Apr 10, 2020
In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein
Journal of Biological Chemistry
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- Volume
- 295
- Number
- 15
- First page
- 5002
- Last page
- 5011
- Language
- Publishing type
- Research paper (scientific journal)
- DOI
- 10.1074/jbc.ra119.012042
- Publisher
- American Society for Biochemistry & Molecular Biology (ASBMB)
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.
- Link information
- ID information
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- DOI : 10.1074/jbc.ra119.012042
- ISSN : 0021-9258
- eISSN : 1083-351X