School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK.
Biochim Biophys Acta Biomembr. 2020 Jun 1;1862(6):183218. doi: 10.1016/j.bbamem.2020.183218. Epub 2020 Feb 11.
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 是人类三磷酸腺苷结合盒转运蛋白之一，具有将多种化学底物从细胞内结合并转运出细胞的能力。这种所谓的“多药”转运具有许多生理后果，包括对药物如何被吸收和从体内消除的影响。了解 ABCG2 如何与多种药物底物相互作用仍然是转运蛋白生物学中的一个重要目标。大多数药物被认为通过疏水性脂质双层与 ABCG2 相互作用，因此 ABCG2 的研究实验系统需要包含这一点。我们利用苯乙烯马来酸将过表达转运蛋白的 HEK293T 细胞中的 ABCG2 溶解，并通过动态光散射和荧光相关光谱（FCS）证实，这导致提取出均一大小的 SMA 脂质共聚物（SMALP）颗粒，其中包含 ABCG2 的二聚体，这是主要的生理状态。FCS 进一步用于测量荧光 ABCG2 底物（BODIPY-prazosin）在存在和不存在纯化的 ABCG2 的 SMALP 颗粒时的扩散。FCS 迹线的自相关分析能够将游离的 BODIPY-prazosin 与与 ABCG2 结合的药物进行数学分离，并使我们能够表明，将 SMALP 提取与 FCS 相结合可用于研究特定的药物：转运蛋白相互作用。
