Characterization of the Transport Properties of Human Multidrug Resistance Protein 7 (MRP7, ABCC10)

Abstract

Human multidrug resistance protein 7 (MRP7, ABCC10) is a recently described member of the C family of ATP binding cassette proteins (Cancer Lett 162:181-191, 2001). However, neither its biochemical activity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP7 using membrane vesicles prepared from human embryonic kidney 293 cells transfected with MRP7 expression vector. It is shown that expression of MRP7 is specifically associated with the MgATPdependent transport of 17␤-estradiol-(17-␤-D-glucuronide) (E 2 17␤G). E 2 17␤G transport was saturable, with K m and V max values of 57.8 Ϯ 15 M and 53.1 Ϯ 20 pmol/mg/min. By contrast, with E 2 17␤G, only modest enhancement of LTC 4 transport was observed and transport of several other established substrates of MRP family transporters was not detectable to any extent. In accord with the notion that MRP7 has a bipartite substrate binding pocket composed of sites for anionic and lipophilic moieties, transport of E 2 17␤G was susceptible to competitive inhibition by both amphiphiles, such as leukotriene C 4 (K i(app) , 1.5 M), glycolithocholate 3-sulfate (K i(app) , 34.2 M) and MK571 (K i(app) , 28.5 M), and lipophilic agents such as cyclosporine A (K i(app) , 14.4 M). Of the inhibitors tested, LTC 4 was the most potent, in agreement with the possibility that it is a substrate of the pump. The determination that MRP7 has the facility for mediating the transport of conjugates such as E 2 17␤G indicates that it is a lipophilic anion transporter involved in phase III (cellular extrusion) of detoxification.

Analysis of the Drug Resistance Profile of Multidrug Resistance Protein 7 (ABCC10)

“…These stable transfectants were previously used for analyzing the substrate selectivity of MRP7 (26). Ectopic expression of MRP7 in the two MRP7-transfected clones is indicated by the intensely immunoreactive bands that migrated with an apparent M r ϳ171,000, but were not present in membranes prepared from the control cells (Fig.…”

“…Generation of HEK293 clones stably transfected with MRP7 expression vector (HEK-MRP7-C17 and HEK-MRP7-C18) and parental vector-transfected control cells (HEK293-pcDNA) was described previously (26). HEK293 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin.…”

“…3). Despite this relatively low degree of amino acid identity, we determined using membrane vesicle transport assays that MRP7 possesses the cardinal biochemical feature of this family, in that it is competent in the transport of amphipathic anions, such as the prototypical MRP family substrate 17␤-estradiol 17-(␤-D-glucuronide) (26). The ability of the pump to transport amphipathic anions such as 17␤-estradiol 17-(␤-Dglucuronide), in combination with the presence of a third membranespanning domain, suggests that like other MRPs that have this structural feature, MRP7 might have the facility for conferring resistance to natural product anticancer agents.…”

ABCC10, ABCC11, and ABCC12

“…However, its functional characteristics were described in two reports from our laboratory [4,5]. These studies established that MRP7 is a lipophilic anion transporter that has the facility for conferring resistance to some natural product anticancer agents.…”

“…The drug resistance properties and substrate selectivity of MRP8 were analyzed in two reports from our laboratory [4,7]. In the first of these studies, the drug resistance profile of MRP8 was determined using LLC-PK1 cells in which the pump was ectopically expressed.…”