Abteilung Vegetative Physiologie und Pathophysiologie, Universitätsmedizin Göttingen, Göttingen, Germany.
Am J Physiol Renal Physiol. 2011 Nov;301(5):F1026-34. doi: 10.1152/ajprenal.00169.2011. Epub 2011 Aug 24.
Organic anions are taken up from the blood into proximal tubule cells by organic anion transporters 1 and 3 (OAT1 and OAT3) in exchange for dicarboxylates. The released dicarboxylates are recycled by the sodium dicarboxylate cotransporter 3 (NaDC3). In this study, we tested the substrate specificities of human NaDC3, OAT1, and OAT3 to identify those dicarboxylates for which the three cooperating transporters have common high affinities. All transporters were stably expressed in HEK293 cells, and extracellularly added dicarboxylates were used as inhibitors of [(14)C]succinate (NaDC3), p-[(3)H]aminohippurate (OAT1), or [(3)H]estrone-3-sulfate (OAT3) uptake. Human NaDC3 was stably expressed as proven by immunochemical methods and by sodium-dependent uptake of succinate (K(0.5) for sodium activation, 44.6 mM; Hill coefficient, 2.1; K(m) for succinate, 18 μM). NaDC3 was best inhibited by succinate (IC(50) 25.5 μM) and less by α-ketoglutarate (IC(50) 69.2 μM) and fumarate (IC(50) 95.2 μM). Dicarboxylates with longer carbon backbones (adipate, pimelate, suberate) had low or no affinity for NaDC3. OAT1 exhibited the highest affinity for glutarate, α-ketoglutarate, and adipate (IC(50) between 3.3 and 6.2 μM), followed by pimelate (18.6 μM) and suberate (19.3 μM). The affinity of OAT1 to succinate and fumarate was low. OAT3 showed the same dicarboxylate selectivity with ∼13-fold higher IC(50) values compared with OAT1. The data 1) reveal α-ketoglutarate as a common high-affinity substrate of NaDC3, OAT1, and OAT3 and 2) suggest potentially similar molecular structures of the binding sites in OAT1 and OAT3 for dicarboxylates.
有机阴离子通过有机阴离子转运体 1 和 3（OAT1 和 OAT3）从血液中摄取到近曲小管细胞中，以交换二羧酸。释放的二羧酸被二羧酸钠协同转运蛋白 3（NaDC3）回收。在这项研究中，我们测试了人源 NaDC3、OAT1 和 OAT3 的底物特异性，以确定这三种协同转运体具有共同高亲和力的二羧酸。所有转运体均稳定表达于 HEK293 细胞中，并用细胞外添加的二羧酸作为[14C]琥珀酸盐（NaDC3）、对[3H]氨基马尿酸（OAT1）或[3H]雌酮-3-硫酸盐（OAT3）摄取的抑制剂。通过免疫化学方法和钠依赖性琥珀酸盐摄取证实人源 NaDC3 稳定表达（钠离子激活的 K0.5 为 44.6 mM；Hill 系数为 2.1；琥珀酸盐的 K(m)为 18 μM）。NaDC3 被琥珀酸盐（IC50 为 25.5 μM）强烈抑制，而被α-酮戊二酸（IC50 为 69.2 μM）和富马酸（IC50 为 95.2 μM）抑制作用较弱。具有较长碳骨架的二羧酸（己二酸、庚二酸、辛二酸）对 NaDC3 的亲和力低或没有。OAT1 对戊二酸盐、α-酮戊二酸和己二酸表现出最高的亲和力（IC50 在 3.3 到 6.2 μM 之间），其次是庚二酸（18.6 μM）和辛二酸（19.3 μM）。OAT1 对琥珀酸盐和富马酸盐的亲和力较低。OAT3 对二羧酸的选择性与 OAT1 相同，但 IC50 值高 13 倍。该数据揭示了 α-酮戊二酸是 NaDC3、OAT1 和 OAT3 的共同高亲和力底物，并且提示 OAT1 和 OAT3 中二羧酸结合位点可能具有相似的分子结构。
