Abstract
P-glycoprotein (P-gp; also known as MDR1 or ABCB1) 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 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. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.
Highlights
P-gp, a member of the ATP-binding cassette (ABC) transporter family, is an ATP-dependent efflux pump that transports various hydrophobic compounds [1,2]
A fluorescence resonance energy transfer (FRET) construct to detect the conformational change of human P-gp in living cells
The distances between the C-terminal of NBD1 and that of NBD2 of human P-gp are estimated to be about 30 Å and 11 Å in the inward-facing and outward-facing structures, respectively (Movie_S01). Because this difference in distance is assumed to be one of the largest between the inward-facing and outward-facing structures, we considered it suitable for FRET analysis in order to detect the conformational change of human P-gp in living cells in real time
Summary
P-gp, a member of the ATP-binding cassette (ABC) transporter family, is an ATP-dependent efflux pump that transports various hydrophobic compounds [1,2]. We analyzed the roles of ATP binding and ATP hydrolysis in the conformational changes of human P-gp in living cells by using fluorescence resonance energy transfer (FRET). A transport substrate of P-gp [14] that stimulates its ATPase activity (Fig. S2), was added to the cells, the proximity ratio of P-gp–FRET increased, whereas that of P-gp–VsCn was unchanged (Fig. 1B).
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