Analysis of ABCG2 pharmacology and oligomerisation by solution based fluorescence correlation spectroscopy

ABCG2 is a human ATP-binding cassette (ABC) transporter. It has a broad substrate range that includes a number of small molecule drugs as well as endogenous metabolites. With its expression in barrier tissues (e.g. kidney epithelium) ABCG2 has a strong influence on pharmacokinetics in addition to other physiological roles (e.g. urate excretion). ABCG2 is also expressed in several tumours and confers resistance to various chemotherapy agents. The detailed interactions and mechanisms that support such polyspecificity are not well-established and progress in understanding ABCG2 pharmacology has been limited. The aim of this project was to develop novel, solution-based analysis methods to investigate ABCG2 pharmacology. Styrene-maleic acid copolymer lipid particles (SMALPs) were used to isolate ABCG2 in nanodiscs from a mammalian expression system (HEK293T). SMALP-ABCG2 particles were then characterised by fluorescence correlation spectroscopy (FCS) to investigate the stoichiometry of ABCG2 complexes and the pharmacology of their substrate binding. ABCG2 tagged with GFP or SNAP-tag was expressed in HEK293T cells. The appropriate plasma membrane localisation was demonstrated by confocal microscopy and the activity of the fusion proteins were validated using mitoxantrone efflux assays. ABCG2 fusion proteins were extracted from HEK293T cell membrane preparations using SMALPs. This process yielded solubilised ABCG2 that was analysed by dynamic light scattering and FCS. These analysis confirmed ABCG2 was extracted into single SMALP particles of 10-20nm diameter with