Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2.
Multidrurg resistance-associated protein 2 (MRP2)/canalicular multispecific organic anion transporter (cMOAT) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane. To identify functional amino acid residues that play essential roles in the substrate transport, each of 13 basic residues around transmembrane regions (TMs) 6-17 were replaced with alanine. Wild type and mutant proteins were expressed in COS-7 cells, and the transport activity was measured as the excretion of glutathione-methylfluorescein. Four mutants, K324A (TM6), K483A (TM9), R1210A (TM16), and R1257A (TM17), showed decreased transport activity, and another mutant, K578A (TM11), showed decreased protein expression. These five mutants were normally delivered to the cell surface similar to the other fully active mutants and wild type MRP2. The importance of TM6, TM16, and TM17 in the transport function of MRP2 is consistent with the previous observation indicating the importance of the corresponding TM1, TM11, and TM12 on P-glycoprotein (Loo, T. W., and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392). Another observation that MRP2 inhibitor, cyclosporine A, failed to inhibit R1230A specifically, indicated the existence of its binding site within TM16.
Ryu
S
S
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Kawabe
T
T
Nada
S
S
Yamaguchi
A
A
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D027321:Anion Transport Proteins; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D016572:Cyclosporine / Q000494:pharmacology; D019793:Fluorescein / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D021381:Protein Transport; D013997:Time Factors
D018435:ATP Binding Cassette Transporter, Subfamily B; D000409:Alanine; D000596:Amino Acids; D027321:Anion Transport Proteins; D001643:Bridged Bicyclo Compounds; D002352:Carrier Proteins; D016572:Cyclosporine; D019793:Fluorescein; D005978:Glutathione; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C000715087:ABCC2 protein, human; C080120:glutathione-bimane; C421109:multidrug resistance-associated protein 1