Background

P-glycoprotein (P-gp) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for the transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side (Kodan et al., 2020). Since the discovery of P-gp (Juliano and Ling, 1976; Chen et al., 1986; Ueda et al., 1986), numerous studies have been conducted to clarify its transport mechanism. Most have utilized artificial membrane environments such as lipid-detergent mixed micelles (Kodan et al., 2014 and 2019; Verhalen et al., 2017), artificial lipid bilayers (Verhalen et al., 2012; Moeller et al., 2015; Zoghbi et al., 2017; Dastvan et al., 2019), or membrane vesicles derived from cells overexpressing P-gp (Liu and Sharom, 1996; Qu and Sharom, 2001). However, these conditions do not reflect physiological membrane environments in terms of lateral pressure, curvature, or constituent lipid species. Because these environmental factors can affect the function of P-gp, the detailed transport mechanism should be investigated in living cells. Accordingly, a conformation-sensitive monoclonal antibody, UIC2, has been used (Bársony et al., 2016). However, because UIC2 binds to the extracellular loops of the inward-facing structure of P-gp, the conformational change of the intracellular side has not been revealed. The distances between the C termini of NBD1 and NBD2 are estimated to be about 30 and 11 Å in the inward-facing and outward-facing structures, respectively (Futamata et al., 2020) and this difference in distance is assumed to be one of the largest between the inward-facing and outward-facing structures. Therefore, we monitored the distance of the two NBDs by FRET analysis. When the donor and acceptor were attached to different domains of the macromolecule, strong FRET occurred when the two domains were in close proximity. The big advantage of FRET analysis is that it can be performed in living cells in real time, which is not true of antibody-based analyses. In this study, two fluorescent proteins, monomeric (m)Cerulean and monomeric (m)Venus, were fused to the N- and C-terminal regions of NBDs, respectively. A change in distance between the two NBDs was evaluated by investigating the sensitized-emission of mVenus (acceptor) elicited during the excitation of mCerulean (donor) in living cells in real time. While the protocol described focuses on human P-gp in living HEK293 cells, it is also applicable to other membrane proteins.