Investigation of interactions between ABC transporters and their lipid environment - Effect of membrane cholesterol content on the function of human ABCG2 (BCRP/MXR)

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2. ABCG2 (also referred to as breast cancer resistance protein and MXR) is one of the most important efflux transporters in endothelial and epithelial cells, modulating absorption distribution metabolism excretion properties of drugs and other xenobiotics (for review, see Mao and Unadkat, 2005). ABCG2 exhibits a broad substrate specificity because it transports hydrophobic, anionic, and cationic drugs (Mao and Unadkat, 2005). Therefore, it is widely believed that ABCG2 plays an important role in intestinal absorption (Polli et al., 2004) and secretion of xenobiotics and metabolites (Ebert et al., 2005), secretion of sulfate conjugates in the liver (Zamek-Gliszczynski et al., 2006), and prevention of penetration of drugs into the brain (Breedveld et al., 2005). ABCG2 may play a pivotal role in placenta as a defensive barrier to drugs as well as reducing steroid levels in fetal tissues (Jonker et al., 2000). ABCG2 knockout mice models shed light on some special functions of ABCG2, such as secretion of xenobiotics into the milk (Jonker et al., 2005) and protection of stem cells from hypoxia-induced protoporphyrin accumulation and damage (Krishnamurthy and Schuetz, 2005). ABCG2 function is commonly studied using high-throughput cellular (Robey et al., 2004) or membrane-based assays (Ozvegy et al., 2001; Janvilisri et al., 2003) (for review, see Glavinas et al., 2004; http://www.solvo.com/). For most applications, the transporter is expressed in insect cell lines (e.g., Sf9), taking advantage of the robust baculovirus insect cell This work was supported by Hungarian government Grants INFRA GVOP3.3.2.-2004-04–0001/3.0, ADME GVOP-3.1.1.-2004-05-0506/3.0, and Asboth XTTPSRT1 and by European Grants EU-FP6 Biosim 005137 and EU FP6 Memtrans 518246. The authors do not have any financial or personal relationships to disclose that could potentially be perceived as influencing the described research. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.119289. ABBREVIATIONS: MXR, mitoxantrone resistance-associated protein; Sf9, Spodoptera frugiperda ovarian; WT, wild type; RAMEB, randomly methylated -cyclodextrin; cholesterol@RAMEB, cholesterol complex of RAMEB. 0022-3565/07/3213-1085–1094$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 321, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 119289/3206609 JPET 321:1085–1094, 2007 Printed in U.S.A.