Abstract
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.
Highlights
The ATP binding cassette (ABC) family of membrane transporter proteins couple the hydrolysis of ATP at intracellular nucleotide binding domains (NBDs) to the binding and transport of substrates across the membrane
Three human MDR-type ABC transporters (Pglycoprotein (ABCB1), multidrug resistance associated protein-1 (ABCC1/MRP1) and breast cancer resistance protein (ABCG2/BCRP)) have been the subject of intensive investigation both to understand their contribution to cancer MDR and to understand the protein biochemical mechanisms of multidrug recognition and export [5,6,7]
fluorescence correlation spectroscopy (FCS) data were analysed using autocorrelation (AC) or photon counting histogram (PCH) analysis using Zen 2012 software (Carl Zeiss, Jena, Germany). Autocorrelation curves for both direct measurement of Styrene maleic acid (SMA) lipid copolymer (SMALP) purified ABCG2 and BODIPY-prazosin binding were fitted using a two-component 3D diffusion model incorporating a pre-exponential triplet term to account for fluorophore photophysics, with triplet lifetime constrained to < 10 μs
Summary
The ATP binding cassette (ABC) family of membrane transporter proteins couple the hydrolysis of ATP at intracellular nucleotide binding domains (NBDs) to the binding and transport of substrates across the membrane. Several family members are capable of exporting a wide range of chemically diverse compounds from the cell This unusual polyspecificity underpins roles in cell, tissue and organ level defence [2], but in disease states these polyspecific transporters can underlie the emergence of a treatment refractory state. A wide range of small molecules appear capable of inhibiting ABCG2 such as tyrosine kinase inhibitors, immunosuppressants, HIV protease inhibitors and calcium channel blockers [14,19] These lists, which include scores of pharmaceutically useful drugs, implicate ABCG2 as a major contributor to drug uptake and elimination. As the majority of ABCG2 transport substrates are hydrophobic and are expected to interact via the lipid milieu it is essential that any system for determining pharmacology includes surrounding lipids This limits studies using detergent solubilised protein as this would remove all but the most tightly associated lipids. The combination of SMALP purification and FCS analysis will enable future solution-based pharmacology of ABCG2 and other membrane proteins
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