Multichannel liquid chromatography-tandem mass spectrometry cocktail method for comprehensive substrate characterization of multidrug resistance-associated protein 4 transporter.
OBJECTIVE
To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4).


METHODS
Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography-tandem mass spectrometry.


RESULTS
From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4.


CONCLUSIONS
The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.
Uchida
Yasuo
Y
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
Kamiie
Junichi
J
Ohtsuki
Sumio
S
Terasaki
Tetsuya
T
D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002513:Cephamycins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009997:Osmotic Pressure; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013678:Technology, Pharmaceutical / Q000379:methods*; D014162:Transfection
D002513:Cephamycins; D008727:Methotrexate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D004364:Pharmaceutical Preparations; C073492:ABCC4 protein, human; C046079:cefminox