title: Substrate translocation and stimulated ATP hydrolysis of human ABC transporter MRP3 show positive cooperativity and are half-coupled.
abstract: ABC transporters are involved in countless processes from lipid excretion over cellular detoxification to multidrug resistance of cancer cells. The latter is especially conferred by the ABCC subfamily also called multidrug resistance-associated proteins (MRPs) that excrete a variety of amphipathics including anticancer drugs by ATP-dependent transport. As the mechanisms of substrate translocation and ATP hydrolysis are still unclear for MRPs, we investigated the kinetics of both processes with focus on cooperativity and coupling between ATPase activity and substrate transport using purified MRP3 in proteoliposomes. Although the ATP-dependent uptake of amphipathics and the hydrophilic 5(6)-carboxy-2'-7'-dichlorofluorescein (CDCF) into the lumen of proteoliposomes showed affinity constants similar to those reported for cell-based assays, the maximal uptake rates were up to 250 times higher. Moreover, all substrates showed cooperative interactions of two subunits. Upon stimulation with amphipathics, ATPase activity of MRP3 increased from 80nmol/(mgmin) to 180nmol/(mgmin) showing positive cooperativity with a Hill coefficient of 2. While Hill coefficient and maximal ATPase activity were found to be substrate independent, the affinity constants are characteristic for a given substrate and correspond to the value for transport. Therefore, cooperative interactions of the two nucleotide binding domains (NBDs) in MRP3 are mediated by substrate binding to the transmembrane domains (TMDs). In contrast to amphipathic substrates, CDCF did not stimulate ATPase activity despite being transported in an ATP-dependent manner. This indicates that ATP hydrolysis and substrate translocation are half-coupled in MRP3 as CDCF shuttles on a basal TMD activity resulting from the basal ATPase activity.
authors.affiliation: Institute for Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Muenster, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Seelheim
authors.forename: Adriane
authors.initials: A
authors.lastname: Wüllner
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Galla
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000255:Adenosine Triphosphate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C114462:multidrug resistance-associated protein 3