MULTIDRUG RESISTANCE PROTEIN: P-GLYCOPROTEIN - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 241

The profile for ATPase activation and inhibition by drugs and modulators changes
when Pgp is moved from detergent solution into a lipid bilayer 25 and also varies
with the nature of the host lipid in which the protein is reconstituted. 65 This suggests
that coupling between drug-binding sites and NB domains is affected by the lipid
environment of the protein.
Pgp-mediated drug transport is also affected in an interesting way by the fluidity
of the membrane. Changes in the fluidity of canalicular membrane vesicles altered
Pgp-mediated transport of daunorubicin and vinblastine. 172 When lipid fluidity was
increased using membrane fluidizers, drug transport was significantly inhibited, sug-
gesting that the physical state of the membrane affects Pgp transport function. This
idea was explored further using Pgp reconstituted into proteoliposomes composed of
two synthetic PCs with different melting points. 105 A real-time fluorescence assay
used to measure the initial rate of transport found a highly unusual biphasic tem-
perature dependence: a high rate of transport in the rigid gel phase, the maximum
transport rate at the melting temperature of the bilayer, and a lower transport rate in the
fluid liquid-crystalline phase. This pattern suggests that the rate of drug transport by
Pgp may be dominated by partitioning of drug into the bilayer, which shows similar
temperature dependence.
10.13. MECHANISM OF ACTION OF P-GLYCOPROTEIN
Much remains to be understood about how Pgp transports (or flips) drugs and how
coupled ATP hydrolysis powers transport. Transport can be broken down into several
steps: entry of substrates into the binding pocket within the cytoplasmic leaflet, con-
formational changes in Pgp driven by ATP binding/hydrolysis, and release of drug to
either the outer leaflet or the extracellular aqueous phase. Many different experimental
approaches, including various biochemical and spectroscopic techniques, have pro-
vided evidence that conformational changes take place during the catalytic cycle of
Pgp and other ABC proteins. 173 It is assumed that release of drug from Pgp involves
reorientation of the drug-binding site from the cytosolic side of the membrane (or
the inner membrane leaflet) to the extracellular side (or the outer membrane leaflet),
accompanied by a switch from high to low drug-binding affinity. Superimposed on the
transport cycle is the ATP hydrolysis cycle, which involves ATP binding, formation of
the nucleotide sandwich dimer, ATP hydrolysis, dissociation of P i , and dissociation of
ADP. A recent review discusses the drug translocation mechanism of Pgp in detail. 174
Substrates may diffuse from the lipid bilayer into the drug-binding pocket through
“gates” formed by TM segments at either end of the pocket. 175 The nature of the
local environment within the drug-binding pocket is still controversial. Loo et al.
tested whether Cys residues within the drug-binding pocket of Pgp were able to
react with charged thiol-reactive compounds and concluded that the drug-binding
pocket is accessible to water. 176 In contrast, the fluorescence properties of drugs
bound to purified Pgp clearly indicate that the local environment of the binding site
is very hydrophobic, with a polarity lower than that of chloroform. 158 Several drugs
(e.g., H33342, LDS-751) show large increases in the intensity of their fluorescence