Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1)
Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1)
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DOI：
10.1124/mol.65.6.1375
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发表时间：
2004-06-01
影响因子：
3.6
通讯作者：
Cole, SPC
中科院分区：
文献类型：
--
作者：
Haimeur, A;Conseil, G;Cole, SPC
多药耐药蛋白1 （MRP1）属于atp结合盒转运蛋白超家族。除药物外，MRP1介导许多偶联和非偶联有机阴离子的主动转运。MRP1由两个跨膜结构域（MSD2和MSD3）组成，每个结构域后跟一个核苷酸结合结构域和第三个nh2末端MSD1。MSD2包含跨膜（TM）螺旋6到11，之前，我们通过表征含有非保守取代的Lys（332）和Asp（336）的突变体，发现TM6中的两个带电残基在MRP1转运活性和底物特异性中具有重要但明显不同的作用。我们现在扩展了这些研究，发现相同电荷的TM6突变体K332R，像非保守取代的Lys（332）突变体一样，表现出白三烯C-4 （LTC4）转运的选择性减少，与K-m和V-max以及LTC4结合的实质性变化有关。同电荷突变体Asp(336) （D336E）的整体有机阴离子运输活性也很低，D336R也是如此。此外，tm6相关的Lys（319）和Lys（347）的非保守替换导致GSH转运的选择性减少。在TM7和TM11附近的8个其他带电残基中，其中3个[Lys(396) (TM7), Asp(436) （TM8）和Arg(593) (TM11)]的非保守取代导致了运输活性的显著降低。然而，与TM6 Asp（336）不同，这些MRP1突变体可以通过保守替换重新建立野生型转运活性。我们得出结论，TM6、TM7、TM8和TM11中或近端带msd2电荷的残基在MRP1转运活性和底物特异性中起着关键但不同的作用。
Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins. In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions. MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH2-terminal MSD1. MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys(332) and Asp(336). We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys(332) mutants, exhibits a selective decrease in leukotriene C-4 (LTC4) transport, associated with substantial changes in both K-m and V-max and LTC4 binding. The overall organic anion transport activity of the same-charge mutant of Asp(336) (D336E) also remained very low, as observed for D336R. In addition, nonconservative substitutions of TM6-associated Lys(319) and Lys(347) resulted in a selective decrease in GSH transport. Of eight other charged residues in or proximal to TM7 to TM11 that were investigated, nonconservative substitutions of three of them [Lys(396) (TM7), Asp(436) (TM8), and Arg(593) (TM11)] caused a substantial and global reduction in transport activity. However, unlike TM6 Asp(336), wild-type transport activity could be re-established in these MRP1 mutants by conservative substitutions. We conclude that MSD2-charged residues in or proximal to TM6, TM7, TM8, and TM11 play critical but differential roles in MRP1 transport activity and substrate specificity.