rat Na+-dicarboxylate cotransporter
Characterization of a rat Na+-dicarboxylate cotransporter
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DOI：
10.1074/jbc.273.33.20972
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发表时间：
1998-08-14
影响因子：
4.8
通讯作者：
Hediger, MA
中科院分区：
文献类型：
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作者：
Chen, XZ;Shayakul, C;Hediger, MA
Krebs循环中间产物的代谢对真核细胞具有重要意义。在肾脏中，这些中间产物被积极地输送到上皮细胞中。由于柠檬酸盐是一种有效的钙结石形成抑制剂，过量摄取会导致低柠檬酸尿导致肾结石。我们报道了一个大鼠肾脏二元酸转运蛋白(SDCT1)的克隆和鉴定。原位杂交显示SDCT1mRNA定位于53段肾近端小管和肠绒毛衬里的肠上皮细胞。当SDCT1在非洲爪哇卵母细胞中表达时，大多数Krebs循环中间产物(K-m=20-60 Elm)的电生和钠依赖转运信号也在肺细管、附睾体和肝脏中检测到，包括柠檬酸、琥珀酸、酮戊二酸和草酰乙酸酯。值得注意的是，酸性氨基酸L-和D-谷氨酸和天冬氨酸也被转运，尽管亲和力较低(K-m=2-18 mM)。柠檬酸盐的转运是pH敏感的。在pH为7.5时，柠檬酸盐的K-m较高(0.64 mm)，而在pH为5.5时，K-m较低(57µM)。这与柠檬酸盐的-2形式是运输物种的概念是一致的。此外，在pH 5.5时的最大电流比在pH 7.5时高70%，我们的数据表明-3形式是一种竞争性抑制物。在电压钳条件下同时测量底物诱发电流和示踪剂摄取，以及热力学方法，给出柠檬酸盐的Na+或琥珀酸的Na+的化学计量比为3：1。根茎维甲素抑制SDCT1介导的电流。这种植物糖苷在没有底物的情况下也抑制了SDCT1特异的钠泄漏，表明至少有一个Na+在底物之前与转运蛋白结合。提供的数据为克隆的二元酸转运蛋白的生物物理特征和生理意义提供了新的见解。
The metabolism of Krebs cycle intermediates is of fundamental importance for eukaryotic cells. In the kidney, these intermediates are transported actively into epithelial cells. Because citrate is a potent inhibitor for calcium stone formation, excessive uptake results in nephrolithiasis due to hypocitraturia. We report the cloning and characterization of a rat kidney dicarboxylate transporter (SDCT1). In situ hybridization revealed that SDCT1 mRNA is localized in 53 segments of kidney proximal tubules and in enterocytes lining the intestinal villi. Signals were also detected in lung bronchioli, the epididymis, and liver, When expressed in Xenopus oocytes, SDCT1 mediated electrogenic, sodium-dependent transport of most Krebs cycle intermediates (K-m = 20-60 ELM), including citrate, succinate, mu-ketoglutarate, and oxaloacetate. Of note, the acidic amino acids L- and D-glutamate and aspartate were also transported, although with lower affinity (K-m = 2-18 mM). Transport of citrate was pH-sensitive. At pH 7.5, the K-m for citrate was high (0.64 mM), whereas at pH 5.5, the K-m was low (57 mu M). This is consistent with the concept that the -2 form of citrate is the transported species. In addition, maximal currents at pH 5.5 were 70% higher than those at pH 7.5, and our data show that the -3 form acts as a competitive inhibitor. Simultaneous measurements of substrate-evoked currents and tracer uptakes under voltage-clamp condition, as well as a thermodynamic approach, gave a Na+ to citrate or a Na+ to succinate stoichiometry of 3 to 1. SDCT1-mediated currents were inhibited by phloretin. This plant glycoside also inhibited the SDCT1-specific sodium leak in the absence of substrate, indicating that at least one Na+ binds to the transporter before the substrate. The data presented provide new insights into the biophysical characteristics and physiological implications of a cloned dicarboxylate transporter.