Chip-Based and Single Protein Transport Assays of Multidrug Resistance Transporte

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

DESCRIPTION (provided by applicant): Multidrug resistance (MDR)-associated transporters contribute to the persistence of many types of cancers during chemotherapy. Three decades of research to inhibit MDR-associated transporters have, so far, produced only limited benefits for cancer treatment. This proposal aims to develop functional assays that will make it possible to characterize better, and hence target effectively, MDR-associated transporters from cancer cells. This research will proceed in three steps. First, it will develop chip-based assay platforms for functional biophysical characterization of multidrug resistance-associated transporters. These assays will be combined with electrophysiology and single molecule fluorescence detection. Second, it will investigate, under well-controlled conditions, the effect of parameters that affect the transport rate, such as the transmembrane potential, the lipid composition, the concentration of ATP and co-transported molecules (e.g. glutathione) to understand better the function of MDR-associated transporters and possible ways to modulate this function. And third, it will develop assays that can monitor the transport rate of individual reconstituted MDR-associated transport proteins in planar lipid bilayers by taking advantage of extremely small volumes (picoliters) in microfabricated recording chips and by combining electrophysiology with single molecule optical detection. We will use these assays to characterize MDR-associated transporters on the single-protein-level in analogy to single ion channel patch clamp recordings. Finally we aim to investigate the activation state and the effect of activation (e.g. by phosphorylation) on the single transporter efflux rates.