Multidrug resistance protein 1 is not associated to detergent-resistant membranes.

Multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette superfamily. Using the energy provided by ATP hydrolysis, it transports a broad spectrum of substrates across the plasma membrane, including hormones, leukotriene C(4), bile salts, and anti-cancer drugs. Recent works have suggested that P-glycoprotein is associated to cholesterol and sphingolipid-rich membrane microdomains and that cholesterol upregulates its ATPase and drug transport activities. Confocal microscopy experiments and Triton X-100 extraction of detergent-resistant membranes provide evidence that MRP1 is not located in raft-like structures and that its activity is downregulated by cholesterol. The data are discussed in terms of cholesterol-protein interaction and topology.

Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis.

MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>>>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.

Mistargeted MRPdeltaF728 mutant is rescued by intracellular GSH.

The most common cystic fibrosis-causing mutation is the deletion of the widely conserved phenylalanine 508 (DeltaF508) of CFTR. The mutant is unable to fold correctly and to transit to the plasma membrane. MRP1 belongs to the same subfamily of ABC proteins as CFTR and confers resistance to a wide range of chemotherapeutic drugs. By analogy, phenylalanine 728 was deleted in MRP1. Our results shown that MRPDeltaF728 is correctly targeted to the plasma membrane, actively transports doxorubicin (DOX) and vincristine (VCR) and shares a structure identical to MRP1. Intracellular GSH depletion however results in a mistargeted mutant that is retained into the cytoplasm, while in the same conditions wild-type MRP1 is correctly routed to the plasma membrane. The GSH-protein complex could adopt a stable conformation protected against proteolytic degradation and correctly targeted to the plasma membrane.

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione.

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.