Permanent Activation of the Human P-glycoprotein by Covalent Modification of a Residue in the Drug-binding Site*
Tip W. Loo,
M. Claire Bartlett and
David M. Clarke
From the
Canadian Institutes of Health Research Group in Membrane Biology,
Department of Medicine and Department of Biochemistry, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
Received for publication, April 10, 2003
, and in revised form, April 21, 2003.
 |
ABSTRACT
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The human multidrug resistance P-glycoprotein (ABCB1) transports a broad
range of structurally diverse compounds out of the cell. The transport cycle
involves coupling of drug binding in the transmembrane domains with ATP
hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used
cysteine-scanning mutagenesis of the transmembrane segments and reaction with
the thiol-reactive substrate analog of verapamil, methanethiosulfonate
(MTS)-verapamil, to test whether it caused permanent activation of ATP
hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase
activity (8-fold higher than untreated) when treated with MTS-verapamil and
isolated by nickel-chelate chromatography. Drug substrates that either enhance
(calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit
(cyclosporin A and trans-(E)-flupentixol) ATPase activity of
Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity
of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the
covalently attached verapamil, and ATPase activity returned to basal levels.
Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil,
vinblastine, or colchicine prevented activation of mutant I306C by
MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a
common drug-binding site. Covalent modification of I306C affects the long
range linkage between the drug-binding site and the distal ATP-binding sites.
This results in the permanent activation of ATP hydrolysis in the absence of
transport. Trapping mutant I306C in a permanently activated state indicates
that Ile-306 may be part of the signal to switch on ATP hydrolysis when the
drug-binding site is occupied.
 |
INTRODUCTION
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The human multidrug resistance P-glycoprotein
(P-gp)1 uses ATP to
transport structurally diverse compounds out of the cell (for recent reviews,
see Refs. 1 and
2). The protein contributes to
the phenomenon of multidrug resistance because many of the drugs used in AIDS
and cancer chemotherapy are substrates of P-gp
(3,
4).
P-gp is a member of the ATP-binding cassette family of transporters. The
1280 amino acids of P-gp are arranged as two repeating units of 610 amino
acids that are joined by a linker region of about 60 amino acids
(5). Each repeat has six
transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding
site (6,
7,
8). The minimum functional unit
is a monomer (9), but the two
halves of the molecule do not have to be covalently linked for function
(10,
11). Both ATP-binding sites
are required for activity (7,
12,
13,
14) and likely function in an
alternating mechanism (15).
Studies on deletion mutants have shown that the TM domains alone are
sufficient to mediate drug binding
(11). Studies on the activity
of cysteine mutants and their inhibition by different thiol-reactive substrate
analogs indicate that residues from multiple TM segments contribute to the
drug-binding site (16,
17,
18,
19,
20,
21).
To determine the mechanism of P-gp, it is important to understand how drug
binding and subsequent efflux are coupled to ATP hydrolysis. A key step in the
reaction cycle is activation of ATP hydrolysis when the drug-binding site is
occupied. It is not known whether drug-stimulated ATPase activity requires
that the drug-binding site alternate between occupied and unoccupied states or
whether ATPase activity is permanently activated when the drug-binding site is
permanently occupied.
In this study we used cysteine-scanning mutagenesis and reaction with the
thiol-reactive analog of verapamil, MTS-verapamil, to test for the presence of
a permanently activated P-gp intermediate.
 |
EXPERIMENTAL PROCEDURES
|
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Construction of MutantsA histidine-tagged Cys-less P-gp
cDNA was constructed (6,
22). The presence of the
histidine tag facilitated purification of the mutant P-gps by nickel-chelate
chromatography (23). Single
cysteine residues were then introduced into the predicted TM segments of
Cys-less P-gp(His)10 as described previously
(18).
Expression, Reaction with MTS-verapamil, Purification, and Measurement
of Drug-stimulated ATPase Activities of P-gp MutantsThe
histidine-tagged P-gp mutants were expressed in HEK 293 cells. After 24 h, the
medium was replaced with fresh medium containing 10 µM
cyclosporin A. Cyclosporin A acts as a specific chemical chaperone to promote
maturation of P-gp and increases the yield of P-gp
(24,
25). After another 24 h, the
transfected cells from 50 10-cm-diameter culture plates were washed three
times with phosphate buffered saline (PBS, pH 7.4) and suspended in 1.5 ml of
PBS. The cells were solubilized by addition of 1 volume of PBS containing 2%
(w/v) n-dodecyl
-D-maltoside. Insoluble material was
removed by centrifugation at 16,000 x g for 15 min at 4 °C.
Half of the supernatant (1.3 ml) was mixed with 8 µl of Me2SO
(control), and the other half was mixed with 8 µl of 50 mM
MTS-verapamil (in Me2SO; final concentration, 0.3 mM).
The mixtures were incubated for 10 min at 22 °C, and cooled to 4 °C,
and then 150 µl of 3 M NaCl and 50 µl of 1 M
imidazole (pH 7.0) were added. P-gp(His)10 was then isolated by
nickel-chelate chromatography as described previously
(23). The recovery of P-gp was
monitored by immunoblot analysis with rabbit anti-P-gp polyclonal antibody
(9). To protect P-gp from
labeling with MTS-verapamil, 0.5 mM cyclosporin A, 5 mM
demecolcine, 5 mM verapamil, 0.5 mM vinblastine, 10
mM colchicine, or 2 mM calcein-AM was added to the
solubilized material before addition of MTS-verapamil. The highest
concentrations possible (solubility in aqueous solution) were chosen to
maximize protection from modification by MTS-verapamil.
The isolated P-gp(His)10 was mixed with lipid and sonicated, and
ATPase activity was determined
(26). A sample of the
P-gp-lipid mixture was mixed with an equal volume of buffer containing 100
mM Tris-HCl, pH 7.4, 100 mM NaCl, 20 mM
MgCl2, 10 mM ATP, and either no drug, 1.2 mM
calcein-AM, 0.3 mM cyclosporin A, 6 mM demecolcine, 1.2
mM trans-(E)-flupentixol, 2 mM
verapamil, or 0.2 mM vinblastine. These concentrations caused
maximal stimulation or inhibition of the ATPase activity of Cys-less P-gp. The
samples were then incubated for 30 min at 37 °C, and the amount of
inorganic phosphate liberated was determined
(27).
Inhibition of ATPase activity by vanadate was done by addition of an equal
volume of buffer containing 100 mM Tris-HCl, pH 7.4, 100
mM NaCl, 20 mM MgCl2, 10 mM ATP,
and 0.2 mM orthovanadate to the P-gp-lipid mixture
(26). Orthovanadate was
prepared from Na3VO4, pH 10 and boiled for 2 min to
break down polymeric species before use
(28).
 |
RESULTS
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Drug transport by P-gp involves the coupling of drug binding with ATP
hydrolysis. The transport cycle predicts the existence of an activated
intermediate. Therefore, the goal in this study was to determine whether P-gp
could be trapped as an activated intermediate if drug substrates could be
prevented from leaving the drug-binding site. An approach to keep the
drug-binding site occupied would be to permanently attach a drug substrate to
a residue in the drug-binding site. Cysteine-scanning mutagenesis and reaction
with a thiol-reactive analog of a drug substrate seemed ideal for these
studies. We have done cysteine-scanning mutagenesis on all the residues in the
TM segments and found that almost all of the single cysteine mutants were
active (16,
17,
18).
A useful thiol-reactive drug substrate is MTS-verapamil
(Fig. 1). It is an analog of
verapamil and contains an alkylthiosulfonate group that reacts selectively
with cysteine residues (29,
30). We showed previously that
MTS-verapamil is a good substrate of Cys-less P-gp as it stimulated its ATPase
activity 10-fold and with a Km of 25
µM (Km for verapamil, 25
µM) (19).
Accordingly a cysteine was introduced at each position in the predicted TM
segments of Cys-less P-gp(His)10. A total of 252 mutants were made,
expressed in HEK 293 cells, and then solubilized with 1% (w/v)
n-dodecyl
-D-maltoside. Insoluble material was
removed by centrifugation, and the samples were treated with or without 0.3
mM MTS-verapamil. This concentration of MTS-verapamil caused
maximum activation of Cysless P-gp
(19). P-gp was then
immobilized on a nickel-chelate column, washed with buffer to remove unbound
MTS-verapamil, and eluted from the column. The ATPase activities of the P-gp
treated with and without MTS-verapamil were determined. One mutant, I306C,
showed an 8-fold increase in ATPase activity after treatment with
MTS-verapamil. Activation by MTS-verapamil was concentration-dependent.
Fig. 1 shows that maximal
stimulation occurred after treatment of mutant I306C with 0.11
mM MTS-verapamil, and half-maximal stimulation was 39
µM. Nickel-chelate chromatography was effective in removing
unreacted MTS-verapamil because the activity of Cys-less P-gp remained at
basal levels even after pretreatment with 1 mM MTS-verapamil
(Fig. 1).

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FIG. 1. Activation of mutant I306C after treatment with MTS-verapamil.
Histidine-tagged Cys-less or mutant I306C P-gps were expressed in HEK 293
cells and solubilized with n-dodecyl -D-maltoside.
Insoluble material was removed by centrifugation, and equivalent amounts of
the supernatant were treated with various concentrations of MTS-verapamil.
P-gp(His)10 was then isolated by nickel-chelate chromatography.
Equivalent amounts of P-gp were mixed with lipid and sonicated, and ATPase
activity was determined in the absence of drug substrate. -Fold stimulation is
the ratio of activity in an untreated sample to that treated with
MTS-verapamil. Each value is the average of two different experiments. The
structure of MTS-verapamil is shown.
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To test whether all of mutant I306C had been modified by MTS-verapamil, we
assayed for stimulation or inhibition of the ATPase activity by other drug
substrates. The rationale is that if a significant amount of unmodified mutant
I306C is present, then the presence of other substrates or inhibitors should
affect the activity of the mutant. We tested compounds that are potent
stimulators (calcein-AM, demecolcine, vinblastine, and verapamil)
(17,
31,
32,
33) or inhibitors (cyclosporin
A and trans-(E)-flupentixol) of P-gp ATPase activity
(33,
34,
35).
Fig. 2 shows the effect of
various stimulators and inhibitors on the ATPase activity of mutant I306C
before and after treatment with MTS-verapamil. Before treatment with
MTS-verapamil, the ATPase activity of mutant I306C was stimulated by
demecolcine, calcein-AM, verapamil, and vinblastine by 11.9-, 9.8-, 7-, and
3-fold, respectively. By contrast, cyclosporin A and
trans-(E)-flupentixol inhibited the ATPase activity (0.9-
and 0.5-fold, respectively). When mutant I306C was pretreated with
MTS-verapamil, the presence of other stimulators or inhibitors of P-gp had
little effect on its ATPase activity (7.78.1-fold versus
8-fold increase) (Fig. 2). This
inability to further stimulate or inhibit the activity of the
MTS-verapamil-treated mutant I306C suggests that most (more than 90%) of the
mutant I306C P-gp was modified and that covalent attachment of verapamil in
the drug-binding site blocks access of other drug substrates to the
drug-binding site.

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FIG. 2. Effect of drug substrates and inhibitors on activity of mutant I306C
before and after labeling with MTS-verapamil. Histidine-tagged mutant
I306C P-gps were expressed in HEK 293 cells, solubilized with
n-dodecyl -D-maltoside, and treated with 0.3
mM MTS-verapamil (+ MTS-verapamil) or without
MTS-verapamil (Untreated). P-gp was then isolated by nickel-chelate
chromatography to remove unreacted MTS-verapamil. The isolated P-gp was mixed
with lipid and sonicated, and ATPase activity was determined in the presence
of no drug, 0.6 mM calcein-AM (CAM), 3 mM
demecolcine (Deme), 1 mM verapamil (Ver), 0.1
mM vinblastine (Vin), 0.15 mM cyclosporin A
(Cyclo), or 0.6 mM trans-(E)-flupentixol
(T-Flu). -Fold stimulation is the ratio of activity with drug
substrate to that without drug substrates.
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It was possible that reaction of the mutant with MTS-verapamil partially
denatured the protein so that the ATP-binding sites are uncoupled from the
drug-binding sites. If modified I306C retained a "native"
structure, then it should be possible to restore basal levels of ATPase
activity if covalent attachment of MTS-verapamil was removed. Accordingly
modified I306C was treated with 20 mM dithiothreitol to reduce the
disulfide bond between P-gp and MTS-verapamil, and then P-gp was reisolated by
nickel-chelate chromatography. Fig.
3 shows that reduction of the disulfide bond by dithiothreitol
reduced the activity of the mutant as its ATPase activity was only slightly
higher (1.5-fold) than the untreated I306C mutant. Mutant I306C remained
active after removal of the covalently bound MTS-verapamil since it retained
the ability to be stimulated by verapamil (6.9-fold).
The characteristics of the ATPase activity of modified I306C mutant were
also examined. We tested for vanadate trapping of nucleotide. P-gp traps
nucleotides in the presence of vanadate plus Mg-ATP and results in the
formation of an inhibitory transition state that inhibits ATP hydrolysis at
the second ATP site (36).
Fig. 3 shows that mutant I306C
that had been labeled with MTS-verapamil was still inhibited by vanadate. In
addition, the modified mutant I306C had a Km for
ATP (1.1 mM) that was very similar to that of Cys-less P-gp (1
mM) (data not shown).
If MTS-verapamil occupied the drug-binding site in mutant I306C, then
pretreatment of the mutant with other drug substrates should protect it from
labeling by MTS-verapamil if they shared a common drug-binding site.
Accordingly mutant I306C was treated with or without the drug substrates
calcein-AM, demecolcine, verapamil, vinblastine, cyclosporin A, or colchicine
before treatment with MTS-verapamil for 10 min at 22 °C. P-gp was isolated
by nickel-chelate chromatography, and ATPase activity was determined.
Fig. 4 shows that all of the
drug substrates protected mutant I306C from labeling with MTS-verapamil since
they prevented modification and activation of I306C by MTS-verapamil by
7085%. These results suggest that the compounds tested likely share a
common drug-binding site.

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FIG. 4. Protection of mutant I306C from labeling by MTS-verapamil with
substrates and inhibitors. Histidine-tagged mutant I306C P-gp was
expressed in HEK 293 cells and solubilized with n-dodecyl
-D-maltoside. Insoluble material was removed by
centrifugation. Equivalent amounts of the supernatant were incubated at 22
°C for 10 min in the presence of no drug, 2 mM calcein-AM
(CAM), 5 mM demecolcine (Deme),5mM
verapamil (Ver), 0.5 mM vinblastine (Vin), 0.5
mM cyclosporin A (Cyclo), or 10 mM colchicine
(Colch). The samples were then incubated with 0.1 mM
MTS-verapamil for 10 min at 22 °C and cooled in an ice bath, and
P-gp(His)10 was isolated by nickel-chelate chromatography.
Equivalent amounts of the isolated P-gps were mixed with lipid and sonicated,
and ATPase activity was determined in the absence of drug substrate. The
activities are expressed relative to a sample treated with MTS-verapamil in
the absence of drug substrate (No Drug).
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DISCUSSION
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Treatment of mutant I306C with MTS-verapamil followed by removal of
unreacted MTS-verapamil caused the mutant to remain in an activated state. ATP
is constantly hydrolyzed at a high rate without concomitant transport,
i.e. ATP hydrolysis is uncoupled from vectorial transport. Addition
of other stimulators or inhibitors of P-gp could not change the ATPase
activity of the activated mutant. An explanation for this observation is that
covalent attachment of MTS-verapamil to the cysteine residue at position 306
in TM5 resulted in the permanent occupation of the drug-binding site in an
orientation similar to that of P-gp with unmodified verapamil. Evidence that
MTS-verapamil tethered to I306C mimics the interaction of the mutant with
verapamil is that both verapamil and MTS-verapamil caused similar activation
of ATPase activity (78-fold). In addition, activation of I306C by
MTS-verapamil was reversed by dithiothreitol, and the dithiothreitol-treated
mutant could rebind verapamil. Both MTS-verapamil and verapamil must bind to
the same drug-binding site because the activity of MTS-verapamil-treated I306C
was not affected by verapamil or other stimulators and inhibitors of P-gp, and
mutant I306C can be protected from labeling by verapamil and the other
substrates and inhibitors.
A model to explain the activated state of P-gp is shown in
Fig. 5. In the resting state
(state I), P-gp can bind ATP with normal affinity, but ATPase activity is low
because the opposing signature "LSGGQ" motifs are farther away
from the Walker A sites in the nucleotide-binding domains (NBDs)
(33,
37). Binding of verapamil in
the TM segments induces conformation changes in the NBDs to bring the LSGGQ
and Walker A sequences closer (state II) and promote ATP hydrolysis
(38,
39). ATP hydrolysis then
changes the conformation of the TM segments
(26,
40) leading to drug release
(state III). Expulsion of verapamil from the drug-binding site allows the
LSGGQ and Walker A sequences to move apart, and P-gp returns to the resting
state (state IV) (41,
42). Covalent attachment of
verapamil, however, results in the permanent occupation of the drug-binding
site (state IIA). The inability of MTS-verapamil to exit the activated state
keeps the LSGGQ and Walker A sequences close together and in a conformation
that ensures continued hydrolysis of ATP and recycling between states IIA and
IIIA (Fig. 5, dotted
arrow). It is possible that Ile-306 may be part of the mechanism that
senses that the drug-binding site is occupied and then initiates
conformational changes in the molecule.

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FIG. 5. Model of interaction between mutant I306C and verapamil or
MTS-verapamil. P-gp in the resting state (I) binds ATP
(blue) but has only basal ATPase activity because the Walker A site
(A) in each NBD is separated from the opposing signature LSGGQ
sequence (Sig). Close opposition of the Walker A and LSGGQ sequences
is required for high levels of ATP hydrolysis. Binding of verapamil (red
diamond) in the transmembrane domains (TMD) induces conformation
changes in the NBDs (II) that results in increased interaction
between the LSGGQ and Walker A sites. As a result, enhanced ATP hydrolysis to
ADP (green) and inorganic phosphate (orange) occurs, which
then causes conformational changes in the drug-binding site, resulting in drug
efflux (III). Release of drug leads to a resetting of the NBDs so
that the Walker A and LSGGQ sites are separated (IV) followed by
return to the resting state (I). Binding of MTS-verapamil (yellow
diamond) by P-gp also increases interaction between the Walker A and
LSGGQ sites and promotes ATP hydrolysis (IIA), but covalent
attachment of verapamil (black line attached to the yellow diamond)
blocks its release after ATP hydrolysis (IIIA). The modified P-gp
cannot return to the resting states (IV and I) but continues
to cycle between state IIA and IIIA (dotted arrow).
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Our model predicts that covalent modification of the drug-binding site can
result in permanent activation of ATP hydrolysis in the absence of transport
because hydrolysis of ATP and release of inorganic phosphate (Pi)
occurs before vectorial displacement of the drug ligand. In this respect,
ATP-binding cassette transporters appear to be different from the P-type
ATPases such as the sarcoplasmic reticulum (SERCA) Ca-ATPase. In the P-type
ATPases, ATP hydrolysis is linked to conformational changes in the
calcium-binding site, but Pi is not released until after vectorial
transport of calcium (43,
44).
This study also shows that TM5 must contribute residues to the drug-binding
site since other substrates and inhibitors could protect I306C from labeling
by MTS-verapamil. We had predicted that I306C likely lined the drug-binding
site because it could be cross-linked to other cysteines in TMs 10, 11, and 12
with thiol-reactive cross-linker substrates
(20).
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FOOTNOTES
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* This work was supported in part by National Institutes of Health Grant
CA80900 and by grants from the Canadian Institutes for Health Research. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Recipient of the Canada Research Chair in Membrane Biology. To whom
correspondence should be addressed: Dept. of Medicine, University of Toronto,
Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario
M5S 1A8, Canada. Tel./Fax: 416-978-1105; E-mail:
david.clarke{at}utoronto.ca.
1 The abbreviations used are: P-gp, P-glycoprotein; TM, transmembrane; HEK,
human embryonic kidney; MTS, methanethiosulfonate; PBS, phosphate-buffered
saline; NBD, nucleotide-binding domain; AM, acetoxymethyl ester. 
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