Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2.

ABCG2 is one of a trio of human ATP binding cassette transporters that have the ability to bind and transport a diverse array of chemical substrates out of cells. This so-called "multidrug" transport has numerous physiological consequences including effects on how drugs are absorbed into and eliminated from the body. Understanding how ABCG2 is able to interact with multiple drug substrates remains an important goal in transporter biology. Most drugs are believed to interact with ABCG2 through the hydrophobic lipid bilayer and experimental systems for ABCG2 study need to incorporate this. We have exploited styrene maleic acid to solubilise ABCG2 from HEK293T cells overexpressing the transporter, and confirmed by dynamic light scattering and fluorescence correlation spectroscopy (FCS) that this results in the extraction of SMA lipid copolymer (SMALP) particles that are uniform in size and contain a dimer of ABCG2, which is the predominant physiological state. FCS was further employed to measure the diffusion of a fluorescent ABCG2 substrate (BODIPY-prazosin) in the presence and absence of SMALP particles of purified ABCG2. Autocorrelation analysis of FCS traces enabled the mathematical separation of free BODIPY-prazosin from drug bound to ABCG2 and allowed us to show that combining SMALP extraction with FCS can be used to study specific drug: transporter interactions.

ABCG2: does resolving its structure elucidate the mechanism?

ABCG2 is one of a few human membrane transporters which display the amazing ability to transport multiple different chemicals out of cells. These multidrug pumps, which have orthologues in all organisms, are important in humans in the context of drug pharmacokinetics, especially with respect to resistance to chemotherapy. In 2016, we presented a mini-review on ABCG2 which identified many areas of exciting research progress as well as many areas of frustrating ignorance. Just 2 years on the field has advanced, particularly with respect to structural biology as the cryo-electron microscopy revolution has brought us new insights into the structure and mechanism of ABCG2. In this update, we evaluate the degree to which new data have enhanced our understanding of the structure and mechanism of ABCG2 and whether we are now in a position to translate some of these findings into inhibitor design and development.

The multidrug transporter ABCG2: still more questions than answers.

ABCG2 is one of at least three human ATP binding cassette (ABC) transporters which can facilitate the export from cells of a wide range of chemically unrelated drug molecules. This capacity for multidrug transport is not only a confounding factor in chemotherapy, but is also one of the more perplexing phenomena in transporter biochemistry. Since its discovery in the last decade of the 20th century much has been revealed about ABCG2's localization, physiological function and its broad substrate range. There have also been many investigations of its structure and molecular mechanism. In this mini review article we take a Rumsfeldian approach to ABCG2 and essentially ask what we do know about this transporter, and what we will need to know about this transporter if we wish to use modulation of ABCG2 activity as a therapeutic approach.