Functional Reconstitution of Substrate Transport by Purified Multidrug Resistance Protein MRP1 (ABCC1) in Phospholipid Vesicles

Qingcheng Mao,Roger G Deeley,Susan P.C Cole

doi:10.1074/jbc.m004584200

Qingcheng Mao, Roger G Deeley + Show 1 more

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https://doi.org/10.1074/jbc.m004584200

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Abstract

The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C(4) (LTC(4)). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69-82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC(4) uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC(4) uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17 beta-estradiol 17-beta-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K(m) for ATP and LTC(4) were 357 +/- 184 microm and 366 +/- 38 nm, respectively, and 2.14 +/- 0.75 microm for 17 beta-estradiol 17-beta-(d-glucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.

Highlights

Resistance of tumors to multiple structurally unrelated anticancer drugs is a major obstacle to successful cancer chemotherapy
Results from these and other studies are consistent with the concept that MRP1 and MRP2 are organic anion and drug efflux pumps that transport their substrates concomitant with ATP hydrolysis
The ability to functionally reconstitute purified ATP-binding cassette (ABC) proteins represents a major step toward understanding the mechanistic relationships between ATP binding and hydrolysis and coupling of these events to translocation of substrates across lipid membranes

Summary

Introduction

Resistance of tumors to multiple structurally unrelated anticancer drugs is a major obstacle to successful cancer chemotherapy. 20 ␮l of reaction mixture was removed, diluted into 80 ␮l of ice-cold transport buffer, and immediately subjected to spin column gel filtration as described under “Experimental Procedures.” A, time course of [3H]LTC4 uptake by the MRP1 proteoliposomes in the presence of 4 mM ATP (XI) or 4 mM AMP-PCP (‚); B, ctiomntercool ulirpsoesoofm[e3Hs (]ŒLT),Cin4 uptake by the MRP1 proteoliposomes (f) or by the presence of 50 nM [3H]LTC4, 10 mM MgCl2, 4 mM ATP, 10 mM creatine phosphate, 100 ␮g mlϪ1 creatine kinase.

Results

Conclusion