TY - JOUR T1 - Multiple Roles of Charged Amino Acids in Cytoplasmic Loop 7 for Expression and Function of the Multidrug and Organic Anion Transporter MRP1 (ABCC1) JF - Molecular Pharmacology JO - Mol Pharmacol SP - 397 LP - 406 DO - 10.1124/mol.108.052860 VL - 75 IS - 2 AU - Gwenaëlle Conseil AU - Alice J. Rothnie AU - Roger G. Deeley AU - Susan P. C. Cole Y1 - 2009/02/01 UR - http://molpharm.aspetjournals.org/content/75/2/397.abstract N2 - Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu1169, Glu1170, and Glu1172 were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, 1169AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and 1169AAQA were accompanied by changes in orthovanadate-induced trapping of [α-32P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1. The American Society for Pharmacology and Experimental Therapeutics ER -