Drug Transport Mediated by ABC Transporters Using Eukaryotic Membrane Vesicles

Drug Transport Mediated by ABC Transporters Using Eukaryotic Membrane Vesicles 

Efflux transporters are often accounted for by the transport of endogenous compounds and pharmaceutical drugs out of the body into bile, urine or gut in order to protect the cell. To study drug efflux, membrane preparations are necessary. Eukaryotic cell lines, tissues as well as insect cells can be used for this application. 

Preparation of Plasma Membrane Vesicles from Eukaryotic Cell Lines 

Selection of the cell line or tissue type influences not only the technique for membrane vesicle preparation but also the resulting percentage of inside-out-oriented plasma membrane vesicles. A sufficient amount of inside-out-oriented vesicles is essential, since only this fraction, with the ATP-binding domains oriented to the outer surface, mediates ATP-dependent transport of a labeled substrate into the vesicle. 

Using LTC4 as substrate, nitrocellulose filters (0.2 pm pore size, 25-mm diameter) are soaked in incubation buffer (0.25 mM sucrose, 10 mM Tris-HCl, pH 7.4). A rapid filtration apparatus from Millipore (Bedford, MA) is prepared. A final volume of 110 pi of transport assay mixture (4 mM ATP (potassium salt), 10 mM creatine phosphate (Tris salt), 10 mM MgCl2, 10 mM Tris/HCl (pH 7.4), 5 mM glutathione (reduced), 0.25 mM sucrose, 100 pg/ml creatine phosphokinase (2 units/110 pi), 50 nM 3H-LTC4 (50 nCi/110 pi) pH 7.4) is preincubated at 37 °C for 1 min. Blanks are prepared by replacing ATP by 5 -AMP. 

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