Multidrug Resistance Protein MRP1 Research Articles

Theoretical Insights into the Cotransport Mechanism of GSH with Anticancer Drugs by MRP1.

The multidrug resistance protein MRP1 is an ATP binding cassette (ABC) transporter that confers resistance to many anticancer drugs and regulates redox homeostasis, inflammation, and hormone secretion. MRP1 actively transports compounds across cell membranes, and the presence of glutathione (GSH) is required in many cases. However, the process of MRP1-mediated substrate transportation has been poorly understood. With extensive molecular dynamics simulations, we have found a sandwich-like structure which is generated by GSH, a transmembrane α-helices 11 (TM11)-TM17 axis, and anticancer drugs. This structure is crucial in MRP1 transportation. It triggers the motion of TM11 and TM17, followed by the movement of nucleotide-binding domains 1 (NBD1) and 2 (NBD2), and finally an occluded structure is formed. Trp1246, Lys332, and Phe594 were identified as the main contributors in the formation of the sandwich-like structure. Our findings clearly explain the synergy of GSH with an anticancer drug in MRP1 transportation and have significant meanings for the rational design of novel inhibitors against MRP1.

ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1

The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellularsubstrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.

Graphene Quantum Dots Downregulate Multiple Multidrug‐Resistant Genes via Interacting with Their C‐Rich Promoters

Multidrug resistance (MDR) is the major factor in the failure of many forms of chemotherapy, mostly due to the increased efflux of anticancer drugs that mediated by ATP-binding cassette (ABC) transporters. Therefore, inhibiting ABC transporters is one of effective methods of overcoming MDR. However, high enrichment of ABC transporters in cells and their broad substrate spectra made to circumvent MDR are almost insurmountable by a single specific ABC transporter inhibitor. Here, this study demonstrates that graphene quantum dots (GQDs) could downregulate the expressions of P-glycoprotein, multidrug resistance protein MRP1, and breast cancer resistance protein genes via interacting with C-rich regions of their promoters. This is the first example that a single reagent could suppress multiple MDR genes, suggesting that it will be possible to target multiple ABC transporters simultaneously with a single reagent. The inhibitory ability of the GQDs to these drug-resistant genes is validated further by reversing the doxorubicin resistance of MCF-7/ADR cells. Notably, GQDs have superb chemical and physical properties, unique structure, low toxicity, and high biocompatibility; hence, their capability of inhibiting multiple drug-resistant genes holds great potential in cancer therapy.

Structural Basis of Substrate Recognition by the Multidrug Resistance Protein MRP1

The multidrug resistance protein MRP1 is an ATP-binding cassette (ABC) transporter that confers resistance to many anticancer drugs and plays a role in the disposition and efficacy of several opiates, antidepressants, statins, and antibiotics. In addition, MRP1 regulates redox homeostasis, inflammation, and hormone secretion. Using electron cryomicroscopy, we determined the molecular structures of bovine MRP1 in two conformations: an apo form at 3.5Å without any added substrate and a complex form at 3.3Å with one of its physiological substrates,leukotriene C4. These structures show that by forming a single bipartite binding site, MRP1 can recognize a spectrum of substrates with different chemical structures. We also observed large conformational changes induced by leukotriene C4, explaining how substrate binding primes the transporter for ATP hydrolysis. Structural comparison of MRP1 and P-glycoprotein advances our understanding of the common and unique properties ofthese two important molecules in multidrug resistance to chemotherapy.

Stem Cell-like Characteristics of MM Plasma Cells Vary By ROS Levels: Implications for Targeted Therapy

Background: Tumor cells exhibit elevated ROS levels and cancer stem cells have lower ROS levels than their normal counterparts [Kobayashi et al., 2011]. However, little is known about the effect of ROS levels on multiple myeloma (MM) plasma cell (PC) chemosensitivity. Previously we found that ROS levels are variable in CD138-selected MMPCs from different cases at diagnosis. However, ROS levels in MMPCs from focal lesions (FL, n=42) are lower than PCs from random bone marrow (BM) aspirates (n=192, p=0.02). ROS levels also vary among MM molecular subgroups, with MF and CD2 subgroups having low ROS levels, suggesting that the microenvironment and genetic factors affect their oxidative capacities. We hypothesized that ROS level may reflect the cancer stemness properties of MM cells. We first sought to establish a model to investigate MM cells with different ROS levels and then determine if this property confers cancer stemness and chemoresistance.Methods: We analyzed gene expression profiles (GEP) of primary myeloma cells from newly diagnosed patients with low (n=48) and high (n=48) ROS level by gene set enrichment analysis. We compared viability, proliferation, cell cycle status, drug efflux ability, chemosensitivity, and clonogenic capacity of MM cell lines H929 and U266 with high and low ROS levels, sorted by FACS using H2DCFDA. We also investigated whether MMPCs with low or high ROS levels would differ in their sensitivity to BBI-608, a drug that is reported to target STAT3 driven gene transcription and cancer stemness properties in solid cancers [Li et al., 2015] and is currently being tested in hematologic cancers [BBI608-103HEME].Results: Pathways enrichment analysis revealed that primary MM cases with low ROS had increased oxidative phosphorylation, proteasome activity, and nucleotide excision repair compared to cases with high ROS that had increased glycolysis, and chemokine and calcium signaling pathways. To explore these differences we separated MMCLs into cells not sorted by ROS level (non-ROS sorted), ROSLo, and ROSHi groups. After sorting, H929 cells retained significant differences in ROS levels for 48-72 hr and U266 cells returned to basal ROS levels within 24hr. ROSLo H929 cells had significantly reduced cell number (p<0.0001) compared to non-ROS sorted and ROSHi cells. Similarly, ROSLo cells had reduced and ROSHi cells had significantly increased proliferation index compared to non-ROS sorted cells (H929-p<0.05, U266-p<0.001). ROSLo cells also have a significantly larger percentage of cells in S phase (p <0.01) and reduced percentage in G2 (p< 0.001) compared to non-ROS sorted cells. While ROSHi cells (but not ROSLo cells) were less sensitive to melphalan compared to non-ROS sorted cells, there was no difference in sensitivity to bortezomib. Examination of ABC transporter efflux activity revealed that ROSHi cells had increased drug efflux capacity via the multidrug resistance protein MRP1 (p<0.01). ROSLo cells showed greater colony forming capacity, however after serial replating, ROSHi cells increase colony forming capacity after each passage eventually matching that of ROSLo cells. Interestingly, if clonogenic cells are re-sorted into ROSLo and ROSHi groups between passages, ROSLo cells retain superior colony forming capacity compared to ROSHi cells. ROSLo (IC50-0.656μM) and ROSHi (IC50-0.56μM) cells had significantly increased sensitivity to BBI-608 compared to non-ROS-sorted cells (IC50-0.991 μM). Cell number was significantly reduced in all 3 groups without significantly affecting viability. The STAT3 target gene, CCND1 is significantly elevated in ROSHi cells and effectively reduced by BBI-608. After low dose BBI-608 (0.6μM) treatment, the remaining cells were subjected to colony forming assays. While BBI-608 reduced colony forming capacity to some extent in all 3 groups, ROSHi cells were most sensitive.Conclusions: Characterization of MM cells with variable ROS levels revealed that ROSHi cells have greater drug efflux capacity and are more proliferative and chemoresistant, whereas ROSLo cells have reduced proliferation and increased clonogenic capacity. ROSLo MMPCs may represent a subpopulation of tumor cells with stemness properties that are enriched in the hypoxic BM niche and in MM focal lesions. DisclosuresMalaviarachchi:University of Arkansas for Medical Sciences: Employment. Turner:University of Arkansas for Medical Sciences: Employment. Stein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; University of Arkansas for Medical Sciences: Employment; CancerNet: Honoraria; Weismann Institute: Honoraria. Epstein:University of Arkansas for Medical Sciences: Employment. Yaccoby:University of Arkansas for Medical Sciences: Employment. Johnson:University of Arkansas for Medical Sciences: Employment.

Regulation of expression and activity of multidrug resistance proteins MRP2 and MDR1 by estrogenic compounds in Caco-2 cells. Role in prevention of xenobiotic-induced cytotoxicity

ABC transporters including MRP2, MDR1 and BCRP play a major role in tissue defense. Epidemiological and experimental studies suggest a cytoprotective role of estrogens in intestine, though the mechanism remains poorly understood. We evaluated whether pharmacologic concentrations of ethynylestradiol (EE, 0.05pM to 5nM), or concentrations of genistein (GNT) associated with soy ingestion (0.1–10μM), affect the expression and activity of multidrug resistance proteins MRP2, MDR1 and BCRP using Caco-2 cells, an in vitro model of intestinal epithelium. We found that incubation with 5pM EE and 1μM GNT for 48h increased expression and activity of both MRP2 and MDR1. Estrogens did not affect expression of BCRP protein at any concentration studied. Irrespective of the estrogen tested, up-regulation of MDR1 and MRP2 protein was accompanied by increased levels of MDR1 mRNA, whereas MRP2 mRNA remained unchanged. Cytotoxicity assays demonstrated association of MRP2 and MDR1 up-regulation with increased resistance to cell death induced by 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor, and by paraquat, an MDR1 substrate. Experiments using an estrogen receptor (ER) antagonist implicate ER participation in MRP2 and MDR1 regulation. GNT but not EE increased the expression of ERβ, the most abundant form in human intestine and in Caco-2 cells, which could lead in turn to increased sensitivity to estrogens. We conclude that specific concentrations of estrogens can confer resistance against cytotoxicity in Caco-2 cells, due in part to positive modulation of ABC transporters involved in extrusion of their toxic substrates. Although extrapolation of these results to the in vivo situation must be cautiously done, the data could explain tentatively the cytoprotective role of estrogens against chemical injury in intestine.

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10–100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3±1.11% and 68.8±1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5–10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.

Drug Metabolism and Pharmacokinetics of Organosulfur Compounds from Garlic

The medicinal activities of garlic organosulfur compounds identified include antitumor, antimicrobial, antifungal, antivirus, anti-atherosclerosis, blood lipids and sugar lowering, antithrombotic, anti-hypotension and immune modulation effects. These multi-targeted new agents may be especially promising since recently developed highly specific anti-cancer agents as well as other disease treatment drugs have failed to live up to the expectations. Despite more than one thousand articles have been published on garlic organosulfur compounds, the drug metabolism and pharmacokinetics of these compounds behind their health-promoting effects are still poorly understood. In this review, we will focus on metabolic pathways and pharmacokinetics of organosulfur compounds from garlic, which is intended to fill the void on the important aspect for further nutraceutical and pharmaceutical product development of this group of compounds. The effects of these organosulfur compounds on various cytochrome P450 enzymes as well as on P-glycoprotein (P-gp) and multidrug resistance proteins (Mrp1 and Mrp 2) will also be discussed.

In Vitro and In Vivo Assessment of Renal Drug Transporters in the Disposition of Mesna and Dimesna

Mesna and its dimer, dimesna, are coadministered for mitigation of ifosfamide- and cisplatin-induced toxicities, respectively. Dimesna is selectively reduced to mesna in the kidney, producing its protective effects. In vitro screens of uptake and efflux transporters revealed saturable uptake by renal organic anion transporters OAT1, OAT3, and OAT4. Efflux transporters breast cancer resistance protein; multidrug and toxin extrusion 1 (MATE1); multidrug resistance proteins MRP1, MRP2, MRP4, and MRP5; and P-glycoprotein (Pgp) significantly reduced dimesna accumulation. Further investigation demonstrated that renal apical efflux transporters MATE1, MRP2, and Pgp were also capable of mesna efflux. Administration of OAT inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure (91% ± 34%) while decreasing the renal clearance due to net secretion (67.0% ± 12.7%) and steady-state volume of distribution (45.2% ± 13.4%). Thus, the kidney represents a significant sink of total mesna, whereas function of renal drug transporters facilitates clearance in excess of glomerular filtration rate and likely the presence of active mesna in the urine. Loss of renal transporter function due to genetic variability or drug-drug interactions may decrease the efficacy of chemoprotectants, increasing the risk of ifosfamide- and cisplatin-induced toxicities.

Folate Decorated Dual Drug Loaded Nanoparticle: Role of Curcumin in Enhancing Therapeutic Potential of Nutlin-3a by Reversing Multidrug Resistance

Retinoblastoma is the most common intraocular tumor in children. Malfunctioning of many signaling pathways regulating cell survival or apoptosis, make the disease more vulnerable. Notably, resistance to chemotherapy mediated by MRP-1, lung-resistance protein (LRP) is the most challenging aspect to treat this disease. Presently, much attention has been given to the recently developed anticancer drug nutlin-3a because of its non-genotoxic nature and potency to activate tumor suppressor protein p53. However, being a substrate of multidrug resistance protein MRP1 and Pgp its application has become limited. Currently, research has step towards reversing Multi drug resistance (MDR) by using curcumin, however its clinical relevance is restricted by plasma instability and poor bioavailability. In the present investigation we tried to encapsulate nutlin-3a and curcumin in PLGA nanoparticle (NPs) surface functionalized with folate to enhance therapeutic potential of nutlin-3a by modulating MDR. We document that curcumin can inhibit the expression of MRP-1 and LRP gene/protein in a concentration dependent manner in Y79 cells. In vitro cellular cytotoxicity, cell cycle analysis and apoptosis studies were done to compare the effectiveness of native drugs (single or combined) and single or dual drug loaded nanoparticles (unconjugated/folate conjugated). The result demonstrated an augmented therapeutic efficacy of targeted dual drug loaded NPs (Fol-Nut-Cur-NPs) over other formulation. Enhanced expression or down regulation of proapoptotic/antiapoptotic proteins respectively and down-regulation of bcl2 and NFκB gene/protein by Fol-Nut-Cur-NPs substantiate the above findings. This is the first investigation exploring the role of curcumin as MDR modulator to enhance the therapeutic potentiality of nutlin-3a, which may opens new direction for targeting cancer with multidrug resistance phenotype.

Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor

Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the Km (Mg·ATP) and Vmax values in Mg·ATP-dependent leukotriene-C4 transport. However, the Vmax values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

The influence of plasma binding on absorption/exsorption in the Caco-2 model of human intestinal absorption.

The Caco-2 cell monolayer has become an increasingly useful in-vitro model of human intestinal absorption. In this study we have determined the effect of plasma on the basolateral side on the absorption as well as exsorption of several drugs that are highly bound to plasma proteins. The drugs used included propranolol and quercetin, which both use the transcellular route of absorption, and taxol and oestradiol 17 beta-D-glucuronide, which are thought to undergo efflux by P-glycoprotein and the multidrug resistance protein MRP, respectively. All experiments were carried out under sink conditions to mimic normal absorption. It was necessary to use heparin anticoagulation for generation of the plasma, as EDTA was found to make the monolayers very leaky. The apparent permeability (P(app)) values for absorption were 1.54 x 10(-6) cm s(-1) for oestradiol 17 beta-D-glucuronide, 3.33 x 10(-6) cm s(-1) for taxol, 20.8 x 10(-6) cm s (-1) for quercetin, and 35.3 x 10(-6) cm s(-1) for propranolol. For these four compounds, plasma on the basolateral side had no influence on absorption. However, plasma on the basolateral side significantly reduced the efflux of oestradiol 17 beta-D-glucuronide by 66%, taxol by 75%, propranolol by 82%, and quercetin by 94%. Failure to consider the effect of plasma binding can result in an overestimate of basolateral to apical efflux and result in misleading net flux calculations.

Magnesium dependent complexation of tri-anionic calix[4]arene detergents by the nucleotide binding domain 1 (NBD1) of multidrug resistance protein MRP1

The tri-carboxylatomethylene-mono-alkoxy calix[4]arenes have been shown by fluorescence spectroscopy to bind to the NBD1 domain of MRP1 protein in a magnesium dependent manner. The observed associations constants are of the same order as that observed for ATP–Mg, the natural substrate for this protein.

Expression of the multidrug resistance protein MRP and the lung-resistance protein LRP in nasal NK/T cell lymphoma: further exploring the role of P53 and WT1 gene

Nasal NK/T-cell lymphoma (NKTL) is relatively common in the adult men of Asia. Many patients with nasal NKTL have poor response to therapy. Some of them show P-glycoprotein over-expression. To investigate the expression of other multidrug resistance proteins (MDR) and possible regulatory factors in nasal NKTL, the clinical and pathological features are described. Thirty Chinese adults with nasal NKTL are presented. Immunohistochemical study was carried out to detect multidrug resistance-associated protein 1 (MRP) and lung resistance-related protein (LRP). The association between possible regulatory proteins (P53 and WT1) and MDR were explored. In situ hybridisation for Epstein-Barr virus (EBV) detection, polymerase chain reaction assay for T-cell receptor gene and direct sequencing for the P53 gene were performed. Seven (23.3%) and eight (26.7%) patients showed immunoreactivity of MRP and LRP, respectively. Positive staining for both markers was identified in 6.7% (2 cases). The EBV was detected in most cases (97%). Twenty-six (86.7%) cases expressed positive nuclear staining of P53. However, of the cases analysed for P53 mutation, none showed a mutaion at the hot spots studied. WT1 protein was not detected in the nasal NKTL. Our study reports expression of MRP and LRP in nasal NKTL. The over-expression of P53 is probably associated with high incidence of EBV infection and unlikely a regulatory protein for the expression of MRP and LRP. Further studies are necessary to validate the association between P53 mutation and expression of MRP and LRP in nasal NKTL.

Metabolism and Renal Elimination of Gaboxadol in Humans: Role of UDP-Glucuronosyltransferases and Transporters

Gaboxadol, a selective extrasynaptic agonist of the delta-containing gamma-aminobutyric acid type A (GABAA) receptor, is excreted in humans into the urine as parent drug and glucuronide conjugate. The goal of this study was to identify the UDP-Glucuronosyltransferase (UGT) enzymes and the transporters involved in the metabolism and active renal secretion of gaboxadol and its metabolite in humans.Methods. The structure of the glucuronide conjugate of gaboxadol in human urine was identified by LC/MS/MS. Human recombinant UGT isoforms were used to identify the enzymes responsible for the glucuronidation of gaboxadol. Transport of gaboxadol and its glucuronide was evaluated using cell lines and membrane vesicles expressing human organic anion transporters hOAT1 and hOAT3, organic cation transporter hOCT2, and the multidrug resistance proteins MRP2 and MRP4.Results. Our study indicated that the gaboxadol-O-glucuronide was the major metabolite excreted in human urine. UGT1A9, and to a lesser extent UGT1A6, UGT1A7 and UGT1A8, catalyzed the O-glucuronidation of gaboxadol in vitro. Gaboxadol was transported by hOAT1, but not by hOCT2, hOAT3, MRP2, and MRP4. Gaboxadol-O-glucuronide was transported by MRP4, but not MRP2.Conlusion. Gaboxadol could be taken up into the kidney by hOAT1 followed by glucuronidation and efflux of the conjugate into urine via MRP4.

Multiple Roles of Charged Amino Acids in Cytoplasmic Loop 7 for Expression and Function of the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

Three C‐terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2

Cardiac ATP-sensitive potassium (K(ATP)) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory--but not inhibitory--transduction processes. These results indicate that, within the K(ATP) channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation.

MRP2 and the DMPS- and DMSA-Mediated Elimination of Mercury in TR− and Control Rats Exposed to Thiol S-Conjugates of Inorganic Mercury

Cysteine (Cys) and homocysteine (Hcy)-S-conjugates of inorganic mercury (Hg2+) are transportable species of Hg2+ that are taken up readily by proximal tubular cells. The metal chelators, 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA), have been used successfully to extract Hg2+ from these cells, presumably via the multidrug resistance protein (Mrp2). In the current study, we tested the hypothesis that Mrp2 is involved in the DMPS- and DMSA-mediated extraction of Hg2+ following administration of Hg2+ as an S-conjugate of Cys or Hcy. To test this hypothesis, control and TR(-) (Mrp2-deficient) rats were injected with 0.5 micromol/kg HgCl2 (containing 203Hg2+) conjugated to 1.25 micromol/kg Cys or Hcy. After 24 and 28 h, rats were treated with saline or 100 mg/kg DMPS or DMSA. Tissues were harvested 48 h after Hg2+ exposure. The renal and hepatic burden of Hg2+ was greater in saline-injected TR- rats than in corresponding controls. Accordingly, the content of Hg2+ in the urine and feces was less in TR- rats than in controls. Following treatment with DMPS or DMSA, the renal content of Hg2+ in both groups of rats was reduced significantly and the urinary excretion of Hg2+ was increased. In liver, the effect of each chelator appeared to be dependent upon the form in which Hg2+ was administered. In vitro experiments provide direct evidence indicating that DMPS and DMSA-S-conjugates of Hg2+ are substrates for Mrp2. Overall, these data support our hypothesis that Mrp2 is involved in the DMPS and DMSA-mediated extraction of the body burden of Hg2+.

Identification of Regions Required for Apical Membrane Localization of Human Multidrug Resistance Protein 2

Multidrug resistance proteins MRP1 and MRP2 transport a wide range of endo- and xenobiotics. However, with the exception of certain parts of the brain, MRP1 traffics to basolateral membranes of polarized cells, whereas MRP2 is apical in location and thus it is particularly important for systemic elimination of such compounds. Different regions of MRP1 and MRP2 seem to target them to their respective membrane locations. In addition to two "core" membrane spanning domains (MSDs) characteristic of ATP-binding cassette transporters, MRP1 and MRP2 have a third NH2-terminal MSD (MSD0), which is not required for basolateral targeting of MRP1, or for transport of at least some substrates. Here, we demonstrate that all elements necessary for apical targeting of MRP2 reside in MSD0 and the adjacent cytoplasmic loop (CL) 3. Furthermore, we show that this region of MRP2 can target the core of MRP1 to an exclusively apical location. Within MRP2 CL3, we identified a lysine-rich element that is essential for apical targeting. When introduced into MRP1, this element alone is sufficient to result in partial apical localization. However, exclusive targeting to the apical membrane seems to require the integrity of the entire region encompassing MSD0 and CL3 of MRP2. Because CL3 of MRP1 is critical for binding, transport, or both of several compounds, we also examined the function of hybrids containing all, or portions of MRP2 MSD0 and CL3. Our results indicate that CL3 is important for interaction with both the glutathione and glucuronide conjugates tested, but that different regions may be involved.