Identification of basic residues involved in drug export function of humanmultidrug resistance-associated protein 2

Multidrurg resistance-associated protein 2 (MRPS)/canalicular multispecific organic anion transporter (cMOAT) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane. To identify functio nal amino acid residues that play essential roles in the substrate transpor t, each of 13 basic residues around transmembrane regions (TMs) 6-17 were r eplaced with alanine. Wild type and mutant proteins were expressed in COS-7 cells, and the transport activity was measured as the excretion of glutath ione-methylfluorescein. Four mutants, K324A (TM6), K483A (TM9), R1210A (TM1 6), and R1257A (TM17), showed decreased transport activity, and another mut ant, K578A (TM11), showed decreased protein expression. These five mutants were normally delivered to the cell surface similar to the other fully acti ve mutants and wild type MRP2. The importance of TM6, TM16, and TM17 in the transport function of MRP2 is consistent with the previous observation ind icating the importance of the corresponding TMI, TM11, and TM12 on P-glycop rotein (Loo, T. W, and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392 ). Another observation that MRP2 inhibitor, cyclosporine A, failed to inhib it R1230A specifically, indicated the existence of its binding site within TM16.