title: Functional importance of three basic residues clustered at the cytosolic interface of transmembrane helix 15 in the multidrug and organic anion transporter MRP1 (ABCC1).
abstract: The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg1138, Lys1141, and Arg1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg1138 and Arg1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[32P]ADP. Mutation of Lys1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter.
authors.affiliation: Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Gwenaëlle
authors.initials: G
authors.lastname: Conseil
authors.forename: Roger G
authors.initials: RG
authors.lastname: Deeley
authors.forename: Susan P C
authors.initials: SP
authors.lastname: Cole
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001386:Azides; D020134:Catalytic Domain; D002460:Cell Line; D003600:Cytosol; D004958:Estradiol / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D017997:Leukotriene C4 / Q000493:pharmacokinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010761:Phosphorus Radioisotopes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014316:Tritium; D014638:Vanadates
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D001386:Azides; D004958:Estradiol; D017997:Leukotriene C4; D008239:Lysine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010761:Phosphorus Radioisotopes; D014316:Tritium; D014638:Vanadates; C021622:8-azidoadenosine diphosphate; C421109:multidrug resistance-associated protein 1