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)

被引:65
作者
Haimeur, A [1 ]
Conseil, G [1 ]
Deeley, RG [1 ]
Cole, SPC [1 ]
机构
[1] Queens Univ, Canc Res Labs, Kingston, ON K7L 3N6, Canada
关键词
D O I
10.1124/mol.65.6.1375
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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.
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页码:1375 / 1385
页数:11
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