Role of two adjacent cytoplasmic tyrosine residues in MRPI (ABM) transport activity and sensitivity to sulfonylureas

The human ATP-binding cassette (ABC) protein MRP1 causes resistance to many anticancer drugs and is also a primary active transporter of conjugated metabolites and endogenous organic anions, including leukotriene C-4 (LTC4) and glutathione (GSH). The sulfonylurea receptors SUR1 and SUR2 are related ABC proteins with the same domain structure as MRP1, but serve as regulators of the K+ channel Kir6.2. Despite their functional differences, the activity of both SUR1/2 and MRP1 can be blocked by glibenclamide, a sulfonylurea used to treat diabetes. Residues in the cytoplasmic loop connecting transmembrane helices 15 and 16 of the SUR proteins have been implicated as molecular determinants of their sensitivity to glibenclamide and other sulfonylureas. We have now investigated the effect of mutating Tyr(1189) and Tyr(1190) in the comparable region of MRP1 on its transport activity and sulfonylurea sensitivity. Ala and Ser substitutions of Tyr(1189) and Tyr(1190) caused a greater than or equal to50% decrease in the ability of MRP1 to transport different organic anions, and a decrease in LTC4 photolabeling. Kinetic analyses showed the decrease in GSH transport was attributable primarily to a 10-fold increase in K-m. In contrast, mutations of these Tyr residues had no major effect on the catalytic activity of MRP1. Furthermore, the mutant proteins showed no substantial differences in their sensitivity to glibenclamide and tolbutamide. We conclude that MRP1 Tyr(1189) and Tyr(1190), unlike the corresponding residues in SUR1, are not involved in its differential sensitivity to sulfonylureas, but nevertheless, may be involved in the transport activity of MRP1, especially with respect to GSH. (C) 2004 Elsevier Inc. All rights reserved.