Cloning of a Human Renal p–Aminohippurate Transporter, hROAT1
Cloning of a Human Renal p–Aminohippurate Transporter, hROAT1
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人肾 p-氨基马尿酸转运蛋白 hROAT1 的克隆
DOI：
10.1159/000025863
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发表时间：
2008
影响因子：
2.8
通讯作者：
G. Burckhardt
中科院分区：
文献类型：
--
作者：
G. Reid;N. Wolff;F. Dautzenberg;G. Burckhardt
几十年来，对氨基马尿酸(PAH)一直作为模型底物来研究两亲性有机阴离子的肾脏排泄[1]。PAH在肾小球中自由过滤，也在近端小管中高效分泌。到目前为止，在所有被研究的物种中，近端小管细胞基侧膜对多环芳烃的摄取是通过多环芳烃/α-酮戊二酸逆向转运体与细胞内的α-酮戊二酸交换而发生的[1-3]。二羧酸盐α-酮戊二酸通过Na+偶联转运机制循环，Na+，K+-ATP酶将Na+-离子泵出细胞外。总体而言，摄取一个多环芳烃分子需要消耗一个ATP分子。对底物特异性的详细研究表明，大鼠近端小管细胞基侧膜上的PAH转运体可以接受大量的两亲性阴离子、不带电荷的化合物甚至一些有机阳离子[4]。三种底侧有机阴离子转运体(多环芳烃/α-酮戊二酸转运体、Na+-二羧酸转运体、硫酸盐/阴离子反向转运体)和位于同一膜上的有机阳离子转运体的特性部分重叠，确保了大多数水溶性异物随尿液排泄。最近，利用非洲爪哇的表达克隆策略，鉴定了介导PAH/α-酮戊二酸逆向转运的蛋白质在大鼠[5，6]和冬牙鲨[7]近端小管细胞的基侧膜上。克隆的肾脏有机阴离子转运蛋白OAT1/ROAT1(大白鼠命名为OAT1/ROAT1)和FROAT(比目鱼命名为FROAT)全长551-562个氨基酸，推测有12个跨膜区。大鼠和比目鱼的氨基酸同源性为47%，与有机阳离子转运蛋白OCT1和OCT2有关[8]。随着来自大鼠OAT1/ROAT1和牙鲆OAT1/ROAT1和FROAT的cRNA在X.laevescell中的表达，丙磺舒可以抑制PAH的饱和摄取[5-7]。α-酮戊二酸和戊二酸可顺式抑制近端小管细胞对多环芳烃的摄取，而这些二羧酸盐可反式刺激近端小管细胞摄取多环芳烃，表明OAT1/ROAT1和FROAT很可能代表近端小管细胞基侧膜上的多环芳烃/α-酮戊二酸逆向转运体。与这一假设相一致的是广泛的底物特异性：除了多环芳烃和α-酮戊二酸外，OAT1还运输标记的甲氨蝶呤、cAMP、cGMP、PGE2和尿酸盐[5]。人肾近端小管细胞的基侧膜上存在多环芳烃/α-酮戊二酸逆向转运蛋白，缺乏直接的实验证据。然而，考虑到基底膜上多环芳烃/α-酮戊二酸逆向转运的进化保守性，我们假设存在人类与OAT1/ROAT1和FROAT的同源基因。本文报道了hROAT1-1基因的PCR克隆，该基因与大鼠OAT1/ROAT1高度同源。肾脏基础研究
For several decades, p-aminohippurate (PAH) has served as a model substrate to study the renal excretion of amphiphilic organic anions [1]. PAH is freely filtered in the glomeruli and also efficiently secreted in the proximal tubules. In all species investigated so far, the uptake of PAH across the basolateral membrane of proximal tubule cells occurs by exchange with intracellular α-ketoglutarate through a PAH/ α-ketoglutarate antiporter [1–3]. The dicarboxylate α-ketoglutarate is recycled by a Na +-coupled transport mechanism and the Na +-ions are pumped out of the cell by the Na +, K+-ATPase. Overall, the uptake of one PAH molecule requires the expenditure of one ATP molecule. Detailed investigations on substrate specificity revealed that the PAH transporter in the basolateral membrane of rat proximal tubule cells accepts a large variety of amphiphilic anions, uncharged compounds and even some organic cations [4]. The specificities of three basolateral organic anion transporters (PAH/ α-ketoglutarate antiporter, Na +-dicarboxylate symporter, sulfate/anion antiporter) and of the organic cation transporter localized in the same membrane partially overlap, ensuring excretion of most water-soluble xenobiotics with the urine. The proteins mediating PAH/ α-ketoglutarate antiport at the basolateral membrane of rat [5, 6] and winter flounder [7] proximal tubule cells have recently been identified by an expression cloning strategy using Xenopus laevisoocytes. The cloned renal organic anion transporters, termed OAT1/ROAT1 for rat and fROAT for flounder, are between 551 and 562 amino acids long with 12 putative transmembrane domains. Rat and flounder ROATs show an amino acid identity of 47% and are related to the organic cation transporters, OCT1 and OCT2 [8]. Following expression of cRNA derived from rat OAT1/ROAT1 and flounder fROAT in X. laevisoocytes, a probenecid-inhibitable, saturable uptake of PAH could be demonstrated [5–7]. PAH uptake was cis-inhibited by α-ketoglutarate and glutarate in the bath, and trans-stimulated by these dicarboxylates loaded previously into the oocytes, indicating that OAT1/ROAT1 and fROAT most likely represent the PAH/ α-ketoglutarate antiporter of the basolateral membrane of proximal tubule cells. In line with this assumption is the broad substrate specificity: besides PAH and α-ketoglutarate, OAT1 transported labeled methotrexate, cAMP, cGMP, PGE 2, and urate [5]. Direct experimental evidence is lacking for the presence of a PAH/α-ketoglutarate antiporter in the basolateral membrane of human renal proximal tubule cells. However, given the evolutionary conservation of PAH/ α-ketoglutarate antiport in the basolateral membrane, we assumed that a human homologue to OAT1/ROAT1 and fROAT exists. Here we report the PCR cloning of hROAT1 1, which is highly homologous to rat OAT1/ROAT1. Basic Renal Research
DOI：
10.1073/pnas.85.23.8998
发表时间：
1988-12-01
影响因子：
11.1
作者：
FROHMAN, MA;DUSH, MK;MARTIN, GR
通讯作者：
MARTIN, GR