Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.     

The contribution of peroxynitrite generation in HIV replication in human primary macrophages

Background

The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability.

Results

The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells.

Conclusion

To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.     

New insights into the P-glycoprotein-mediated effluxes of rhodamines.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.     

Different roles of TM5, TM6, and ECL3 in the oligomerization and function of human ABCG2

ABCG2 is a member of the ATP-binding cassette transporter superfamily, and its overexpression causes multidrug resistance (MDR) in cancer chemotherapy. ABCG2 may also protect cancer stem cells by extruding cytotoxic materials. ABCG2 has previously been shown to exist as a high-order homo-oligomer consisting of possibly 8-12 subunits, and the oligomerization domain was mapped to the C-terminal domain, including TM5, ECL3, and TM6. In this study, we further investigate this domain in detail for the role of each segment in the oligomerization and drug transport function of ABCG2 using domain swapping and site-directed mutagenesis. We found that none of the three segments (TM5, TM6, and ECL3) is essential for the oligomerization activity of ABCG2 and that any one of these three segments in the full-length context is sufficient to support ABCG2 oligomerization. While TM5 plays an important role in the drug transport function of ABCG2, TM6 and ECL3 are replaceable. Thus, each segment in the TM5-ECL3-TM6 domain plays a distinctive role in the oligomerization and function of ABCG2.     

Caspase-dependent cleavage of 170-kDa P-glycoprotein during apoptosis of human T-lymphoblastoid CEM cells

Multidrug resistance (MDR) mediated by the drug efflux protein, 170-kDa P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape cell death induced by chemotherapeutic drugs. Moreover, evidence suggests that cell lines expressing high levels of 170-kDa P-gp are less sensitive to caspase-mediated apoptosis induced by a wide range of death stimuli, including Fas ligand, tumor necrosis factor, and ultraviolet irradiation. However, the fate of 170-kDa P-gp during apoptosis is unknown. In this study, we demonstrate for the first time that 170-kDa P-gp is cleaved during apoptosis of VBL100 human T-lymphoblastoid CEM cells. Apoptotic cell death was induced by LY294002 (a pharmacological inhibitor of the phosphoinositide 3-kinase/Akt survival pathway), H2O2, and Z-LEHD-FMK (a caspase-9 inhibitor which has been recently reported to induce apoptosis in CEM cells). Using an antibody to a common epitope present in both the third and the sixth extracellular loop of P-gp, two cleavage products were detected, with an apparent molecular weight of 80 and 85 kDa. DEVD-FMK (a caspase-3 inhibitor), but not VEID-CHO (a caspase-6 inhibitor), blocked 170-kDa P-gp cleavage. Recombinant caspase-3 was able to cleave in vitro 170-kDa P-gp yielding two fragments of equal size to those generated in vivo. Considering the size of the cleaved fragments and their reactivity with antibodies, which recognize either the N-half or the C-half region of the protein, it is conceivable that the cleavage occurs intracytoplasmically. Since 170-kDa P-gp has been reported to counteract apoptosis, its cleavage may be a mechanism aimed at blocking an important cell survival component.     

Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151

Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186-217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology.     

Histone deacetylase inhibitor apicidin-mediated drug resistance: involvement of P-glycoprotein

Multidrug resistance (MDR), which is a significant impediment to the success of cancer chemotherapy, is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein (P-gp), resulting in an increased drug efflux. In this study, we show that the histone deacetylase (HDAC) inhibitor apicidin leads to resistance of HeLa cells to paclitaxel through the induction of P-gp expression. Furthermore, apicidin dramatically increases the release of a fluorescent P-gp substrate, rhodamine 123, from cells. In parallel, apicidin resistance to the apoptotic potential of paclitaxel is associated with induction of P-gp expression in HeLa cells, as evidenced by specific inhibition of P-gp function using either the pharmacological inhibitor verapamil or RNA silencing. We also demonstrate the contribution of apicidin-induced functional P-gp expression to drug resistance using KB cells. Failure of P-gp induction by apicidin does not reverse paclitaxel-induced cytotoxicity in the cells. Although HDAC inhibitors are widely appreciated as a new class of anti-tumor agent, our findings clearly demonstrate that apicidin treatment may lead to P-gp-mediated resistance to other anti-tumor agents, suggesting a need for careful design of clinical applications using HDAC inhibitors.     

Cholesterol interaction with the daunorubicin binding site of P-glycoprotein

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp.     

Genes of multidrug resistance in haematological malignancies

Since the early 1970s, multiple drug resistance has been known to exist in cancer cells and is thought to be attributable to a membrane-bound, energy-dependent pump protein (P-glycoprotein [P-gp]) capable of extruding various related and unrelated chemotherapeutic drugs. The development of refractory disease in haematological malignancies is frequently associated with the expression of one or several multidrug resistance (MDR) genes. MDR1, multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP) have been identified as important adverse prognostic factors. Recently it has become possible to reverse clinical MDR by blocking P-gp-mediated drug efflux. The potential relevance of these reversal agents of MDR as well as the potential new approaches to treat the refractory disease are discussed in this article. In addition, an array of different molecules and mechanisms by which resistant cells can escape the cytotoxic effect of anticancer drugs has now been identified. These molecules and mechanisms include apoptosis-related proteins and drug inactivation enzymes. Resistance to chemotherapy is believed to cause treatment failure in more than 50% patients. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanism of drug resistance in haematological malignancies with emphasis on molecules involved in MDR. In addition, it brings the survey of methods involved in determination of MDR, in particular P-gp/MDR1, MRP and LRP.     

MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [alpha-(32)P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with one MDR3 null allele).     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding.     

The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment

The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.     

Designed P-glycoprotein inhibitors with triazol-tetrahydroisoquinoline-core increase doxorubicin-induced mortality in multidrug resistant K562/A02 cells

Multidrug resistance (MDR) refers to the cross-resistance of cancer cells to one drug, accompanied by other drugs with different mechanisms and structures, which is one of the main obstacles of clinical chemotherapy. Overexpression of P-glycoprotein (P-gp) was an extensively studied cause of MDR. Therefore, inhibiting P-gp have become an important strategy to reverse MDR. In this study, two series of triazole-tetrahydroisoquinoline-core P-gp inhibitors were designed and synthesized. Among them, compound I-5 had a remarkable reversal activity of MDR activity and the preliminary mechanism study was also carried out. All the results proved that compound I-5 was considered as a promising P-gp-mediated MDR reversal candidate.     

P-glycoprotein preferentially effluxes anthracyclines containing free basic versus charged amine.

The multidrug resistant (MDR) tumor phenotype, characterized by a decreased cellular drug accumulation is achieved by ATP-dependent extrusions of drugs from cells by P-glycoprotein (P-gp) and/or by multidrug resistance protein (MRP1). Despite the huge amount of research that has been performed on the mechanisms of P-gp-mediated efflux of drug, it is not yet known what the molecular parameters are required for a molecule to be recognized and pumped out by P-gp. Anthracyclines are weak bases and, depending on the pH, can exist either in the neutral or in the positively charged form. The aim of the work reported here was to determine which molecular form is actively pumped out by P-gp (the neutral form, the protonated form, or both), and if both, the relative efficiencies of pumping. We used spectrofluorometric methods to determine the efflux of anthracyclines in K562/Adr cells, at different intracellular and extracellular pH levels. Using 3'-deamino, 3'-hydroxyl doxorubicin (OH-DOX), which is permanently neutral, we first verified that our methodologies were accurate and that the P-gp-mediated efflux of OH-DOX would not depend on the pH being in the range 6.6--8.4. The P-gp-mediated efflux of daunorubicin (DNR) and 3'-hydroxy-4-amino (WP608) was determined at different pH values. These two drugs were chosen because: (a) the lipophilicity of the neutral forms of these two molecules is so similar that any difference in the P-gp-mediated efflux cannot be assigned to lipohilicity variation, and (b) their pKa values are different (8.4 and 7.7 for DNR and WP608, respectively), which makes it easy to obtain a large variation in the proportions of the neutral and positively charged forms. Our data show that both forms are recognized by P-gp but the neutral form is pumped about three times more efficiently than the charged form. This is corroborated by results showing the active efflux (checked at pH(i) 7.3 only) of five other anthracycline containing a basic center. We interpret these data to mean that: (a) the positive charge of anthracycline is not a necessary requirement for P-gp recognition, but that (b) the presence of a protonable basic nitrogen facilitates the processing of these compounds by MDR efflux system.     

Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.     

Loop diuretic and ion-binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition.     

(+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone, a derivative of (+/-)-praeruptorin A, reverses P-glycoprotein mediated multidrug resistance in cancer cells

P-glycoprotein (Pgp) is an ATP-driven membrane exporter for a broad spectrum of hydrophobic xenobiotics. Pgp-overexpression is a common cause of multidrug resistance (MDR) in cancer cells and could lead to chemotherapeutic failure. Through an extensive herbal drug screening program we previously showed that (+/-)-praeruptorin A (PA), a naturally existing pyranocumarin isolated from the dried root of Peucedanum praeruptorum Dunn., re-sensitizes Pgp-mediated MDR (Pgp-MDR) cancer cells to cancer drugs. A number of PA derivatives were synthesized and one of these, (+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone (DCK), was more potent than PA or verapamil in the reversal of Pgp-MDR. In Pgp-MDR cells DCK increased cellular accumulation of doxorubicin without affecting the expression level of Pgp. In Pgp-enriched membrane fractions DCK moderately stimulated basal Pgp-ATPase activity, suggesting some transport substrate-like function. However, DCK also inhibited Pgp-ATPase activity stimulated by the standard substrates verapamil or progesterone with decreased V(max)s but K(m)s were relatively unchanged, suggesting a primarily non-competitive mode of inhibition. While the binding of substrates to active Pgp would increase the reactivity of the Pgp-specific antibody UIC2, DCK decreased UIC2 reactivity. These results suggest that DCK could bind simultaneously with substrates to Pgp but perhaps at an allosteric site and thus affect Pgp-substrate interactions.     

Studies of Human MDR1-MDR2 Chimeras Demonstrate the Functional Exchangeability of a Major Transmembrane Segment of the Multidrug Transporter and Phosphatidylcholine Flippase

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 and MDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).