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INTRODUCTION |
Cellular expression of human P-glycoprotein
(Pgp),1 the product of the
MDR1 gene, confers resistance to a broad variety of structurally unrelated chemotherapeutic agents and restricts
bioavailability of many therapeutic drugs in experimental models (1,
2). Pgp is a 1280-amino acid plasma membrane protein that has two homologous halves separated by a linker region of about 80 amino acids
(3). Each half of the protein contains a hydrophobic region with six
putative transmembrane (TM) helices, followed by a cytoplasmic
consensus ATP-binding/hydrolysis site (3). The TM regions are presumed
to form the drug-translocating pathway (4), whereas the ATP sites,
through ATP hydrolysis, provide the necessary driving force for
transport (5, 6).
Pgp-mediated drug transport is inhibited by a number of structurally
unrelated compounds known as reversing agents or modulators (for review
see Ref. 2). Although some of the modulators are currently being tested
for their clinical effectiveness, there remains a growing need for
molecules with higher efficacy (7-9). To develop such compounds, a
clear knowledge of the mechanisms of action of the existing repertoire
is essential. Some of the Pgp modulators themselves, such as verapamil
(10) and cyclosporin A (11), are substrates of the pump and inhibit
drug transport in a competitive manner without interrupting the
catalytic turnover (catalytic cycle) of Pgp (12-15). However, for many
others, the inhibitory mechanisms are yet to be fully understood.
Recent studies on the mechanism of action of Pgp modulators indicated
an allosteric mode of action for several compounds. Martin et
al. (16) demonstrated that inhibition of vinblastine transport by
the anthranilic acid derivative XR9576 is not through direct physical
competition for the drug translocating pathway, indicating an
allosteric effect on substrate recognition or ATP hydrolysis (17). A
similar study suggested that the indolizin sulfone SR33557 affected
vinblastine binding to Pgp through interaction with a site distinct
from the site of substrate recognition (17). Boer et al.
(18) and Ferry et al. (19) have shown that modulators like
dexniguldipine and prenylamine inhibited vinblastine interaction with
Pgp by a non-competitive mechanism. Based on these and other similar
studies, drug interaction sites of Pgp have been categorized into the
following two discrete types: 1) transport sites, where translocation
of drug across the lipid bilayer can occur, and 2) regulatory sites,
which modulate Pgp function (20, 21). Most of these studies
investigated the effect of the modulators on substrate binding in
isolated membranes, and relied exclusively on the kinetic parameters of
substrate interaction with Pgp. However, little is known about the
site(s) of modulator interaction with Pgp and the molecular mechanism
by which the allosteric site(s) communicate with the other domains to
inhibit drug transport.
Based on our prior studies (21, 22) and studies of other groups (23),
we have proposed an allosteric mode of action for Pgp inhibitors with a
thioxanthene backbone. In this study using a photoaffinity compound
[125I]iodoarylazidoprazosin ([125I]IAAP) as
a transport substrate, we provide experimental evidence for an
allosteric modulator site in Pgp through which drug transport is
inhibited by preventing substrate translocation and dissociation, without interfering with the initial step of substrate recognition.
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EXPERIMENTAL PROCEDURES |
Chemicals--
cis-(Z)-Flupentixol was
from Research Biochemicals International. Sodium orthovanadate,
vincristine, sodium azide, and 2-deoxyglucose were purchased from
Sigma. Cyclosporin A and rapamycin were supplied by Calbiochem.
[125I]IAAP (2200 Ci/mmol) was supplied by PerkinElmer
Life Sciences. cis-(Z)-[3H]Flupentixol was
custom-labeled by American Radiochemical Inc., and
[3H]cyclosporin A was purchased from Amersham
Biosciences. Monoclonal antibody UIC2 was obtained from Immunotech
(Westbrook, ME), and FITC-labeled anti-mouse IgG2a secondary antibody
was purchased from Pharmingen. The human Pgp-specific polyclonal
antibody PEPG13 was a generous gift from the laboratory of Dr. Michael
M. Gottesman, NCI, National Institutes of Health. Goat anti-rabbit IgG
conjugated with horseradish peroxidase was obtained from Invitrogen.
Cell Lines and Plasmid Construction--
Previously
characterized mouse cell line NIH3T3 fibroblasts (drug-sensitive cell
line) and the drug-resistant wild-type human Pgp-expressing
NIHMDR1 cells (14) were used for this study. In addition,
two cell lines NIHMDR1-WT and NIHMDR1-F983A were generated. NIHMDR1-WT and NIHMDR1-F983A were
created by stepwise selection of NIH3T3 cells transfected with
pHaMDR1 and pHaMDR1-F983A plasmids, respectively.
The plasmid pHa-MDR1, which contains the human
MDR1 cDNA in its entirety, was kindly provided by S. Kane (City of Hope, Duarte, CA) (24). To construct the vector
pHaMDR1-F983A, the NsiI-XhoI fragment
of the previously described plasmid pTM1MDR1 containing the F983A
mutation (25) was cloned into the corresponding sites of pHaMDR1. The
region corresponding to the NdeI-PstI fragment within pHaMDR1-F983A including the insertion points and the flanking regions was sequenced in its entirety in both directions by automated sequencing (PRISM Ready Reaction DyeDeoxy Terminator Sequencing Kit,
PerkinElmer Life Sciences). The plasmids pHaMDR1 and
pHaMDR1-F983A were calcium phosphate-transfected into NIH3T3
cells and selected with vincristine. Clones were then picked and
cultured to confluency. A stepwise selection was carried out with
increasing concentrations of vincristine to generate
NIHMDR1-WT and NIHMDR1-F983A cell lines that were
able to grow in the presence of 1 µM vincristine. For maintenance of the cultures, cells were grown in monolayers at 37 °C
in the presence of 5% CO2, in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as described earlier (15). Pgp-expressing cells were maintained in the
presence of respective drugs that were used for their selection.
[125I]IAAP Accumulation in Intact
Cells--
0.5 × 106 cells/well were grown in
monolayers in a 24-well tissue culture plate at 37 °C in the
presence of 5% CO2 in DMEM supplemented with 10% FBS
(DMEM + 10% FBS). Cells were washed once with 1 ml/well of DMEM + 10%
FBS 15 min prior to the initiation of the assay. The assay was
initiated by incubating cells with 1.5 nM
[125I]IAAP in 0.3 ml of DMEM + 10% FBS under subdued
light (to avoid photocross-linking) at 37 °C in the presence of 5%
CO2. After incubation for varying times, cells were washed
twice with 1 ml/well ice-cold PBS. Washed cells were harvested by
treatment with 0.5 ml/well trypsin/EDTA at 37 °C for 15 min.
Harvested cells were diluted in 5 ml of Biosafe II scintillation fluid
and mixed well by vortexing. Radioactivity associated with the cells
was measured in a scintillation counter. Cells washed with ice-cold PBS
immediately after addition of the assay mix was used as the "0"-min
time point, and the value for accumulated [125I]IAAP was
subtracted from each data point as nonspecific binding of
[125I]IAAP to the cells. The counts/min values were
converted to pmol/million cells. Accumulation of
[3H]cyclosporin A and
cis-(Z)-[3H]flupentixol in intact
cells was measured following the same procedure as for
[125I]IAAP, except the concentrations of
[3H]cyclosporin A and
cis-(Z)-[3H]flupentixol in the
assay mix were 0.1 and 1 µM, respectively. For metabolic
starvation, cells were incubated in a glucose-free DMEM containing 10 mM sodium azide and 10 mM 2-deoxyglucose for 30 min at room temperature.
[125]IAAP Efflux from Intact Mammalian
Cells--
Similar to the drug accumulation assay, 0.5 × 106 cells/well were incubated for a period of 60 min in an
assay mix containing 1.5 nM [125I]IAAP
supplemented with either 5 µM cyclosporin A or 5 µM cis-(Z)-flupentixol. Following
incubation cells were washed twice with 1 ml/well ice-cold PBS and
incubated for varying times at 37 °C (and 5% CO2) in
0.6 ml of DMEM + 10% FBS either in the presence or in the absence of
modulators. The incubations were stopped by washing the cells twice in
1 ml/well ice-cold PBS. The radioactivity associated with the cells was
determined as described. The rate constants for
[125I]IAAP release from cells were determined by
non-linear regression (in GraphPad PRISM program) using a first order
rate equation [I]t = [I]0·e
kt,
where [I]t and [I]0 denote the amounts of
intracellular [125I]IAAP at times t and 0 min,
respectively, and k represents the rate constant of
[125I]IAAP release from the cells. The values for
t1/2 (time for half-maximal release) were calculated
from the relationship t1/2 = 0.69/k.
Isolation of Crude Membranes Form Mammalian Cells--
Crude
membranes were prepared according to Dey et al. (22) except
that membranes were collected by centrifugation at 300,000 × g for 25 min instead of 100,000 × g for
1 h.
[125I]IAAP Photocross-linking of Pgp in Intact
Cells--
For [125I]IAAP photocross-linking of Pgp in
intact cells, 0.5 × 106 cells/well were grown in
monolayers as for the transport assay. Cells were washed once with 1 ml/well DMEM + 10% FBS and incubated at 37 °C for 60 min with 0.3 ml of IMEM + 10% FBS containing 1.5 nM
[125I]IAAP either in the presence or in the absence of 5 µM cyclosporin A, 5 µM
cis-(Z)-flupentixol, or 1 mM sodium
orthovanadate. Cells were exposed to UV light (SPECTROLINE, model
XX-15A, 365 nm) for 5 min at room temperature. After
photocross-linking, cells were resuspended in 100 µl/well (0.5 × 106 cells/100 µl) of cell lysis buffer containing 10 mM Tris, pH 8.0, 0.1% (v/v) Triton X-100, 10 mM MgSO4, 2 mM CaCl2, 1 mM dithiothreitol, 2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, and 50 units/ml micrococcal
nuclease (Staphylococcus aureus). Resuspended cells were
lysed by three cycles of freezing (on dry ice) and thawing (at
37 °C), and resolved by SDS-PAGE. The gels were dried and exposed to
an x-ray film or to a PhosphorImager screen. For photocross-linking during [125I]IAAP efflux, cells at varying times were
washed twice with 1 ml/well ice-cold PBS and photocross-linked for 5 min in the absence of any medium to minimize dissociation of
Pgp-[125I]IAAP during photocross-linking.
Photocross-linking was carried out at room temperature.
[125]IAAP Photoaffinity Labeling of Pgp in Isolated
Membranes--
Photo-affinity labeling of crude membranes was carried
out according to Dey et al. (22) in the presence of 3 nM [125I]IAAP.
SDS-PAGE and Immunoblot Analysis--
Electrophoresis and
immunoblot analyses were performed as described previously (22). Human
Pgp-specific polyclonal antibody PEPG13 was used at a dilution of
1:4000 for detection of the wild-type and the mutant Pgps. A goat
anti-rabbit IgG conjugated with horseradish peroxidase, at a dilution
of 1:10000, was used as secondary antibody. Horseradish
peroxidase-conjugated secondary antibody bound to the nitrocellulose
was detected by using the horseradish peroxidase-catalyzed luminal-based chemiluminescence reaction (ECL Western blotting system)
kit from Amersham Biosciences. The light emission signal was captured
on a Kodak Bio-Max MR film.
Quantification of Radioactivity in Protein Bands--
To
determine the amount of [125I]IAAP photocross-linked to
Pgp, the radioactivity associated with each band was quantified from the dried gels by exposing to a PhosphorImager screen and analyzed using a STORM 860 PhosphorImaging system (Amersham Biosciences). Values were expressed either in arbitrary units or as percentage of a
control experiment.
UIC2 Reactivity Shift Assay--
Cells grown in monolayers were
harvested by trypsinization, washed, and resuspended in IMDM
supplemented with 5% FBS. 0.5 × 106 cells were
incubated at 37 °C for 30 min with 5 µg of the monoclonal antibody
UIC2 in 0.4 ml of IMDM + 5% FBS. Following incubation, cells were
diluted with IMDM to 4.5 ml and centrifuged at 200 × g
for 7 min. Washed cell pellets were resuspended in 0.4 ml of IMDM + 10% FBS containing 1 µg of the FITC-conjugated anti-mouse IgG and
incubated at 37 °C for 30 min under subdued light. Cells were washed
two times with IMDM + 5% FBS and resuspended in 0.4 ml of cold PBS and
analyzed in a fluorescence-assisted cell sorter (FACS). Wherever
mentioned, cells were preincubated at 37 °C for 3 min with 5 µM cyclosporin A, 5 µM
cis-(Z)-flupentixol, or 1 mM sodium
orthovanadate prior to addition of UIC2. The fluorescence intensity
associated with cells was expressed on a log scale.
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RESULTS |
cis-(Z)-Flupentixol Induces an Elevated Level of Substrate
([125]IAAP) Association with Pgp-expressing
Cells--
cis-(Z)-Flupentixol and certain
structurally related analogs are potent inhibitors of Pgp-mediated drug
transport (26-28). Characterization of its effect on substrate binding
(23) suggested an allosteric mode of action for the modulator (21, 22);
however, the exact mechanism of action remained unresolved. To
understand the mechanism better, we developed a cell-based assay
suitable for studying both Pgp-mediated transport and Pgp-substrate
interaction, simultaneously, without altering experimental conditions.
Drug-sensitive NIH3T3 cells and drug-resistant NIHMDR1 (NIH3T3 cells
transfected with human MDR1 cDNA) cells were incubated with the Pgp substrate analog [125I]IAAP. The
steady-state level of [125I]IAAP accumulation in NIHMDR1
cells (0.15 pmol/million cells), expressing human Pgp, was 2-fold less
than that of the NIH3T3 cells (0.3 pmol/million cells) lacking any
detectable Pgp expression, indicating a Pgp-mediated efflux of
[125I]IAAP from the cells (Fig.
1A). Inclusion of 5 µM cyclosporin A, a competitive inhibitor of Pgp-mediated
drug transport (29), increased the steady-state level of
[125I]IAAP accumulation in NIHMDR1 cells (0.3 pmol/million cells) to that of the control NIH3T3 cells (Fig.
1A), substantiating the role of Pgp in
[125I]IAAP efflux from cells. Vanadate, a phosphate
analog, inhibits Pgp-ATPase activity by stabilizing a catalytic
transition state conformation of the protein with the substrate site
poorly accessible (21, 30). Addition of 1 mM sodium
orthovanadate induced a similar increase in [125I]IAAP
accumulation in NIHMDR1 cells, further supporting the role of Pgp in
[125I]IAAP transport. Neither cyclosporin A nor vanadate
had any effect on the [125I]IAAP accumulation in NIH3T3
cells indicating Pgp as the only transporter responsible for
[125I]IAAP efflux from NIHMDR1 cells.

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Fig. 1.
Effect of modulators on
[125I]IAAP transport by Pgp (A) and
Pgp-[125I]IAAP interaction (B).
A, drug-sensitive NIH3T3 (open symbols) and
drug-resistant NIHMDR1 (closed symbols) cells were incubated
at 37 °C for indicated times with 1.5 nM
[125I]IAAP in the absence ( and ) or presence of
either 5 µM cyclosporin A ( and ), 5 µM cis-(Z)-flupentixol ( and
), or 1 mM sodium orthovanadate ( ). Intracellular
accumulation of [125I]IAAP was measured and expressed as
pmol per million cells. Data presented are average of three independent
experiments. B, effect of modulators on
Pgp-[125I]IAAP interaction in NIHMDR1 cells. Monolayers
of NIHMDR1 cells were incubated at 37 °C with 1.5 nM
[125I]IAAP for 60 min either in the presence of 5 µM cyclosporin A (CsA) (2nd lane),
5 µM cis-(Z)-flupentixol
(Cis(Z)) (3rd lane), 1 mM
sodium orthovanadate (Vi) (4th lane), or in the
absence of any modulator (None) (1st lane).
Following incubation, cells were exposed to UV irradiation for 5 min,
lysed, and resolved by SDS-PAGE (80,000 cells per well) as indicated
under "Experimental Procedures." Radioactivity associated with Pgp
was detected in an autoradiogram (lower panel) and
quantified in a PhosphorImager (upper panel). Data are
expressed as fold stimulation of the basal [125I]IAAP
binding to Pgp in the absence of any modulator.
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Interestingly, in the presence of 5 µM
cis-(Z)-flupentixol, cellular association of
[125I]IAAP with NIHMDR1 cells (0.8 pmol/million cells)
increased to a level 2.5-fold higher than that of cyclosporin A- or
vanadate-treated NIHMDR1 cells (0.3 pmol/million cells) (Fig.
1A). No such increase was observed in NIH3T3 cells,
indicting that the phenomenon of elevated association was
Pgp-dependent. Therefore, the results indicated that in the
presence of cis-(Z)-flupentixol the amount of
[125I]IAAP molecules associated with Pgp-expressing cells
exceeded (2.5-fold) the level usually reached in the absence of
Pgp-mediated outward transport (Fig. 1A). The increased
amount of this excess of [125I]IAAP molecules associated
with NIHMDR1 cells varied with increasing concentrations of
cis-(Z)-flupentixol and reached a maximum at around 30 µM (data not shown). Under optimal conditions,
an excess of about 7 pmol of [125I]IAAP (relative to that
achieved by simple inhibition of Pgp-mediated transport by cyclosporin
A) was associated per million NIHMDR1 cells, which was
equivalent to 0.4 × 106 molecules per cell.
Interestingly, this value approximates the number of Pgp molecules
(1.5 × 106 molecules per cell) present on the cell
surface of each NIHMDR1 cell (31), indicating a possible complex
formation of [125I]IAAP with Pgp in NIHMDR1 cells, in the
presence of cis-(Z)-flupentixol. Preincubation
for 60 min with 5 µM cyclosporin A (which competes for
the substrate-binding site) or 1 mM sodium orthovanadate
(which lowers the substrate binding affinity), prior to the addition of
cis-(Z)-flupentixol in the assay, effectively
inhibited (by 80 and 100%, respectively) the elevated association of
[125I]IAAP with NIHMDR1 cells induced by
cis-(Z)-flupentixol without altering the level of
intracellular [125I]IAAP accumulation resulting from
inhibition of transport (data not shown). These results indicated that
cis-(Z)-flupentixol-induced elevated association
of [125I]IAAP with NIHMDR1 cells can be prevented by
blocking access of the substrate molecule to the substrate-binding site
of Pgp.
cis-(Z)-Flupentixol-induced Elevated Association of
[125I]IAAPIs Due to Formation of a Stable Complex
between Pgp and [125I]IAAP--
To determine the
possibility of a complex formation between the excess
[125I]IAAP molecules associated with the NIHMDR1 cells
and Pgp expressed in the cells, in the presence of
cis-(Z)-flupentixol, cells were briefly exposed
to UV irradiation at the end of the accumulation assay, and
photocross-linking of [125I]IAAP to cellular proteins
was determined. In the absence of a modulator, where
[125I]IAAP was efficiently transported out of the cells,
a detectable amount of [125I]IAAP was photocross-linked
to Pgp, indicating a momentary association of the substrate molecule
with Pgp during transport (Fig. 1B, 1st lane). In
the presence of 5 µM cyclosporin A or 1 mM
sodium orthovanadate, which inhibited [125I]IAAP
transport by Pgp, no measurable photocross-linking was observed (Fig.
1B, 2nd and 4th lanes). On the other
hand, in the presence of 5 µM
cis-(Z)-flupentixol, which induced an elevated level of [125I]IAAP association with NIHMDR1 cells, an
80-fold increase in the amount of [125I]IAAP
photocross-linked was observed (Fig. 1B, 3rd
lane). This result indicated that in the presence of
cis-(Z)-flupentixol, a large fraction of the
accumulated [125I]IAAP in NIHMDR1 cells remained
physically associated with Pgp. Consistent with that, preincubation of
the cells with either 5 µM cyclosporin A or 1 mM sodium orthovanadate prior to addition of
cis-(Z)-flupentixol reduced the stimulatory
effect on photocross-linking by 80 and 98%, respectively (data not
shown). Therefore, the excess [125I]IAAP associated with
NIHMDR1 cells apparently remained in a stable
Pgp-[125I]IAAP complex, presumably in the plasma membrane.
cis-(Z)-Flupentixol-induced Pgp-[125I]IAAP Complex Is
Formed Prior to the Translocation Step--
Stabilization of
Pgp-[125I]IAAP complex by
cis-(Z)-flupentixol could occur either prior to
or following the ATP-dependent translocation step. To
distinguish between the two possibilities, we investigated the effect
of cis-(Z)-flupentixol on
[125I]IAAP accumulation in NIHMDR1 cells that were
metabolically starved using NaN3 and 2-deoxyglucose. Energy
depletion of NIHMDR1 cells resulted in a 2-fold increase in the
intracellular accumulation of [125I]IAAP (Fig.
2A), indicating a requirement
for cellular ATP in Pgp-mediated [125I]IAAP extrusion.
However, cis-(Z)-flupentixol-mediated elevated association of [125I]IAAP with NIHMDR1 cells remained
unaffected by metabolic starvation (Fig. 2A). Because
substrate recognition by Pgp does not require ATP, whereas the
translocation step does, the results suggested that the
cis-(Z)-flupentixol-induced
Pgp-[125I]IAAP complex formation occurs prior to the
translocation step. The elevated [125I]IAAP association
with NIHMDR1 cells induced by cis-(Z)-flupentixol was efficiently reversed by 1 mM vanadate but not in the
energy-depleted cells (Fig. 2A). Because vanadate trapping
requires ATP hydrolysis, the lack of reversal in starved cells
suggested that the cells were effectively depleted of ATP.

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Fig. 2.
Effect of deenergization on
cis-(Z)-flupentixol-mediated
Pgp-[125I]IAAP complex formation. A,
NIHMDR1 cells were incubated with ( and ) or without ( and
) sodium azide and 2-deoxyglucose at room temperature for 30 min
(for deenergization), prior to incubation with 1.5 nM
[125I]IAAP in the presence ( and ) or absence ( and ) of 5 µM
cis-(Z)-flupentixol. Intracellular accumulation
of [125I]IAAP was measured at indicated time intervals
and expressed as pmol/million cells. As indicated by the
arrow, 1 mM sodium orthovanadate (Vi)
was added to the assay at the 30-min time point, and intracellular
accumulation of [125I]IAAP was measured for another 30 min at different intervals. Data presented are average of three
independent experiments. B, in a similar experiment as
described above, after 30 (1st and 2nd
lanes) and 90 min (3rd lane) of incubation, normal
(upper panel) and energy-depleted (lower panel)
cells were exposed to UV irradiation for 5 min. Cells were lysed and
resolved in an SDS-PAGE, and [125I]IAAP cross-linked
to Pgp was detected in an autoradiogram.
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To investigate directly the Pgp-[125I]IAAP interaction
(under the condition of elevated [125I]IAAP association),
cells were subjected to photocross-linking immediately prior to and 60 min after addition of vanadate. Energy depletion that inhibited
[125I]IAAP extrusion (Fig. 2A) had no effect
on Pgp-[125I]IAAP interaction and its stimulation by 5 µM cis-(Z)-flupentixol (Fig.
2B), further suggesting that stabilization of the
Pgp-[125I]IAAP complex by
cis-(Z)-flupentixol occurred prior to the
translocation step. On the other hand, addition of vanadate effectively
destabilized the Pgp-[125I]IAAP complex induced by
cis-(Z)-flupentixol (Fig. 2B),
presumably by stabilizing the nucleotide-trapped transition state of
Pgp. Consistent with the requirement of ATP hydrolysis for vanadate trapping, no such destabilization of the complex was observed in the
energy-depleted cells.
Stabilization of the Pgp-[125I]IAAP Complex Blocks
Pgp-mediated [125I]IAAP Transport from the
Cells--
It is conceivable that stabilization of the
Pgp-[125I]IAAP complex prior to the translocation step
would result in inhibition of transport and accumulation of
[125I]IAAP molecules inside the cells. This would lead to
two distinct [125I]IAAP pools in the cell, one bound to
Pgp in a complex and the other a free [125I]IAAP pool. To
experimentally distinguish between the two populations, NIHMDR1 cells
were loaded with [125I]IAAP in the presence of
modulators, and the kinetics of [125I]IAAP release from
the cells in a drug-free medium was determined. Cells loaded in
presence of cyclosporin A showed a one-phase exponential release of
[125I]IAAP with a rate constant (k) of
0.21 ± 0.0027/min (t1/2 = 3.2 min)
(R2 = 0.986) (Fig.
3A). Inclusion of 5 µM cyclosporin A in the efflux medium had no effect
on the rate of [125I]IAAP release (0.189 ± 0.028/min) (t1/2 = 3.6 min)
(R2 = 0.981), indicating no involvement of Pgp
in the process. Under the same experimental conditions, a similar
one-phase kinetics of [125I]IAAP release was also
observed in NIH3T3 cells (data not shown), which has no detectable
level of Pgp expression, further suggesting that the single phase
kinetics represented passive diffusion of the accumulated drug analog
across the lipid bilayer. In contrast, [125I]IAAP release
from cells loaded in the presence of 5 µM
cis-(Z)-flupentixol was clearly biphasic (Fig.
3B). The initial phase was more rapid with a rate constant
(k) of 0.18 ± 0.028/min (t1/2 = 3.8 min) (R2 = 0.99) comparable with
[125I]IAAP release from NIH3T3 cells or from the
cyclosporin A-treated NIHMDR1 cells, and likely represented
transmembrane diffusion of [125I]IAAP molecules
accumulated inside the cells. In contrast, the second phase of
[125I]IAAP release was considerably (10-fold) slower with
a rate constant k of 0.0158 ± 0.0011/min
(t1/2 = 43.6 min) (R2 = 0.99). Inclusion of 5 µM
cis-(Z)-flupentixol in the efflux medium
selectively reduced the rate of release during the second phase to a
considerable extent (k = 0.0058 ± 0.0003/min, t1/2 = 118 min)
(R2 = 0.989) with a negligible effect on
the initial phase (k = 0.131 ± 0.021/min,
t1/2 = 5.2 min) (R2 = 0.989).
When similar experiments were carried out in NIH3T3 cells,
[125I]IAAP release from the cells followed a single
exponential kinetics, irrespective of inclusion of
cis-(Z)-flupentixol in the efflux medium (data
not shown). This suggested that the second phase of
[125I]IAAP release from
cis-(Z)-flupentixol-treated NIHMDR1 cells likely
reflected dissociation of [125I]IAAP molecules from a
Pgp-[125I]IAAP complex.

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Fig. 3.
A and B, extrusion of
[125I]IAAP from NIHMDR1 cells. NIHMDR1 cells were
incubated with 1.5 nM [125I]IAAP in DMEM
either in the presence of 5 µM cyclosporin A ( and
) (A) or 5 µM
cis-(Z)-flupentixol ( and ) (B)
for 60 min. Following incubation, cells were transferred to DMEM
without [125I]IAAP either in the absence ( and ) or
in the presence of 5 µM cyclosporin A ( ) or 5 µM cis-(Z)-flupentixol ( ). At
the indicated time points the amount of [125I]IAAP
associated with the cells was measured and expressed as pmol of
[125I]IAAP retained per million cells. Values are average
of three independent experiments. C, status of
Pgp-[125I]IAAP complex during [125I]IAAP
efflux from cells. NIHMDR1 cells were preincubated with 1.5 nM [125I]IAAP and 5 µM
cis-(Z)-flupentixol for 60 min prior to
transferring into DMEM containing 5 µM
cis-(Z)-flupentixol and no
[125I]IAAP. After 0, 5, and 90 min of incubation, cells
were exposed to UV light, lysed, resolved by SDS-PAGE, and exposed to
an x-ray film (upper panel). The amount of
[125I]IAAP photocross-linked to Pgp was quantified using
a PhosphorImager and expressed as percentage of the amount incorporated
at 0 min (lower panel). Values represent an average of three
independent experiments. The presence of an equal amount of Pgp in all
three samples was confirmed by immunoblot analysis using polyclonal
antibody PEPG13 (middle panel).
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To determine definitely the nature of the two [125I]IAAP
pools, the status of the Pgp-[125I]IAAP complex during
[125I]IAAP release was studied by briefly exposing the
cells to UV irradiation at three different time points. A reduction of
only 5% was observed in Pgp-bound [125I]IAAP at the end
of the initial phase (5 min) (Fig. 3C,
autoradiogram), during which almost 20% of the total
[125I]IAAP molecules associated with NIHMDR1 cells was
released to the extracellular medium. This suggested that most of the
[125I]IAAP released during the initial rapid phase was
not associated with Pgp but instead remained free inside the cell.
These molecules, not in complex with Pgp, most likely accumulated
because of inhibition of outward transport. On the other hand, at 90 min, the amount of photocross-linked [125I]IAAP was
reduced to 58% compared the initial level (at 0 min) (Fig.
3C, autoradiogram). This closely matched the
percentage of [125I]IAAP molecules released from the
cells (58%) during the same period. Immunoblotting with Pgp-specific
polyclonal antibody PEPG13 of the same samples showed equal amounts of
Pgp present in each lane (Fig. 3C, immunoblot).
This result strongly suggested that the initial phase of
[125I]IAAP release from the
cis-(Z)-flupentixol-treated NIHMDR1 cells represented release of free [125I]IAAP accumulated inside
the cells due to inhibition of outward transport, whereas the latter
phase reflected dissociation of [125I]IAAP from its
complex with Pgp.
A Single Amino Acid Substitution That Alters the Inhibitory
Potential of cis-(Z)-Flupentixol Also Affects
cis-(Z)-Flupentixol-induced Formation of Pgp-[125I]IAAP
Complex--
To determine the functional significance of the
Pgp-[125I]IAAP complex formation and the elevated level
of [125I]IAAP associated with Pgp-expressing cells in the
presence of cis-(Z)-flupentixol, both phenomena
were tested in the Pgp mutant F983A, which is largely insensitive to
modulation by cis-(Z)-flupentixol (22). The
intracellular accumulation of [125I]IAAP in cells
expressing either the wild-type Pgp (NIHMDR1-WT) or the mutant F983A
Pgp (NIHMDR1-F983A) was considerably lower than that of the control
NIH3T3 cells, suggesting efficient transport of
[125I]IAAP by both the wild-type and F983A mutant Pgp.
However, the elevated level of cellular [125I]IAAP
association, in the presence of 5 µM
cis-(Z)-flupentixol, was selectively abrogated in
NIHMDR1-F983A (Fig. 4A).
Photocross-linking for 5 min at the end of the uptake assay showed no
increase in interaction (photocross-linking) between
[125I]IAAP and F983A by
cis-(Z)-flupentixol, although under the same conditions increased complex formation was observed between wild-type Pgp and [125I]IAAP in NIHMDR1-WT cells (Fig.
4B, upper panel). In the absence of
cis-(Z)-flupentixol, there was no significant
difference in the interaction with [125I]IAAP between the
wild-type and the F983A mutant. Cyclosporin A (5 µM), a
competitive inhibitor of Pgp, inhibited both transport and
[125I]IAAP binding by wild-type and F983A with similar
efficiency (Fig. 4B, upper panel). Similar
results were obtained from experiments with membranes isolated from
NIHMDR1-WT and NIHMDR1-F983A cells (Fig. 4B, lower
panel).

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Fig. 4.
A, [125I]IAAP
association with NIHMDR1WT and NIHMDR1F983A cells. NIH3T3, NIHMDR1-WT
(wild-type Pgp), and NIHMDR1-F983A (mutant F983A) cells were
incubated with 1.5 nM [125I]IAAP in the
presence and absence of 5 µM
cis-(Z)-flupentixol for 60 min at 37 °C. Cells
were washed, and intracellular accumulation of [125I]IAAP
was measured in a scintillation counter. Data were expressed as
picomoles of [125I]IAAP accumulated per million cells.
Data points represent average value of three identical experiments.
B, interaction of [125I]IAAP with
wild-type (WT) Pgp and F983A, in intact cells (upper
panel) and in isolated membranes (lower panel). For the
experiment with intact cells (upper panel), NIHMDR1WT and
NIHMDR1F983A cells in monolayer were incubated with IMEM
containing 1.5 nM [125I]IAAP in the presence
or absence (None) of 5 µM cyclosporin A
(CsA) and 5 µM
cis-(Z)-flupentixol
(Cis(Z)) for 60 min at 37 °C. After
incubation, cells were exposed to UV irradiation, lysed, and subjected
to SDS-PAGE. Alternatively, membranes (lower panel) were
isolated from NIHMDR1WT and NIHMDR1F983A cells and incubated with 1.5 nM [125I]IAAP for 10 min at room temperature
with or without (None) preincubation for 3 min in the
presence of 5 µM cyclosporin A (CsA) or 5 µM cis-(Z)-flupentixol
(Cis(Z)). Photocross-linked samples were resolved
by SDS-PAGE. [125I]IAAP photocross-linked to Pgp was
identified by exposing the gels to an x-ray film.
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cis-(Z)-Flupentixol Induces a Conformational Change in Pgp That Is
Distinct from the Change Induced by Substrates and Competitive
Inhibitors--
Substrates and competitive inhibitors induce a
conformational change in Pgp, which causes an increased reactivity to
UIC2, a monoclonal antibody specific for a conformation-sensitive Pgp external epitope (32). A similar increase in reactivity was observed
with Pgp-expressing NIHMDR1 cells in the presence of the competitive
inhibitor cyclosporin A (32) (Fig.
5A) or the Pgp substrate
vinblastine (data not shown), suggesting a conformational change in the
protein-enhancing accessibility of the UIC2 epitope. Interestingly,
cis-(Z)-flupentixol induced a clear decrease in the level of UIC2 binding to NIHMDR1 cells (Fig. 5A)
indicating a conformational change distinct from that induced by the
competitive inhibitor cyclosporin A. 1 mM sodium
orthovanadate induced a more profound reduction in UIC2 binding (Fig.
5A). Because vanadate inhibits Pgp-mediated drug transport
by trapping Pgp in a transition-state conformation without physically
competing for the substrate site, the result confirmed that reduced
binding of UIC2 represented a mechanism of Pgp modulation that was
distinct from competitive inhibition. To investigate the mechanistic
significance of this conformational change, UIC2 reactivity was studied
in cells expressing the Pgp mutant F983A, which has impaired
sensitivity to inhibition by cis-(Z)-flupentixol
(Fig. 5B). cis-(Z)-Flupentixol was
unable to induce any negative effect on UIC2 binding to NIHMDR1-F983A cells (Fig. 5B), suggesting that inhibition of transport and
the conformational change induced by
cis-(Z)-flupentixol could be mechanistically
related. Because increased or decreased UIC2 reactivity induced by
cyclosporin A or vanadate, respectively, was unaffected in
NIHMDR1-F983A (Fig. 5B), compared with that observed in
NIHMDR1-WT cells, the possibility of a nonspecific effect could be
ruled out.

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Fig. 5.
A, monoclonal antibody UIC2 reactivity
of Pgp in NIHMDR1 cells. NIHMDR1 cells expressing Pgp were incubated
with monoclonal antibody UIC2 in the presence and absence (filled
histogram) of 5 µM cyclosporin A (thin
line), 5 µM
cis-(Z)-flupentixol (thick line), or 1 mM sodium orthovanadate (dotted line) as
described under "Experimental Procedures." Cells were then stained
with a FITC-conjugated secondary antibody and subjected to FACS
analysis. The fluorescence intensity (x axis) is plotted
against cell counts (y axis). B, UIC2
reactivity of Pgp mutant F983A. UIC2 binding to NIHMDR1-WT and
NIHMDR1-F983A cells was carried out as mentioned above either in the
presence of 1 mM sodium orthovanadate (Vi), 5 µM cis-(Z)-flupentixol
(Cis(Z)), 5 µM cyclosporin A
(CsA), or in the absence of any modulators. Values were
expressed as mean fluorescence and represent average of two independent
experiments.
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cis-(Z)-Flupentixol Is Not a Transport Substrate for
Pgp--
Competitive inhibitors such as cyclosporin A or verapamil are
high affinity substrates of Pgp. When NIH3T3 and NIHMDR1 cells were
incubated with
cis-(Z)-[3H]flupentixol, no
difference in intracellular accumulation of the radioactive compound
was observed between the two cell types (Fig.
6A), suggesting no
Pgp-mediated outward transport of the modulator from the cells.
Inclusion of the competitive inhibitor cyclosporin A did not have any
effect (Fig. 6A), further suggesting lack of
cis-(Z)-[3H]flupentixol transport
by Pgp. Under similar experimental conditions, the steady-state level
of [3H]cyclosporin A accumulation in NIHMDR1 cells was
6-fold lower than that of the NIH3T3 cells (Fig. 6B). This
low level of [3H]cyclosporin accumulation in NIHMDR1
cells was effectively increased to that of the control NIH3T3 cells by
inclusion of 5 µM rapamycin, a Pgp modulator, in the
assay (Fig. 6B). No comparable increase in
[3H]cyclosporin accumulation was observed in NIH3T3 cells
(Fig. 6B), demonstrating that cyclosporin A extrusion from
NIHMDR1 cells was Pgp-dependent.

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Fig. 6.
Effect of Pgp on intracellular accumulation
of
cis-(Z)-[3H]flupentixol
(A) and [3H]cyclosporin A
(B). NIH3T3 ( and ) and NIHMDR1 ( and
) cells, in monolayers, were incubated either with 0.5 µM
cis-(Z)-[3H]flupentixol
(A) or with 0.1 µM
[3H]cyclosporin A (B) for indicated times.
Cells were washed, and radioactivity associated with the cells was
determined in a scintillation counter. Values are expressed as
nanomoles of radioactive drug accumulated per million cells.
A, cells were incubated either in the presence ( ) or in
the absence ( ) of 5 µM cyclosporin A; B
cells were incubated in the presence ( ) and absence ( ) of 5 µM rapamycin. Values are average of three independent
experiments.
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DISCUSSION |
An allosteric mode of action has been proposed for a number of Pgp
modulators (16-20). However, the exact mechanism by which these
compounds inhibit Pgp-mediated drug transport is not yet fully
understood. Knowledge about the mode of allosteric modulation will
benefit rational design of Pgp inhibitors with higher efficacy. In the
absence of high resolution structural information about Pgp, such
studies rely heavily on biochemical and molecular biology approaches.
In this study, we demonstrate that
cis-(Z)-flupentixol, a thioxanthene derivative,
allosterically inhibits Pgp-mediated drug transport by blocking
translocation of substrate molecules and their subsequent dissociation
from the transporter, without affecting the initial recognition of the substrate.
cis-(Z)-Flupentixol and its structurally related analogs are potent
inhibitors of Pgp-mediated drug transport (26-28). A number of these
thioxanthene derivatives do not physically compete for the
substrate-binding site of Pgp (21, 23), but instead they seem to
interact at a distinct (allosteric) site within the protein (22),
indicating an allosteric mode of action. However, the molecular events
leading to inactivation of the pump remained unresolved. To determine
experimentally the mechanism of action of these modulators, we
developed a cell-based assay that allows independent but simultaneous
monitoring of substrate binding and substrate translocation by Pgp,
without the need for changing experimental conditions. Because Pgp
recruits substrate molecules directly from the lipid bilayer (33, 34),
the integrity and polarity of the plasma membrane were kept unperturbed
using intact cells instead of inside-out membrane vesicles. The
drug-sensitive NIH3T3 cells and the drug-resistant NIHMDR1 cells
(NIH3T3 cells transfected with human MDR1 cDNA) provided
a good signal to noise ratio in both drug extrusion and drug binding
assays. The use of the photoaffinity analog of prazosin
([125I]IAAP) as a substrate in the assay provided two
distinct advantages. The high specific activity of the 125I
group in [125I]IAAP allowed convenient detection of the
molecule, whereas the photoactivable-N3 group provided an
efficient means to covalently immobilize the drug analog to its site of
interaction, whenever required. Therefore, assessing both substrate
recognition and substrate transport by Pgp could be monitored using the
same experimental set up.
Although the photoaffinity analog [125I]IAAP has been
extensively used for studying substrate-binding properties of Pgp (35, 36), no prior report has demonstrated Pgp-mediated transport of the
molecule. In this study, we demonstrate that [125I]IAAP
is transported by Pgp out of the cells affecting a 2-fold reduction in
its intracellular steady-state level (Fig. 1A).
Photocross-linking of [125I]IAAP with Pgp demonstrated a
transient association between the two during transport (Fig.
1B). Cyclosporin A, which competes for the Pgp
substrate-binding site (21), and vanadate, which traps Pgp in a
conformation with reduced affinity for substrates (21, 30),
independently blocked both recognition as well as transport of
125I]IAAP by Pgp (Fig. 1). On the other hand, metabolic
starvation that depletes cells of intracellular ATP inhibited transport
(Fig. 2A) but not [125I]IAAP recognition (Fig.
2B) by Pgp. Because ATP (hydrolysis) is specifically
required for the translocation step but not for substrate recognition,
the results are in clear agreement with the proposed mechanism of
action of Pgp. Therefore, the assay proves to be a reliable tool for
studying the mode of action of Pgp modulators with unknown mechanisms
of action.
By using this assay, we observed that the thioxanthene derivative
cis-(Z)-flupentixol inhibits Pgp-mediated drug
transport by a distinct mechanism. Unlike most other Pgp modulators,
cis-(Z)-flupentixol did not compete for substrate
binding or translocation, but instead induced formation of a stable but
reversible complex between Pgp and its substrate,
[125I]IAAP. Because Pgp is present in the plasma membrane
of the cells, formation of this complex resulted in an additional build
up of [125I]IAAP molecules in Pgp-expressing cells (Fig.
1A). Sequestration of these [125I]IAAP
molecules by Pgp led to a 2-3-fold higher cellular association than
the steady-state level achieved by simple inhibition of Pgp-mediated outward transport. Consistent with this, photocross-linking of Pgp-bound [125I]IAAP revealed an 80-fold increase in
direct interaction between cellular Pgp and [125I]IAAP in
the presence of cis-(Z)-flupentixol (Fig.
1B). Although the association between Pgp and
[125I]IAAP was stabilized by
cis-(Z)-flupentixol, the phenomenon was reversible, because removal of the cells to a drug-free medium resulted
in dissociation of the complex (Fig. 3, B and C).
The fact that cis-(Z)-flupentixol neither
stimulated Pgp-[125I]IAAP photocross-linking nor induced
any build up of excess [125I]IAAP in the cells, when the
substrate-site of Pgp was blocked by cyclosporin A or made poorly
accessible by vanadate trapping (data not shown), suggested a direct
involvement of the Pgp substrate site in the complex formation.
ATP-dependent transport by Pgp involves three major steps
as follows: 1) substrate recognition, 2) substrate translocation (coupled to ATP hydrolysis), and 3) substrate dissociation. It is
conceivable that interference with any of these three steps may lead to
inhibition of transport. Depletion of cellular ATP specifically
inhibited the translocation step without affecting substrate
recognition by Pgp (Fig. 2), substantiating that ATP hydrolysis is
required for substrate translocation and not for substrate binding.
Interestingly, ATP depletion had no effect on stabilization of the
Pgp-[125I]IAAP complex by
cis-(Z)-flupentixol (Fig. 2). Because substrate translocation by Pgp has an absolute requirement for ATP
(binding/hydrolysis), this result clearly indicates that
cis-(Z)-flupentixol-induced stabilization of the
Pgp-[125I]IAAP complex occurs prior to the translocation
step. It is likely that stabilization of the Pgp-substrate association
preceding the translocation step stalls further progression of the
catalytic cycle and could be the key event in inhibition by
cis-(Z)-flupentixol of Pgp-mediated transport.
According to this model, there should be two distinct pools of
[125I]IAAP molecules in the
cis-(Z)-flupentixol-inhibited cells as follows:
one bound to Pgp molecules in the plasma membrane as Pgp-[125I]IAAP complex, and the other as free
intracellular [125I]IAAP accumulated due to inhibition of
transport. When [125I]IAAP release from NIHMDR1 cells was
determined, biphasic kinetics did in fact indicate the existence of two
distinct [125I]IAAP pools in
cis-(Z)-flupentixol-treated cells (Fig.
3B). Consistent with that, a rapid initial phase, which had
a rate constant comparable with that of the single phase
[125I]IAAP release from NIH3T3 cells (data not shown) or
from the cyclosporin A-inhibited NIHMDR1 cells (Fig. 3A),
represented transmembrane diffusion of the free
[125I]IAAP molecules accumulated due to inhibition of
transport. In contrast, the second slower phase reflected the
dissociation of [125I]IAAP from the
Pgp-[125I]IAAP complexes in cells, because inclusion of
cis-(Z)-flupentixol in the medium reduced the
rate of [125I]IAAP release selectively during the latter
phase (Fig. 3B). A direct investigation of the status of the
Pgp-[125I]IAAP interaction, by photocross-linking
during the biphasic release of the [125I]IAAP molecules
from cis-(Z)-flupentixol-treated cells,
demonstrated that dissociation of the Pgp-[125I]IAAP
complex mainly occurred during the latter phase (Fig. 3C). Interestingly, formation of the stable Pgp-[125I]IAAP
complex induced by cis-(Z)-flupentixol was
remarkably affected in the Pgp mutant F983A (Fig. 4B), drug
transport function by which is not inhibited by
cis-(Z)-flupentixol (Fig. 4A). This emphasizes the mechanistic significance of the stable complex formation
in inhibition of transport by
cis-(Z)-flupentixol. Because the mutation F983A
did not affect [125I]IAAP transport or the ability of
cyclosporin A to block the substrate site (Fig. 4B) and
inhibit transport (Fig. 4A), any global effect of the
mutation can be ruled out.
Recently, by using bivalent cross-linking agents and cysteine-scanning
mutagenesis, Loo and Clarke (4, 37) have mapped the substrate-binding
site of Pgp. The proposed model shows the relative disposition of the
putative transmembrane helices of Pgp and their contribution in
constituting the substrate-binding site. Interestingly, residue
Phe-983, which is necessary for
cis-(Z)-flupentixol interaction with Pgp, maps
well outside the substrate interaction site (4, 37). This is in clear
agreement with our experimental data showing no physical competition
for the substrate site by cis-(Z)-flupentixol
(Fig. 1B) and no transport of
cis-(Z)-flupentixol by Pgp (Fig. 6A).
However, the spatial distinctness of the putative interaction site for
cis-(Z)-flupentixol indicated a requirement for
communication between the allosteric modulator site and the substrate
translocating pathway. Communication of this nature often requires
conformational changes in the protein. By using the monoclonal antibody
UIC2, which is specific to a conformation-sensitive extracellular
epitope of Pgp, we demonstrate that
cis-(Z)-flupentixol induces a remarkable change
in Pgp conformation, which is distinct from the changes induced by Pgp
substrates (data not shown) or competitive inhibitors (Fig.
5A). No such conformational change was induced by
cis-(Z)-flupentixol in the Pgp mutant F983A,
whereas the ability of the competitive inhibitor cyclosporin A to
induce its characteristic change in conformation remained unaltered
(Fig. 5B), underscoring the functional distinctness of the
allosteric site.
Allosteric regulations of ion channels and carrier proteins often play
important functional roles in regulating ion fluxes across cellular
membranes, and these have important implications in cell signaling and
neuronal transmission (38). Although allosteric modulation of
ligand-gated channels is well documented, allosteric modulation of ABC
transporters is a novel and emerging concept. The basic structural plan
of ABC transporters includes two hydrophobic transmembrane domains and
two cytosolic nucleotide-binding moieties per functional unit (39).
According to the most widely accepted model, the transmembrane regions
associate with each other to form a single drug translocating pathway
across the lipid bilayer, whereas the two nucleotide-binding sites
constitute the catalytic domains that hydrolyze ATP to provide the
driving force for the translocation step (1). The coupling of ATP
hydrolysis to vectorial translocation of the drug substrate requires
precise communication between the two domains. Stimulation of ATP
hydrolysis by substrate binding to Pgp (6) and occlusion of the
drug-binding site upon ATP hydrolysis (21, 30) suggested direct
communication between the two domains via conformational changes
(40-42). In this study, we demonstrate the presence of an allosteric
modulator site within the Pgp capable of communicating with the
substrate-binding/translocating site evidently through conformational
change(s). Studying the details of the conformational change will
reveal important information on the molecular events responsible for
this communication. On the other hand, mapping the amino acid residues
constituting the modulator site and the structural moieties of the
modulator in contact with the site are likely to provide valuable clues
for designing more efficient Pgp modulators. These avenues are
currently being explored in our laboratory.