Substrate- and cell contact-dependent inhibitor affinity of human organic cation transporter 2: studies with two classical organic cation substrates and the novel substrate cd2+.
Substrate- and cell contact-dependent inhibitor affinity of human organic cation transporter 2: studies with two classical organic cation substrates and the novel substrate cd2+.
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DOI：
10.1021/mp400113d
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发表时间：
2013-06
影响因子：
4.9
通讯作者：
F. Thévenod;G. Ciarimboli;M. Leistner;N. Wolff;Wing Lee;Irina Schatz;T. Keller;Rouvier Al-Monajjed;V. Gorboulev;H. Koepsell
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文献类型：
--
作者：
F. Thévenod;G. Ciarimboli;M. Leistner;N. Wolff;Wing Lee;Irina Schatz;T. Keller;Rouvier Al-Monajjed;V. Gorboulev;H. Koepsell
来自大鼠的多特异性有机阳离子转运蛋白Oct 2（基因Slc 22 A2）先前已被证明转运Cs（+）。在这里，我们报告了人OCT 2（hOCT 2）能够转运Cd（2+），显示底物饱和，米氏常数（Km）为54 ± 5.8 μM。典型的hOCT 2配体（未标记的底物和抑制剂）抑制hOCT 2对Cd（2+）的摄取，hOCT 2底物结合结构域中的点突变降低了摄取速率。过表达hOCT 2的人胚肾293细胞（HEK-hOCT 2-C）或表达rOct 2的大鼠肾近端小管细胞（NRK-52 E-C）与Cd（2+）孵育导致细胞凋亡水平升高，而OCT 2抑制配体西咪替丁（+）可降低细胞凋亡水平。当HEK-hOCT 2-C融合或通过去除Ca（2+）和Mg（2+）而解离时，它们表现出不同的功能特性。只有融合的HEK-hOCT 2-C转运Cd（2+），融合和解离的细胞对Cd（2+）、MPP（+）、四乙铵（+）、西咪替丁（+）和皮质酮摄取1-甲基-4-苯基吡啶（+）（MPP（+））的抑制效力不同。在融合HEK-hOCT 2-C中，使用远低于相应Km值的底物浓度比较Cd（2+）摄取、4-[4-（二甲氨基）苯乙烯基]-N-甲基吡啶（+）（ASP（+））摄取和MPP（+）摄取的抑制后，获得了差异很大的抑制效力。在先前确定的底物结合位点的大鼠Oct 1采用点突变产生的证据表明，底物和抑制剂的结合位点之间的短距离变构效应参与底物依赖性抑制剂的效力。底物依赖性抑制剂亲和力可能是OCT的共同特性。为了预测由OCT转运的药物与抑制性药物之间的相互作用，有必要采用特定的转运药物而不是模型底物进行体外测量。
Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 μM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.