6.1.1. Membrane Vesicles

For the study of substrate transport and inhibition by drugs and metabolites, membrane vesicles represent the most commonly used model system. Vesicles are either prepared from BSEP-transfected insect cell lines (Sf9 and Sf21), which give higher protein yields, or mammalian cell lines (including CHO, HeLa, MDCK, LLC-PK1 and HEK cells). Despite having lower protein expression, mammalian cells are often preferred for functional studies, as insect cells show a different lipid membrane composition and only core glycosylated protein is produced. In order to overcome lower expression levels in mammalian cells, the Bac/Mam gene transfer system has been advocated [72]. For experimental details on the preparation of membrane vesicles, readers are referred to [13,69,73]. As a mixture of inside-out and right-side-out vesicles is obtained, a protocol for increasing the yield of inside-out vesicles has been published [74]. In addition, a protocol for preparation of membrane vesicles from canalicular membranes of rat hepatocytes has been

reported [73]. These vesicles were used for the identification and characterization of BSEP substrates and inhibitors [75,76].

High-quality membrane vesicles represent an ideal experimental system for transport and transport inhibition studies, but cannot be used to address aspects of trafficking or cellular metabolism [77]. As in inside-out vesicles, the BSEP NBDs are exposed towards the medium, ATP and substrates can be added into the transport medium and accumulation of substrates in vesicles can be monitored by rapid filtration. We previously used membrane vesicles from plasmid-transfected HEK cells to study the domain interaction and the roles of the canonical and non-canonical NBS in supporting BSEP substrate transport [67].
