Functional Analysis of Human Multidrug Resistance Protein 8 (MRP8, AABCC11)
Zhe-Sheng Chen, Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences
We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509-29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-β-D-glucuronide (E217βG), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E217βG, and methotrexate were transported with K(m) and V(max) values of 13.0 +/- 0.8 µM and 34.9 +/- 9.5 pmol/mg/min, 62.9 +/- 12 µM and 62.0 +/- 5.2 pmol/mg/min, and 957 +/- 28 µM and 317 +/- 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E217βG, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E217βG, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.