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Originally published In Press as doi:10.1074/jbc.C300154200 on April 23, 2003

J. Biol. Chem., Vol. 278, Issue 23, 20449-20452, June 6, 2003
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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 {ddagger}

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
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Mutants—A 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 Mutants—The 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 {beta}-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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 {beta}-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 0.1–1 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 {beta}-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.

 

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 (7.7–8.1-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 {beta}-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.

 

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).



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  		FIG. 3.
Reversal of MTS-verapamil activation of mutant I306C. Histidine-tagged mutant I306C P-gps were expressed in HEK 293 cells, solubilized with n-dodecyl {beta}-D-maltoside, and treated without MTS-verapamil (Untreated) or with 0.3 mM MTS-verapamil (+ MTS-verapamil) The proteins were isolated by nickel-chelate chromatography and then incubated on ice with 20 mM dithiothreitol (+ DTT) or without dithiothreitol (None) for 20 min. The samples were diluted 10-fold with buffer containing 50 mM sodium phosphate, pH 8, 500 mM NaCl, and 0.1% (w/v) n-dodecyl {beta}-D-maltoside to reduce the concentration of dithiothreitol and imidazole, and P-gp was reisolated by nickel-chelate chromatography. The eluted P-gps were mixed with lipid and sonicated, and ATPase activity was determined in the presence (+ DTT/Ver) or absence (+ DTT)of1mM verapamil. The ATPase activity of an equivalent amount of the MTS-verapamil-treated I306C P-gp was determined in the presence of 0.2 mM sodium vanadate (+Vi). -Fold stimulation is the ratio of activity of the treated sample to that of an untreated sample.

 

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 70–85%. 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 {beta}-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).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 (7–8-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).

 

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).


   FOOTNOTES
 
* 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. Back

{ddagger} 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. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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