MULTIDRUG RESISTANCE PROTEIN: P-GLYCOPROTEIN - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 239

site within the cytoplasmic membrane leaflet. 148 The binding site for another fluores-
cent substrate, LDS-751, was also localized by FRET to the cytoplasmic half of the
bilayer, although closer to the membrane surface in the interfacial region. 158 H33342
binds to the H-site and LDS-751 to the R-site, so it appears that both of these functional
drug transport sites are in Pgp domains within the cytoplasmic membrane leaflet.
It was proposed that Pgp may operate as a drug flippase, moving hydrophobic
drug molecules from the inner to the outer leaflet of the membrane (Figure 10.2 a ). 149
Given the high level of amino acid similarity between Pgp and its close relative
the MDR3/ABCB4 protein, which functions as a PC-specific phospholipid flippase,
this suggestion seems reasonable. The location of the drug-binding pocket in the
cytoplasmic leaflet of the membrane is also compatible with this idea. If Pgp maintains
a higher drug concentration in the outer leaflet than in the inner leaflet, equilibration
of drug between the membrane and the aqueous phase on each side would result
in the observed drug concentration gradient. Such partitioning and equilibration of
nonpolar drugs between lipid bilayers and water is a very fast process, limited only
by diffusion. 159 In fact, it is not possible to distinguish experimentally between a
transport process in which drugs are moved from the inner to the outer leaflet, followed
by rapid partitioning into the aqueous phases on each side, and one in which drugs
are moved from the inner leaflet directly to the extracellular aqueous phase, followed
by repartitioning of drug into the outer leaflet.
Several studies have indicated that Pgp can move fluorescent NBD-labeled phos-
pholipid derivatives from the inner to the outer leaflet of the plasma membrane in intact
cells overexpressing the protein, 160 , 161 and glycosphinglipids (GSLs) have also been
considered as substrates. 162 , 163 Since then, reconstituted Pgp in lipid bilayer vesicles
has been shown to act as an outwardly-directed flippase for NBD-labeled phospho-
lipids and simple GSLs such as glucosyl-, galactosyl-, and lactosylceramide. 164 , 165
The lipid translocation process shares many biochemical features with drug transport:
it requires ATP hydrolysis, it is inhibited by V i , and drug substrates compete with
flippase activity. 164 , 165 Thus, both drugs and membrane lipids appear to follow the
same transport route through the Pgp molecule, increasing the likelihood that drug
transport takes place via a flippase-like mechanism. It is possible that Pgp plays a
physiological role in flipping glucosylceramide from the cytoplasmic leaflet to the
luminal leaflet of the Golgi apparatus, which is a required step in the biosynthesis of
complex GSL. 165 , 166
10.12. ROLE OF THE LIPID BILAYER
IN P-GLYCOPROTEIN FUNCTION
The hydrophobic vacuum cleaner model proposes that drugs and modulators partition
into the membrane before interacting with the transporter. Pgp substrates generally
have high lipid:water partition coefficients 152 , 167 and accumulate within the mem-
brane to high concentrations (Figure 10.2 b ). Pgp would thus experience a much
higher drug concentration than that nominally added to the aqueous phase, by 300- to