ABCMdb

PMID: 12711602

Loo TW, Bartlett MC, Clarke DM
Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site.
J Biol Chem. 2003 Jun 6;278(23):20449-52. Epub 2003 Apr 23., 2003-06-06 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCB1 p.Ile306Cys ABCB1 p.Ile306Cys 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. Login to comment 7 ABCB1 p.Ile306Cys Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. Login to comment 8 ABCB1 p.Ile306Cys The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Login to comment 9 ABCB1 p.Ile306Cys Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. Login to comment 11 ABCB1 p.Ile306Cys 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. Login to comment 70 ABCB1 p.Ile306Cys One mutant, I306C, showed an 8-fold increase in ATPase activity after treatment with MTS-verapamil. Login to comment 72 ABCB1 p.Ile306Cys 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 ␮M. 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). Login to comment 73 ABCB1 p.Ile306Cys 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. Login to comment 74 ABCB1 p.Ile306Cys 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. Login to comment 76 ABCB1 p.Ile306Cys Fig. 2 shows the effect of various stimulators and inhibitors on the ATPase activity of mutant I306C before and after treatment with MTS-verapamil. Login to comment 77 ABCB1 p.Ile306Cys 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. Login to comment 79 ABCB1 p.Ile306Cys 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). Login to comment 80 ABCB1 p.Ile306Cys ABCB1 p.Ile306Cys 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. Login to comment 82 ABCB1 p.Ile306Cys Activation of mutant I306C after treatment with MTS-verapamil. Login to comment 83 ABCB1 p.Ile306Cys 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. Login to comment 90 ABCB1 p.Ile306Cys Effect of drug substrates and inhibitors on activity of mutant I306C before and after labeling with MTS-verapamil. Login to comment 91 ABCB1 p.Ile306Cys 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 (ϩ MTS-verapamil) or without MTS-verapamil (Untreated). Login to comment 96 ABCB1 p.Ile306Cys 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. Login to comment 97 ABCB1 p.Ile306Cys 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. Login to comment 98 ABCB1 p.Ile306Cys 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. Login to comment 99 ABCB1 p.Ile306Cys 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). Login to comment 100 ABCB1 p.Ile306Cys The characteristics of the ATPase activity of modified I306C mutant were also examined. Login to comment 103 ABCB1 p.Ile306Cys Fig. 3 shows that mutant I306C that had been labeled with MTS-verapamil was still inhibited by vanadate. Login to comment 104 ABCB1 p.Ile306Cys 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). Login to comment 105 ABCB1 p.Ile306Cys 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. Login to comment 106 ABCB1 p.Ile306Cys 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. Login to comment 107 ABCB1 p.Ile306Cys ABCB1 p.Ile306Cys 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%. Login to comment 109 ABCB1 p.Ile306Cys DISCUSSION Treatment of mutant I306C with MTS-verapamil followed by removal of unreacted MTS-verapamil caused the mutant to FIG. 3. Login to comment 110 ABCB1 p.Ile306Cys Reversal of MTS-verapamil activation of mutant I306C. Login to comment 111 ABCB1 p.Ile306Cys 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 (ϩ MTS-verapamil) The proteins were isolated by nickel-chelate chromatography and then incubated on ice with 20 mM dithiothreitol (ϩ DTT) or without dithiothreitol (None) for 20 min. Login to comment 114 ABCB1 p.Ile306Cys 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 (ϩVi). Login to comment 117 ABCB1 p.Ile306Cys Protection of mutant I306C from labeling by MTS-verapamil with substrates and inhibitors. Login to comment 118 ABCB1 p.Ile306Cys 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. Login to comment 124 ABCB1 p.Ile306Cys Model of interaction between mutant I306C and verapamil or MTS-verapamil. Login to comment 136 ABCB1 p.Ile306Cys 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). Login to comment 137 ABCB1 p.Ile306Cys In addition, activation of I306C by MTS-verapamil was reversed by dithiothreitol, and the dithiothreitol-treated mutant could rebind verapamil. Login to comment 138 ABCB1 p.Ile306Cys ABCB1 p.Ile306Cys 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. Login to comment 150 ABCB1 p.Ile306Cys 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. Login to comment 151 ABCB1 p.Ile306Cys 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). Login to comment