MULTIDRUG RESISTANCE PROTEIN: P-GLYCOPROTEIN - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 237

Mutagenesis studies indicated that residues in TM 4, 5, and 6 in the N-terminal half
of Pgp and TM 9, 10, 11, and 12 in the C-terminal half were involved in forming the
drug-binding pocket. 143 Loo and Clarke systematically inserted single Cys residues
by site-directed mutagenesis into 252 positions in the TM segments, and then reacted
them with either a thiol-reactive substrate or a drug analog. 131 , 132 , 144 Overall, the drug-
binding pocket is envisioned as funnel-shaped, narrower at the cytoplasmic side of the
membrane, 145 where TM2/TM11 and TM5/TM8 come together. 47 , 48 They concluded
that the drug-binding pocket is found at the interface between the two TM “halves”
of Pgp. This was confirmed by Pleban et al., who used propafenone-type substrate
photoaffinity ligands, in conjunction with matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry, to define the substrate-binding site(s) of Pgp. 146
They observed the highest labeling in TM 3, 5, 8, and 11, and when this pattern was
projected onto a three-dimensional homology model of Pgp, labeling was found to
occur at the interface formed by TM3 and 11 in one half of the protein, and TM5 and
8 in the other half.
Pawagi et al. proposed that aromatic amino acid residues may play an important
role in the binding and transport path for drug substrates. 147 Studies of intrinsic Pgp
fluorescence also showed that Trp residues were highly quenched by binding of certain
drugs, and FRET from Trp to substrate molecules took place with high efficiency, 81 , 148
suggesting that Trp residues are located close to the sites of drug-binding.
10.11. P-GLYCOPROTEIN AS A HYDROPHOBIC
VACUUM CLEANER OR DRUG FLIPPASE
Pgp substrates are typically hydrophobic and are expected to partition into the mem-
brane. The substrate-binding sites of Pgp appear to be contained within its TM regions,
and drugs gain access to these sites after partitioning into the lipid bilayer (Figure
10.2 a ). 34 The idea that the transporter acts as a “vacuum cleaner” for hydrophobic
molecules present in the membrane was first suggested by Higgins and Gottesman 149
and has found widespread acceptance. In intact cells, Pgp substrates entering the cell
from the extracellular medium are intercepted at the plasma membrane and extruded
to the exterior without entering the cytosol. 150 Lipid bilayers are amphipathic multi-
layered structures and do not behave like an organic solvent such as octanol in terms of
drug partitioning. After entering the membrane, Pgp substrates (which are generally
amphipathic in nature) are not distributed uniformly in the hydrophobic core of the
lipid bilayer but tend to concentrate in the interfacial regions of the membrane. 151
Several studies suggested that the drug-binding pocket of Pgp is probably located
within the cytoplasmic leaflet of the membrane. Drugs appear to gain access to this
binding site after moving to the cytoplasmic leaflet by spontaneous “flip-flop” from
the outer leaflet, which can be a slow process for many compounds that are Pgp
substrates. 152 , 153 Transport by reconstituted Pgp of the fluorescent dyes H33342 and
LDS-751 suggested that they were probably extracted from the cytoplasmic leaflet
of the membrane. 154 , 155 The positively charged compound N -methyldexniguldipine,
which is unable to flip-flop to the inner leaflet, could interact with Pgp if added to cell
fragments and membrane vesicles where the cytoplasmic membrane face is accessible,