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T361:Meszaros:MRP1Sphingolipids

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Improvement of MRP1 activity for early ADME studies

Péter Mészáros, Karin Klappe, Jan Willem Kok

University of Groningen, Department of Cell Biology, A. Deusinglaan 1, 9713AV Groningen, The Netherlands


Introduction
ABC (ATP Binding Casette) transporters are transmembrane proteins, which influence the drugs ADME (Absorption, Distribution, Metabolism, Extretion) properties. ABC proteins can perform a directed transmembrane movement of their substrates, including elimination of certain xenobiotics and make cancer cells multidrug resistant as well. There are several factors which influence the localization and/or activity of ABC proteins. Among these are interactions with other proteins, such as actin and the direct lipid environment, e.g. localization in lipid rafts, which are enriched in sphingolipids (SL) and cholesterol (Kok JW, Meszaros P, et.al. Trends in glycoscience and glycotechnology, 2008).
Aim
With the improvement of MRP1 activity and signal to noise ratio, more efficient membrane vesicles could be made to study drug-transporter interaction for early ADME studies without expensive intervention to the manufacturing process. Our aim is to study the sphingolipid dependence of the Multidrug Resistance Protein 1 (MRP1) activity.
Approach
In addition to intact BHK cells, we used cell-derived membrane vesicles containing MRP. We modified sphingolipid levels with ISP-1 and studied the effect on the specific activity of MRP1. Activity was measured using the fluorescent substrate CFDA for intact cells, vesicular transport assay and ATPase assay for the vesicle system.

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