Differential Coupling of Binding, ATP Hydrolysis, and Transport of Fluorescent Probes with P-Glycoprotein in Lipid Nanodiscs

The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-...