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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)

Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada

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 Lys³³² and Asp³³⁶. We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys³³² mutants, exhibits a selective decrease in leukotriene C₄ (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 Asp³³⁶ (D336E) also remained very low, as observed for D336R. In addition, nonconservative substitutions of TM6-associated Lys³¹⁹ and Lys³⁴⁷ 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 [Lys³⁹⁶ (TM7), Asp⁴³⁶ (TM8), and Arg⁵⁹³ (TM11)] caused a substantial and global reduction in transport activity. However, unlike TM6 Asp³³⁶, 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.

Address correspondence to: Dr. Susan P. C. Cole, Cancer Research Laboratories, 3rd Floor, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6 Canada. E-mail: coles{at}post.queensu.ca

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