Characterization of the Role of Polar Amino Acid Residues within Predicted Transmembrane Helix 17 in Determining the Substrate Specificity of Multidrug Resistance Protein 3

Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRPI, MRP3 confers resistance to etoposide (VP-16) and actively transports 17β-estradiol 17-(β-D-glucuronide) (E 2 17βG), cysteinyl leukotriene 4 (LTC4), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E 2 17βG transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM 17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S 1231 A and N1241A decreased resistance to VP-16 and transport of E 2 17βG and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E 2 17βG but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser 1 2 2 9 , Ser 1 2 3 1 , Gln 1 2 3 5 , and Asn 1 2 4 1 play an important role in determining the specificity of MRP3, while mutation of Tyr 1 2 3 2 , Ser 1 2 3 3 , and Thr 1 2 3 7 affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr 1 2 3 7 , markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.