Nano-scale analysis of the drug transport mechanism of MRP1

胰蛋白酶在MRP 1的第17个跨膜结构域和C-末端NBD之间形成一个胞质环。我们发现，在谷胱甘肽（GSH）的存在下，限制消化被抑制。加入AG-A或阿糖胞苷后，GSH的抑制作用增强。L_0/ICL 3结构域对于GSH的抑制作用是不可或缺的。结果表明，GHS与L_0/ICL 3结构域相互作用，导致MRP 1C端结构发生改变。在ICL 5和ICL 7中发现了谷氨酰胺（Glutamine，507和1157位氨基酸）和甘氨酸（Glycine，511和1161位氨基酸）的保守序列。这些突变蛋白结构没有明显变化，但失去了转运LTC_4的活性，不能被azidoAG-A光亲和标记。ICL 5突变体MRP 1蛋白具有NBD 1的ATP结合活性，但不具有NBD 2的ATP结合活性。ICL 7突变体MRP 1蛋白在两种NBD中都失去了ATP结合活性。这些保守氨基酸在识别GSH、ATP结合和ATP酶活性等方面具有重要作用，并已报道了一些不含GSH的底物可以与MRP 1一起转运。然而，GSH依赖性和非依赖性转运与MRP 1之间的差异尚不清楚。SN-38可以在不存在GSH的情况下与MRP 1一起转运。我们发现一个L_0/ICL 3突变体MRP 1不能转运LTC_4，但能转运SN-38。这些数据表明，MRP 1的底物识别位点可能是不同的GSH依赖和独立的运输至少部分。我们还表明，它可能是可能的区分GHS依赖或独立的化学结构的基础上的底物。

Trypsin digests a cytoplasmic loop between the 17th membrane spanning domain and the C-terminal NBD of MRP1. We found that the limited digestion was inhibited in the presence of glutathione (GSH). The inhibitory effect of GSH enhanced by adding of AG-A or Vincristine. The L_0/ICL3 domain was indispensable for the inhibitory effect of GSH. These data suggested GHS interact with the L_0/ICL3 domain and induce a structural change of the C-terminal part of MRP1.We found conserved pairs of Glutamine (507th and 1157th AA(amino acid)) and Glycine (511th and 1161st AA) in the ICL5 (intracellular loop) and the ICL7. These mutant proteins of MRP1 did not have significant structural changes, however lost the transport activity of LTC_4 and could not be photoaffinity labeled with azidoAG-A. The ICL5 mutant MRP1 protein had ATP binding activity of NBD1 but did not that of NBD2. The ICL7 mutant MRP1 protein had lost ATP binding activity in both NBDs. These data suggested that these conserved AAs have important roles in recognition of GSH, binding of ATP and ATPase activity.Some substrates which can be transported with MRP1 without GSH had been reported. However the difference between GSH dependent and independent transport with MRP1 was not clear. SN-38 can be transported with MRP1 in the absence of GSH. We showed one L_0/ICL3 mutant MRP1 which could not transport LTC_4, could transport SN-38. This data suggested the substrates recognition sites of MRP1 may be different between GSH dependent and independent transport at least in part. We also demonstrated that it might be possible to distinguish between GHS dependent or independent substrate on the base of chemical structures.