Cell free expression and functional reconstitution of eukaryotic drug transporters
Cell free expression and functional reconstitution of eukaryotic drug transporters
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DOI：
10.1021/bi800060w
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发表时间：
2008-04-15
期刊：
影响因子：
2.9
通讯作者：
Koepsell, Hermann
中科院分区：
文献类型：
--
作者：
Keller, Thorsten;Schwarz, Daniel;Koepsell, Hermann
SLC 22蛋白家族的多特异性有机阳离子和阴离子转运蛋白与药物的吸收和排泄密切相关。为了阐明运输机制，纯化的转运蛋白的功能和生物物理特性是必需的，必须确定三级结构。在此，我们合成了大鼠有机阳离子转运蛋白OCT 1和OCT 2以及大鼠有机阴离子转运蛋白OAT 1。我们用2%1-肉豆蔻酰-2-羟基-sn-甘油基-3-[磷酸-rac-（1 -甘油）]（LMPG）溶解沉淀物，在1%3-[（3-胆酰胺丙基）二甲基铵-1-丙磺酸盐（CHAPS）或辛基葡糖苷存在下纯化转运蛋白，并将它们重构成脂蛋白体。从1 mL反应容器中0.纯化13-0.36 mg转运蛋白。因此，从5至10个1 mL反应容器中获得了足够用于结晶的蛋白质。在1%LMPG和0. 5%CHAPS、OCT 1和OAT 1形成同源寡聚体，但不形成异源寡聚体。将OCT 1、OCT 2和OAT 1复溶至脂蛋白体后，分别测定了有机阳离子和阴离子转运蛋白对1-甲基-4-苯基吡啶鎓和对氨基马尿酸盐（PAH（-））的摄取的Michaelis-Menten Km值，与转运蛋白在细胞中表达后相似。使用，重建系统，获得的证据表明，OAT 1作为强制性和电中性PAH-/二羧酸逆向转运蛋白，并含有低亲和力的氯离子结合位点，刺激营业额。仅在反式侧使用（x-酮戊二酸（KG（2-））观察到PAH-摄取，并且反式-KG（2-）使电压钳位的脂蛋白体中的PAH-浓度瞬时高于平衡。Cl-使PAH（-）/KG（2-）反向转运的Vmax增加，但不依赖于膜电位梯度，而PAH-/KG 2-反向转运在Cl-存在或不存在时均不依赖于膜电位。
Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1 -glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1mL reaction vessels 0. 13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0. 5 % CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis -Menten Km values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH(-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using, the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH-/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH- uptake was observed only with (x-ketoglutarate (KG(2-)) on the trans side, and trans-KG(2-) increased the PAH- concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V-max Of PAH(-)/KG(2-) antiport was increased by Cl- in a manner independent of gradients, and PAH-/KG2- antiport was independent of membrane potential in the absence or presence of Cl-.