Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein

Several fluorescent probes have been used in functional studies to analyze drug transport in multidrug-resistant cells by fluorescent microscopy. Because many of these molecules have some drawbacks, such as toxicity, nonspecific background, or accumulation in mitochondria, new fluorescent compounds have been proposed as more useful tools. Among these substances, Bodipy-FL-Verapamil, a fluorescent conjugate of the drug efflux blocker verapamil, has been used to study P-glycoprotein activity in different cell types. In this study we tested by fluorescent microscopy the accumulation of Bodipy-FL- Verapamil in cell lines that overexpress either P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1). Expression of P-gp and MRP1 was evaluated at the mRNA level by RT-PCR technique and at the protein level by flow cytometric analysis using C219 and MRP-m6 monoclonal antibodies. Results indicate that Bodipy-FL-Verapamil is actually a substrate for both proteins. As a consequence, any conclusion about P-gp activity obtained by the use of Bodipy-FL-Verapamil as fluorescent tracer should be interpreted with caution.

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Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Journal of Cellular Physiology ◽

10.1002/jcp.27402 ◽

2018 ◽

Vol 234 (5) ◽

pp. 6611-6623 ◽

Cited By ~ 6

Author(s):

Qi Chen ◽

Xueqiang Liu ◽

Zhesi Luo ◽

Shisi Wang ◽

Jialin Lin ◽

...

Keyword(s):

Multidrug Resistance ◽

Chloride Channel ◽

P Glycoprotein

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Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling

Journal of Cell Science ◽

10.1242/jcs.107.12.3281 ◽

1994 ◽

Vol 107 (12) ◽

pp. 3281-3290

Author(s):

A. Sardini ◽

G.M. Mintenig ◽

M.A. Valverde ◽

F.V. Sepulveda ◽

D.R. Gill ◽

...

Keyword(s):

Multidrug Resistance ◽

Chloride Channels ◽

Drug Transport ◽

Atp Hydrolysis ◽

Cell Swelling ◽

Drug Efflux ◽

Fluorescent Substrate ◽

Membrane Stretch ◽

P Glycoprotein ◽

In Cells

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

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Spectroscopic and biophysical approaches for studying the structure and function of the P-glycoprotein multidrug transporter

Biochemistry and Cell Biology ◽

10.1139/o98-075 ◽

1998 ◽

Vol 76 (5) ◽

pp. 695-708 ◽

Cited By ~ 16

Author(s):

Frances J Sharom ◽

Ronghua Liu ◽

Yolanda Romsicki

Keyword(s):

Electron Microscopy ◽

Circular Dichroism ◽

Multidrug Resistance ◽

Fluorescence Spectroscopy ◽

Lipid Bilayers ◽

Drug Transport ◽

Circular Dichroism Spectroscopy ◽

P Glycoprotein ◽

Infra Red ◽

Circular Dichroïsm

Multidrug resistance is a serious obstacle to the successful chemotherapeutic treatment of many human cancers. A major cause of multidrug resistance is the overexpression of a 170-kDa plasma membrane protein, known as P-glycoprotein, which appears to function as an ATP-driven efflux pump with a very broad specificity for hydrophobic drugs, peptides, and natural products. P-Glycoprotein is a member of the ABC superfamily and is proposed to consist of two homologous halves, each comprising six membrane-spanning segments and a cytosolic nucleotide binding domain. In recent years, P-glycoprotein has been purified and functionally reconstituted into lipid bilayers, where it retains both ATPase and drug transport activity. The availability of purified active protein has led to substantial advances in our understanding of the molecular structure and mechanism of action of this unique transporter. This review will focus on the recent application of fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, electron microscopy, and other biophysical techniques to the study of P-glycoprotein structure and function.Key words: multidrug resistance, P-glycoprotein, fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry, electron microscopy.

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Radiopharmaceuticals for Assessment of Multidrug Resistance P-Glycoprotein-Mediated Drug Transport Activity

Bioconjugate Chemistry ◽

10.1021/bc0498469 ◽

2004 ◽

Vol 15 (6) ◽

pp. 1464-1474 ◽

Cited By ~ 42

Author(s):

Vijay Sharma

Keyword(s):

Multidrug Resistance ◽

Drug Transport ◽

Transport Activity ◽

P Glycoprotein

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Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.277-286.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 277-286

Author(s):

M Raymond ◽

S Ruetz ◽

D Y Thomas ◽

P Gros

Keyword(s):

Multidrug Resistance ◽

Mammalian Cells ◽

Drug Transport ◽

Function Analysis ◽

Expression System ◽

Functional Expression ◽

Yeast Cells ◽

Mutant Form ◽

Cellular Resistance ◽

P Glycoprotein

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

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DIFLOXACIN REVERSES MULTIDRUG-RESISTANCE IN P388 ADR CELLS VIA A MECHANISM INDEPENDENT OF P-GLYCOPROTEIN AND WITHOUT CORRECTING DRUG TRANSPORT OR SUBCELLULAR DRUG DISTRIBUTION

International Journal of Oncology ◽

10.3892/ijo.7.3.475 ◽

1995 ◽

Author(s):

S GUPTA ◽

F THADEPALLI ◽

S GOLLAPUDI

Keyword(s):

Multidrug Resistance ◽

Drug Transport ◽

Drug Distribution ◽

P Glycoprotein

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Molecular dissection of the human multidrug resistance P-glycoprotein

Biochemistry and Cell Biology ◽

10.1139/o99-014 ◽

1999 ◽

Vol 77 (1) ◽

pp. 11-23 ◽

Cited By ~ 33

Author(s):

Tip W Loo ◽

David M Clarke

Keyword(s):

Multidrug Resistance ◽

Drug Transport ◽

Metal Chelate ◽

Physiological Role ◽

Clinical Importance ◽

The Body ◽

Cytotoxic Agents ◽

Protein Chemistry ◽

Disulfide Crosslinking ◽

P Glycoprotein

The human multidrug resistance P-glycoprotein is an ATP-dependent drug pump that extrudes a broad range of cytotoxic agents from the cell. Its physiological role may be to protect the body from endogenous and exogenous cytotoxic agents. The protein has clinical importance because it contributes to the phenomenon of multidrug resistance during chemotherapy. In this review, we discuss some of the results obtained by using molecular biology and protein chemistry techniques for studying this important and intriguing protein.Key words: P-glycoprotein, ABC transporters, drug transport, dibromobimane, mutagenesis, disulfide crosslinking, metal-chelate chromatography, ATPase activity.

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