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Updated: Sep 20, 2026
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Published on: December 3, 2010
Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)
Chantarawan Saengkhae1, Chatchanok Loetchutinat, Arlette Garnier-Suillerot
1Laboratoire de Physicochimie Biomoléculaire et Cellulaire, Université Paris Nord, Bobigny, France.
Abstract:
Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.
Insights
This study quantifies rhodamine dye transport by multidrug resistance-associated protein 1 (MRP1), revealing glutathione-dependent kinetics. These findings provide the first quantitative data on rhodamine efflux mediated by MRP1.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) is often mediated by efflux pumps like P-glycoprotein and MRP1.
- Rhodamine 123 (Rh123) is used as an assay for MDR, with suggestions it's an MRP1 substrate.
- Quantitative studies on MRP1-mediated rhodamine efflux were lacking.
Purpose of the Study:
- To quantitatively characterize the MRP1-mediated efflux of four rhodamine dyes.
- To determine the kinetic parameters of rhodamine transport by MRP1.
- To compare rhodamine efflux kinetics with other MRP1 substrates.
Main Methods:
- Utilized a continuous fluorescence assay with four rhodamine analogs (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, tetramethylrhodamine methyl ester).
- Quantified drug transport by MRP1 in living GLC4/ADR cells.
- Measured substrate concentration gradients and glutathione dependence.
Main Results:
- Demonstrated MRP1-mediated transport of rhodamine dyes.
- Observed glutathione-dependent transport of rhodamine by MRP1.
- Calculated kinetic parameters (k(a)) for rhodamine analogs, finding them similar to each other but ~10-fold lower than for anthracyclines.
Conclusions:
- Provided the first quantitative kinetic parameters for MRP1-mediated efflux of rhodamine dyes.
- Established that rhodamine transport by MRP1 is a glutathione-dependent process.
- Highlighted differences in kinetic efficiency between rhodamine dyes and anthracyclines for MRP1.
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