ABCB1 p.Ile306Cys
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PMID: 12711602
[PubMed]
Loo TW et al: "Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site."
No.
Sentence
Comment
5
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.
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ABCB1 p.Ile306Cys 12711602:5:197
status: NEWX
ABCB1 p.Ile306Cys 12711602:5:278
status: NEW7 Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil.
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ABCB1 p.Ile306Cys 12711602:7:134
status: NEW8 The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site.
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ABCB1 p.Ile306Cys 12711602:8:51
status: NEW9 Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites.
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ABCB1 p.Ile306Cys 12711602:9:25
status: NEW11 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.
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ABCB1 p.Ile306Cys 12711602:11:16
status: NEW70 One mutant, I306C, showed an 8-fold increase in ATPase activity after treatment with MTS-verapamil.
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ABCB1 p.Ile306Cys 12711602:70:12
status: NEW72 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).
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ABCB1 p.Ile306Cys 12711602:72:73
status: NEW73 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.
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ABCB1 p.Ile306Cys 12711602:73:30
status: NEW74 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.
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ABCB1 p.Ile306Cys 12711602:74:67
status: NEW76 Fig. 2 shows the effect of various stimulators and inhibitors on the ATPase activity of mutant I306C before and after treatment with MTS-verapamil.
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ABCB1 p.Ile306Cys 12711602:76:95
status: NEW77 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.
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ABCB1 p.Ile306Cys 12711602:77:67
status: NEW79 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).
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ABCB1 p.Ile306Cys 12711602:79:12
status: NEW80 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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ABCB1 p.Ile306Cys 12711602:80:96
status: NEWX
ABCB1 p.Ile306Cys 12711602:80:151
status: NEW82 Activation of mutant I306C after treatment with MTS-verapamil.
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ABCB1 p.Ile306Cys 12711602:82:21
status: NEW83 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.
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ABCB1 p.Ile306Cys 12711602:83:36
status: NEW90 Effect of drug substrates and inhibitors on activity of mutant I306C before and after labeling with MTS-verapamil.
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ABCB1 p.Ile306Cys 12711602:90:63
status: NEW91 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).
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ABCB1 p.Ile306Cys 12711602:91:24
status: NEW96 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.
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ABCB1 p.Ile306Cys 12711602:96:12
status: NEW97 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.
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ABCB1 p.Ile306Cys 12711602:97:21
status: NEW98 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.
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ABCB1 p.Ile306Cys 12711602:98:181
status: NEW99 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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ABCB1 p.Ile306Cys 12711602:99:7
status: NEW100 The characteristics of the ATPase activity of modified I306C mutant were also examined.
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ABCB1 p.Ile306Cys 12711602:100:55
status: NEW103 Fig. 3 shows that mutant I306C that had been labeled with MTS-verapamil was still inhibited by vanadate.
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ABCB1 p.Ile306Cys 12711602:103:25
status: NEW104 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).
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ABCB1 p.Ile306Cys 12711602:104:33
status: NEW105 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.
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ABCB1 p.Ile306Cys 12711602:105:58
status: NEW106 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.
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ABCB1 p.Ile306Cys 12711602:106:19
status: NEW107 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%.
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ABCB1 p.Ile306Cys 12711602:107:62
status: NEWX
ABCB1 p.Ile306Cys 12711602:107:153
status: NEW109 DISCUSSION Treatment of mutant I306C with MTS-verapamil followed by removal of unreacted MTS-verapamil caused the mutant to FIG. 3.
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ABCB1 p.Ile306Cys 12711602:109:31
status: NEW110 Reversal of MTS-verapamil activation of mutant I306C.
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ABCB1 p.Ile306Cys 12711602:110:47
status: NEW111 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.
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ABCB1 p.Ile306Cys 12711602:111:24
status: NEW114 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).
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ABCB1 p.Ile306Cys 12711602:114:73
status: NEW117 Protection of mutant I306C from labeling by MTS-verapamil with substrates and inhibitors.
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ABCB1 p.Ile306Cys 12711602:117:21
status: NEW118 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.
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ABCB1 p.Ile306Cys 12711602:118:24
status: NEW124 Model of interaction between mutant I306C and verapamil or MTS-verapamil.
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ABCB1 p.Ile306Cys 12711602:124:36
status: NEW136 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).
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ABCB1 p.Ile306Cys 12711602:136:40
status: NEW137 In addition, activation of I306C by MTS-verapamil was reversed by dithiothreitol, and the dithiothreitol-treated mutant could rebind verapamil.
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ABCB1 p.Ile306Cys 12711602:137:27
status: NEW138 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.
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ABCB1 p.Ile306Cys 12711602:138:119
status: NEWX
ABCB1 p.Ile306Cys 12711602:138:211
status: NEW150 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.
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ABCB1 p.Ile306Cys 12711602:150:133
status: NEW151 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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ABCB1 p.Ile306Cys 12711602:151:22
status: NEW
PMID: 12909621
[PubMed]
Loo TW et al: "Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein."
No.
Sentence
Comment
180
Residue I306C covalently binds to methanethiosulfonate-verapamil, and the P-gp ATPase activity is permanently activated.
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ABCB1 p.Ile306Cys 12909621:180:8
status: NEW
PMID: 14670948
[PubMed]
Loo TW et al: "Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane."
No.
Sentence
Comment
74
Transmembrane segments 5 and 6 are particularly interesting because labeling of mutants I306C (TM5) and F343C(TM6) with methanethiosulfonate (MTS)-verapamil or MTS-rhodamine, respectively, caused permanent activation of P-gp ATPase activity (23, 44).
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ABCB1 p.Ile306Cys 14670948:74:88
status: NEW92 Residues that react with the MTS derivatives of drug substrates verapamil (I306C) and rhodamine (F343C) to stimulate ATPase activity are shown as closed circles.
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ABCB1 p.Ile306Cys 14670948:92:75
status: NEW
PMID: 15379547
[PubMed]
Loo TW et al: "The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium."
No.
Sentence
Comment
92
A good candidate is mutant I306C in TM5 (Figure 1A).
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ABCB1 p.Ile306Cys 15379547:92:27
status: NEW93 Cys-306 is close to the verapamil-binding site because treatment of mutant I306C with thiol-reactive MTS-verapamil resulted in covalent attachment of the verapamil to Cys-306 (46).
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ABCB1 p.Ile306Cys 15379547:93:75
status: NEW102 Histidine-tagged mutant I306C was expressed in HEK 293 cells, isolated by nickel-chelate chromatography, and mixed with lipid.
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ABCB1 p.Ile306Cys 15379547:102:24
status: NEW103 Mutant I306C was then treated with 1 mM MTSET or 5 mM MTSES and assayed for verapamil-stimulated ATPase activity after removal of untreated MTSET or MTSES using gel filtration columns.
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ABCB1 p.Ile306Cys 15379547:103:7
status: NEW107 Figure 2 shows that treatment of mutant I306C with either MTSET or MTSES profoundly affected the verapamil-stimulated ATPase activity.
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ABCB1 p.Ile306Cys 15379547:107:40
status: NEW108 Treatment of mutant I306C with MTSET reduced the apparent affinity of P-gp for verapamil from 24 µM to greater than 600 µM. Treatment of mutant I306C with MTSES caused about a 10-fold reduction in apparent affinity (260 µM) for verapamil, as well as about 50% decrease in ATPase activity.
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ABCB1 p.Ile306Cys 15379547:108:20
status: NEWX
ABCB1 p.Ile306Cys 15379547:108:154
status: NEW109 The change in activity was apparently due to modification of residue I306C since 1 mM MTSET or 5 mM MTSES had no effect on basal or verapamil-stimulated ATPase activity of cysteine-less P-gp (data not shown).
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ABCB1 p.Ile306Cys 15379547:109:69
status: NEW116 Labeling of residue I306C with MTS-verapamil permanently activated P-gp ATPase activity (46).
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ABCB1 p.Ile306Cys 15379547:116:20
status: NEW132 Wild-type P-gp showed an 8.2-fold maximal activation and FIGURE 2: Effect of MTSES or MTSET on verapamil-stimulated ATPase activity of mutant I306C.
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ABCB1 p.Ile306Cys 15379547:132:142
status: NEW133 Histidine-tagged mutant I306C P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography.
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ABCB1 p.Ile306Cys 15379547:133:24
status: NEW196 Accordingly, cells expressing mutants L531C(NBD1)/C1074C(NBD2), I306C- (TM5)/I868C(TM10), L339C(TM6)/F942C(TM12), and S222C(TM4)/G872C(TM10) were treated with MTSEA, MTSES, or MTSET before cross-linking.
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ABCB1 p.Ile306Cys 15379547:196:64
status: NEW201 Cross-linking of mutants with cysteines within the drug-binding pocket (mutants I306C- (TM5)/I868C(TM10), L339C(TM6)/F942C(TM12), and S222C(TM4)/G872C(TM10)) was also completely inhibited by MTSEA.
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ABCB1 p.Ile306Cys 15379547:201:80
status: NEW212 Samples of mutants L339C/(TM6)/F942C(TM11), I306C- (TM5)/I868C(TM10), and S222C(TM4)/G872C(TM10) were then treated with (+) or without (-) M17M cross-linker for 15 min at 22 °C. Mutant G300C(TM5)/F770C(TM8) was cross-linked with 1 mM copper (phenanthroline)3 (CuP) for 15 min at 22 °C. In panel B, whole cells expressing mutants L531C(NBD1)/C1074C(NBD2), I306C(TM5)/I868C(TM10), L339C(TM6)/F942C(TM11), or S222C- (TM4)/G872C(TM10) were incubated for 10 min at 22 °C in the presence of 2.5 mM MTSEA, 10 mM MTSES, or 1 mM MTSET.
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ABCB1 p.Ile306Cys 15379547:212:44
status: NEW229 Modification of mutant I306C by these compounds resulted in a change in affinity for verapamil (Figure 2).
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ABCB1 p.Ile306Cys 15379547:229:23
status: NEW272 The mutant, however, could still interact with hydrophobic substrates such as cyclosporin A (Figure 6B) These results are consistent with the labeling studies involving I306C with MTS-rhodamine and MTS-verapamil that show distinct binding sites for the verapamil and rhodamine B (30).
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ABCB1 p.Ile306Cys 15379547:272:169
status: NEW
PMID: 16472108
[PubMed]
Srinivas E et al: "Recent advances in molecular modeling and medicinal chemistry aspects of phospho-glycoprotein."
No.
Sentence
Comment
156
It is also reported that MTS-verapamil is responsible for the permanent activation of ATP-hydrolysis by covalent modification of a residue I306C in the drug-binding site [80].
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ABCB1 p.Ile306Cys 16472108:156:139
status: NEW
PMID: 16492138
[PubMed]
Loo TW et al: "Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket."
No.
Sentence
Comment
72
The locations of residues L65C in TM1, I306C in TM5 and F343C in TM6 are shown.
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ABCB1 p.Ile306Cys 16492138:72:39
status: NEW212 While the ATPase activities of both mutants L65C and I306C modified by MTS-verapamil could not be further stimulated by calcein-AM or demecolcine, the inhibition of their activities were different.
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ABCB1 p.Ile306Cys 16492138:212:53
status: NEW213 The ATPase activity of mutant I306C modified by MTS-verapamil could not be inhibited by cyclosporin A or trans-(E)-flupentixol [40], whereas that of MTS-verapamil- modified mutant L65C was inhibited only by cyclosporin A (Figure 5).
X
ABCB1 p.Ile306Cys 16492138:213:30
status: NEW229 Therefore the presence of a permanently bound verapamil molecule at I306C may interfere with movement between TM1 and TM11 during ATP hydrolysis (Figure 1B), resulting in inhibition of ATPase activity.
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ABCB1 p.Ile306Cys 16492138:229:68
status: NEW
PMID: 16813563
[PubMed]
Loo TW et al: "Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket."
No.
Sentence
Comment
90
We have previously shown that modification of specific cysteines in TM1 (L65C) and in TM5 (I306C) with MTS-verapamil caused permanent activation of P-gp ATPase activity (an 8to 11-fold increase in activity compared with untreated P-gp) [27,36].
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ABCB1 p.Ile306Cys 16813563:90:91
status: NEW161 cysteines facing the drug-binding pocket that may be cross-linked with F728C are L65C(TM1), I306C(TM5) and F343C(TM6) because they were covalently modified with MTS-verapamil (L65C and I306C) or with MTS-Rhodamine (F343C).
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ABCB1 p.Ile306Cys 16813563:161:185
status: NEW
PMID: 22380603
[PubMed]
Sager G et al: "Novel cGMP efflux inhibitors identified by virtual ligand screening (VLS) and confirmed by experimental studies."
No.
Sentence
Comment
85
This is in accordance with a study on ABCB1 where cysteine-scanning mutagenesis and reaction with a methanethiosulfonate (MTS) thiol reactive analogue of verapamil (MTS verapamil) showed that mutants Leu65Cys (TMH1) and Ile306Cys (TMH5) modified with MTS verapamil have slightly different characteristics, indicating that the bound verapamil molecules in these mutants have different orientations and that the protein can function quite well with the substrate bound in different orientations.28 Theoretically, even though 10 different conformations of each ligand were evaluated by ICM during VLS, focusing on only the ligand orientation with best score may lead to missing out better inhibitors oriented in a pose yielding a poorer score.
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ABCB1 p.Ile306Cys 22380603:85:220
status: NEW
PMID: 22700974
[PubMed]
Loo TW et al: "The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together."
No.
Sentence
Comment
254
For example, covalent labeling of F728C (TM7) (22), L65C (TM1) (57), or I306C (TM5) (24) with a thiol-reactive derivative of verapamil increased basal ATPase activity of P-gp by 7-12-fold.
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ABCB1 p.Ile306Cys 22700974:254:72
status: NEW247 For example, covalent labeling of F728C (TM7) (22), L65C (TM1) (57), or I306C (TM5) (24) with a thiol-reactive derivative of verapamil increased basal ATPase activity of P-gp by 7-12-fold.
X
ABCB1 p.Ile306Cys 22700974:247:72
status: NEW
PMID: 24083983
[PubMed]
Loo TW et al: "Drug rescue distinguishes between different structural models of human P-glycoprotein."
No.
Sentence
Comment
13
It was found that labeling of the I306C mutant with a thiol-reactive derivative of the substrate verapamil activated ATPase activity ~8-fold12 and labeling was blocked by verapamil.
X
ABCB1 p.Ile306Cys 24083983:13:34
status: NEW