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).

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 NH 2 -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 (LTC 4 ) transport, associated with substantial changes in both K m and V max and LTC 4 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 reestablished 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.