Pharmacogenetic, functional and toxicological studies of the human multidrug and organic anion transporters MRP1, MRP2 and MRP3

The multidrug resistance proteins MRP1, MRP2 and MRP3 mediate ATP-dependent cellular efflux of a wide variety of compounds including toxicants and chemotherapeutic agents. The overall goal of my research was to study the activity of these transporters and their modulation by mutations and chemicals. In a first study, eleven non-synonymous polymorphisms (nsSNPs) of MRP1/ABCC1 were recreated in vitro and their expression levels and transport activities were analyzed. The Ala989Thr substitution was found to selectively decrease estradiol 17-(D-beta-glucuronide) (E217betaG) transport, while the activities of the other mutants were comparable to wild-type MRP1. The ability of two different algorithms to predict the phenotype of MRP1 nsSNPs was determined to be poor, probably due to the complexity of MRP1 structure and function. In a second study, the phenotype of MRP1-Pro1150Ala mutant was further explored. First, Pro1150 was mutated to Gly, Ile, Leu and Val, and found that only the Val substitution supported vanadate-induced [alpha32P]8N3ADP trapping to a level similar to wild-type MRP1, in spite of the fact that all mutants displayed the same transport phenotype as MRP1-Pro1150A1a. Data obtained from experiments in which 8N3ADP trapping condition were modified and led to the conclusion that vanadate-induced trapping of [alpha32P]8N 3ADP was decreased in the MRP1-Pro1150Ala mutant due to an increased ability to release ADP after hydrolysis. The corresponding Pro residue in MRP2 and MRP3 were also mutated and functional characterization of MRP2-Pro1158Ala and MRP3-Pro1147Ala revealed that transport activity phenotype was dependent on both the substrates and the transporter. However, as observed for MRP1-Pro1150Ala, vanadate-induced [alpha32P]8N3ADP trapping was reduced in the MRP2 and MRP3 mutants. The modulation of MRP1-, MRP2- and MRP3-mediated transport of organic anions by nicotine glucuronide metabolites, nicotine- N-glucuronide, cotinine-N-glucuronide and trans-hydroxycotinine-O-glucuronide was also examined. Only MRP3 could be modulated by trans-hydroxycotinine- O-glucuronide. These results suggest that the nicotine glucuronide metabolites are not substrates of MRP1 and MRP2, while trans-hydroxycotinine- O-glucuronide may be a MRP3 substrate. Finally, our results suggest that none of MRP1 nsSNPs are likely to affect function of MRP1, ATP hydrolysis and transport activity are at least in part, uncoupled, and that knowledge from MRP1 can be extended to MRP2 and MRP3 with caution.