ABCB1 p.Ile306Arg
| Predicted by SNAP2: | A: D (63%), C: D (53%), D: D (85%), E: D (85%), F: N (53%), G: D (75%), H: D (80%), K: D (85%), L: D (63%), M: N (82%), N: D (71%), P: D (85%), Q: D (80%), R: D (85%), S: D (66%), T: D (63%), V: N (72%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] The drug-binding pocket of the human multidrug res... Biochemistry. 2004 Sep 28;43(38):12081-9. Loo TW, Bartlett MC, Clarke DM
The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium.
Biochemistry. 2004 Sep 28;43(38):12081-9., 2004-09-28 [PMID:15379547]
Abstract [show]
P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates. Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the drug-binding pocket is accessible to water.
Comments [show]
None has been submitted yet.
No. Sentence Comment
48 Histidine-tagged wild-type P-gp and mutants I306E and I306R were constructed as described previously (35, 36).
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ABCB1 p.Ile306Arg 15379547:48:54
status: NEW77 Stable cell lines expressing wild-type P-gp or mutant I306R were generated using the Flp-In system (Invitrogen, Canada).
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ABCB1 p.Ile306Arg 15379547:77:54
status: NEW78 Briefly, the full-length cDNAs of histidine-tagged wild-type P-gp and mutant I306R were subcloned into pcDNA5/FRT vector (Invitrogen, Canada) and cotransfected with the Flp recombinase vector pOG44 into the Flp-In-293 cells.
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ABCB1 p.Ile306Arg 15379547:78:77
status: NEW81 Cells expressing wild-type or mutant I306R P-gp were then grown in 24-well plates in the presence of various concentrations (0-2000 nM) of colchicine and in the presence or absence of 25 µM verapamil or 5 µM cyclosporin A.
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ABCB1 p.Ile306Arg 15379547:81:37
status: NEW84 Flp-In-293 cells stably expressing wild-type or mutant I306R P-gps were washed twice with PBS, pH 7.4, and incubated with PBS containing 10 mM sodium periodate for 30 min at 4 °C in the dark.
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ABCB1 p.Ile306Arg 15379547:84:55
status: NEW113 Therefore, an alternative approach to introduce a charged group at position 306 would be to mutate Ile306 to glutamic acid or arginine.
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ABCB1 p.Ile306Arg 15379547:113:99
status: NEW123 Histidine-tagged wild-type and mutants I306E and I306R P-gps were expressed in HEK 293 cells, isolated by nickel-chelate chromatography and mixed with lipid, and verapamil-stimulated ATPase activity was determined.
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ABCB1 p.Ile306Arg 15379547:123:49
status: NEW124 Figure 4 shows that mutation of Ile306 to glutamic acid or arginine significantly affected the apparent affinity for verapamil.
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ABCB1 p.Ile306Arg 15379547:124:32
status: NEW125 The wild-type P-gp and mutants I306R and I306E showed maximal stimulation of 16.1-, >10.8-, and 7-fold and S50 (concentration required for 50% stimulation) of 44, >2200, and 305 µM, respectively.
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ABCB1 p.Ile306Arg 15379547:125:31
status: NEW126 Since mutant I306R showed the largest change (50-fold) in affinity for verapamil (Figure 4), we tested whether the interaction of this mutant with drug substrates rhodamine B, vinblastine, or colchicine were also changed.
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ABCB1 p.Ile306Arg 15379547:126:13
status: NEW128 Figure 5 shows that the ATPase activity of mutant I306R in the presence of rhodamine B and colchicine was similar to that of the wild-type P-gp.
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ABCB1 p.Ile306Arg 15379547:128:50
status: NEW129 Both wild-type and mutant I306R showed a 6-7-fold maximal stimulation in the presence of rhodamine B and S50 concentrations of 58 and 67 µM, respectively.
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ABCB1 p.Ile306Arg 15379547:129:26
status: NEW130 Similarly, in the presence of colchicine, both wild-type and mutant I306R had maximal stimulation of about 6-6.5-fold and S50 concentrations of about 1 mM.
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ABCB1 p.Ile306Arg 15379547:130:68
status: NEW139 HEK 293 cells were transfected with wild-type P-gp or with P-gp mutants I306E or I306R (in wild-type background) cDNAs. After 24 h, the medium was replaced with fresh medium.
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ABCB1 p.Ile306Arg 15379547:139:81
status: NEW143 FIGURE 4: Verapamil-stimulated ATPase activity of wild-type and mutant I306R and I306E P-gps.
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ABCB1 p.Ile306Arg 15379547:143:71
status: NEW144 Histidine-tagged wild-type, mutant I306R or mutant I306E P-gps were expressed in HEK 293 cells and isolated by nickel-chelate chromatography.
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ABCB1 p.Ile306Arg 15379547:144:35
status: NEW146 FIGURE 5: Drug-stimulated ATPase activity of wild-type and mutant I306R in the presence of vinblastine, rhodamine B, or colchicine.
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ABCB1 p.Ile306Arg 15379547:146:66
status: NEW147 Histidine-tagged wild-type (filled symbols) and mutant I306R (open symbols) P-gps were expressed in HEK 293 cells and isolated by nickel-chelate chromatography.
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ABCB1 p.Ile306Arg 15379547:147:55
status: NEW151 The mutant I306R, however, showed little activation (<2-fold) even at 300 µM vinblastine.
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ABCB1 p.Ile306Arg 15379547:151:11
status: NEW152 These results indicate that mutation of Ile-306 to arginine interferes with its ability to interact with verapamil and vinblastine but not with rhodamine B or colchicine and that Ile-306 must contribute to the verapamil and vinblastine binding sites.
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ABCB1 p.Ile306Arg 15379547:152:40
status: NEW153 We then determined whether verapamil could interfere with ability of mutant I306R to confer resistance in transfected cells.
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ABCB1 p.Ile306Arg 15379547:153:76
status: NEW154 Mutant I306R was selected because it showed the largest decrease in apparent affinity for verapamil (Figure 4).
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ABCB1 p.Ile306Arg 15379547:154:7
status: NEW158 We avoided problems associated with generating stable cell lines that involve direct selection with cytotoxic compounds (potential for selecting a clone in which P-gp has additional mutations or problems with integration of P-gp into different chromosomal sites) by generating stable cell lines expressing wild-type or mutant I306R P-gps using the Flp-In system (Invitrogen, Carlsbad, CA).
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ABCB1 p.Ile306Arg 15379547:158:326
status: NEW159 Wild-type and mutant I306R P-gps were subcloned into the pcDNA5/FRT vector and cotransfected with the Flp recombinase expression vector pOG44 into the Flp-In-293 cells that contained a single Flp recombinase target (56).
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ABCB1 p.Ile306Arg 15379547:159:21
status: NEW162 To test whether mutation I306R affected folding and trafficking of the mutant protein to the cell surface, we performed cell-surface labeling experiments.
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ABCB1 p.Ile306Arg 15379547:162:25
status: NEW163 HEK 293 cells expressing wild-type and mutant I306R P-gp were treated with sodium periodate to oxidize the carbohydrate groups on proteins and then reacted with biotin hydrazide.
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ABCB1 p.Ile306Arg 15379547:163:46
status: NEW165 The cell-surface labeling experiments showed that equivalent levels of wild-type and mutant I306R P-gps were present at the cell surface (Figure 6A).
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ABCB1 p.Ile306Arg 15379547:165:92
status: NEW166 These results indicate that mutation of Ile-306 to arginine does not affect folding or trafficking of the mutant P-gp to the cell surface.
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ABCB1 p.Ile306Arg 15379547:166:40
status: NEW167 The cells expressing wild-type or mutant I306R P-gps were then incubated with various concentrations of colchicine and verapamil for several days.
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ABCB1 p.Ile306Arg 15379547:167:41
status: NEW169 In the absence of verapamil, cells expressing wild-type or mutant I306R showed about (D50 of 200-225 nM) 30-fold increase in resistance to colchicine relative to that of mock-transfected Flp-In-293 cells (D50 of 7 nM).
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ABCB1 p.Ile306Arg 15379547:169:66
status: NEW170 In the presence of 25 µM verapamil, however, cells expressing wild-type P-gp were much more sensitive to colchicine than cells expressing mutant I306R (Figure 6B).
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ABCB1 p.Ile306Arg 15379547:170:150
status: NEW172 By contrast, the relative resistance of mutant I306R to colchicine in the presence of verapamil was only slightly decreased (27.7-fold).
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ABCB1 p.Ile306Arg 15379547:172:47
status: NEW174 Mutation of Ile-306 to arginine had little effect on the ability of P-gp to interact with colchicine but greatly affected the ability of the enzyme to interact with verapamil.
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ABCB1 p.Ile306Arg 15379547:174:12
status: NEW175 To test whether mutant I302R could still interact with the hydrophobic substrate cyclosporin A, we incubated the cells expressing wild-type or mutant I306R P-gp with colchicine and 5 µM cyclosporin A. Figure 6 B shows that cyclosporin A inhibited the ability of the mutant P-gps to confer resistance to colchicine (D50`s of 28 and 35 nM for wild-type and mutant I306R, respectively).
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ABCB1 p.Ile306Arg 15379547:175:150
status: NEWX
ABCB1 p.Ile306Arg 15379547:175:367
status: NEW176 These results show that mutant I306R was still able to interact with hydrophobic substrates.
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ABCB1 p.Ile306Arg 15379547:176:31
status: NEW177 FIGURE 6: Cell-surface labeling and effect of verapamil and cyclosporin A on wild-type and mutant I306R P-gp-mediated colchicine resistance.
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ABCB1 p.Ile306Arg 15379547:177:98
status: NEW178 In panel A, Flp-In-293 cells stably expressing wild-type P-gp (wild-type), mutant I306R P-gp (I306R), or vector only (control) were treated with sodium periodate followed by biotin hydrazide.
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ABCB1 p.Ile306Arg 15379547:178:82
status: NEWX
ABCB1 p.Ile306Arg 15379547:178:94
status: NEW181 The positions of the mature (170 kDa) and core-glycosylated (140 kDa) P-gps are indicated. In panel B, Flp-In-293 cells lines stably expressing wild-type or mutant I306R P-gp were incubated with various concentrations of colchicine and with (+) or without (-) 25 µM verapamil or with or without 5 µM cyclosporin A (cyclo A).
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ABCB1 p.Ile306Arg 15379547:181:164
status: NEW270 Mutant I306R affected interaction of P-gp with only some drug substrates (Figures 4 and 5).
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ABCB1 p.Ile306Arg 15379547:270:7
status: NEW275 The verapamil-stimulated ATPase activity was altered in mutant I306R and was reflected in decreased ability of verapamil to modulate the resistance of the mutant to colchicine.
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ABCB1 p.Ile306Arg 15379547:275:63
status: NEW[hide] Suppressor mutations in the transmembrane segments... J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11. Loo TW, Bartlett MC, Clarke DM
Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.
J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PMID:17848563]
Abstract [show]
Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.
Comments [show]
None has been submitted yet.
No. Sentence Comment
1 The folding defect in ⌬F508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (⌬Y490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R).
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ABCB1 p.Ile306Arg 17848563:1:332
status: NEW7 The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold.
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ABCB1 p.Ile306Arg 17848563:7:106
status: NEWX
ABCB1 p.Ile306Arg 17848563:7:127
status: NEW40 Printed in the U.S.A. NOVEMBER 2, 2007•VOLUME 282•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 32043 essing mutants by a direct mechanism because suppressor mutations (arginines) introduced into TM segments 5 (I306R) and 6 (F343R) also promoted maturation of the protein (21).
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ABCB1 p.Ile306Arg 17848563:40:222
status: NEW69 For disulfide cross-linking analysis, the cDNA of mutant L339C(TM6)/ F728C(TM7) (34) was modified to also encode the L65R, T199R, I306R, or F343R mutations.
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ABCB1 p.Ile306Arg 17848563:69:130
status: NEW86 Disulfide Cross-linking Analysis-The double cysteine mutants L339C(TM6)/F728C(TM7), L65R(TM1)/L339C(TM6)/ F728C(TM7), T199R(TM3)/L339C(TM6)/F728C(TM7), I306R (TM5)/L339C(TM6)/F728C(TM7), or F343R(TM6)/L339C (TM6)/F728C(TM7) were transiently expressed in HEK 293 cells (32).
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ABCB1 p.Ile306Arg 17848563:86:152
status: NEW103 Therefore, the presence of arginine mutations in TM5 (I306R) or TM6 (F343R) might have promoted maturation of ⌬Y490 P-gp by influencing distal folding events.
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ABCB1 p.Ile306Arg 17848563:103:54
status: NEW165 Processing mutant G251V was chosen because it yields very low levels of mature 170-kDa protein and could be rescued by the presence of arginine in TM5 (I306R) or in TM6 (F343R) (21).
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ABCB1 p.Ile306Arg 17848563:165:152
status: NEW198 The presence of the I306R mutation, however, caused a large reduction in the apparent affinity for vinblastine (42 Ϯ 9 M; 60-fold) and for cyclosporin A (15 Ϯ 3 M; 25-fold) and had little effect on the apparent affinity for rhodamine B (96 Ϯ 11 M; 1.1-fold).
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ABCB1 p.Ile306Arg 17848563:198:20
status: NEW217 The reactions were stopped by the addition of trypsin inhibitor, and the samples were subjected to SDP-PAGE on 6% gels followed A B I306 I306R X-link 170 kDa X-link 170 kDa 0 0.08 0.7 2.2 6.6 19.8 59 178 533 1600 0.24 [Vin] µM 0.1 1 10 100 [Vinblastine] (µM) PercentCross-linked 20 40 60 80 100 0 I306R I306 1000 C D I306 I306R X-link 170 kDa X-link 170 kDa 0 0.007 0.06 0.18 0.54 1.6 4.9 14.6 44 132 0.02 [Cyclo] µM 0.01 0.1 1 10 [Cyclosporin] (µM) PercentCross-linked 20 40 60 80 100 0 I306R I306 100 E F I306 I306R X-link 170 kDa X-link 170 kDa 0 0.6 5.5 16.5 49 148 444 1333 4000 1.8 [Rhod B] µM 1 10 100 1000 [Rhodamine B] (µM) PercentCross-linked 20 40 60 80 100 0 I306R I306 10000 FIGURE 6.
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ABCB1 p.Ile306Arg 17848563:217:137
status: NEWX
ABCB1 p.Ile306Arg 17848563:217:307
status: NEWX
ABCB1 p.Ile306Arg 17848563:217:332
status: NEWX
ABCB1 p.Ile306Arg 17848563:217:508
status: NEWX
ABCB1 p.Ile306Arg 17848563:217:532
status: NEWX
ABCB1 p.Ile306Arg 17848563:217:701
status: NEW276 For example, it appeared that vinblastine interactions with mutant I306R were completely abolished because it did not stimulate ATPase activity at vinblastine concentrations of up to 200 M (45).
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ABCB1 p.Ile306Arg 17848563:276:67
status: NEW278 The results from the cross-linking protection assay, however, showed that mutant I306R could still interact with vinblastine but with reduced affinity (Fig. 6A).
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ABCB1 p.Ile306Arg 17848563:278:81
status: NEW[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]
Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.
Comments [show]
None has been submitted yet.
No. Sentence Comment
50 Previously identified suppressor mutations in TMD1 (I306R- (TM5) or F343R(TM6)) (40) did not promote maturation of TMD1 þ 2 (unpublished data).
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ABCB1 p.Ile306Arg 21182301:50:52
status: NEW[hide] Mutational analysis of ABC proteins. Arch Biochem Biophys. 2008 Aug 1;476(1):51-64. Epub 2008 Mar 5. Loo TW, Clarke DM
Mutational analysis of ABC proteins.
Arch Biochem Biophys. 2008 Aug 1;476(1):51-64. Epub 2008 Mar 5., [PMID:18328253]
Abstract [show]
The 49 human members of the ATP-binding cassette (ABC) family of proteins are involved in a wide range of activities such as active transport of compounds across membranes, extraction of compounds out of membranes, functioning as ion channels, or regulators of channel activity. Mutations and/or overexpression of many of the proteins can have adverse effects on health. A goal in the study of ABC proteins is to understand their mechanisms of action. This review will focus on the mutational approaches that have been used to study the structure and mechanisms of some ABC proteins.
Comments [show]
None has been submitted yet.
No. Sentence Comment
370 In drug-stimulated ATPase assays, it was found that the I306R mutation completely abolished vinblastine-stimulated ATPase activity but had little effect on rhodamine-stimulated ATPase activity [112].
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ABCB1 p.Ile306Arg 18328253:370:56
status: NEW374 The effects of I306R mutation on vinblastine interactions were similar in the ATPase and cross-linking assays.
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ABCB1 p.Ile306Arg 18328253:374:15
status: NEWX
ABCB1 p.Ile306Arg 18328253:374:33
status: NEWX
ABCB1 p.Ile306Arg 18328253:374:89
status: NEW376 For example, the L65R, T199R and I306R mutations inhibited vinblastine interactions, the I306R and F343R changes inhibited binding of cyclosporin A and the F343R mutation inhibited binding of rhodamine.
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ABCB1 p.Ile306Arg 18328253:376:33
status: NEWX
ABCB1 p.Ile306Arg 18328253:376:89
status: NEW368 In drug-stimulated ATPase assays, it was found that the I306R mutation completely abolished vinblastine-stimulated ATPase activity but had little effect on rhodamine-stimulated ATPase activity [112].
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ABCB1 p.Ile306Arg 18328253:368:56
status: NEW372 The effects of I306R mutation on vinblastine interactions were similar in the ATPase and cross-linking assays.
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ABCB1 p.Ile306Arg 18328253:372:15
status: NEW[hide] New light on multidrug binding by an ATP-binding-c... Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20. Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW
New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20., [PMID:16545467]
Abstract [show]
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.
Comments [show]
None has been submitted yet.
No. Sentence Comment
53 The G341A mutation almost completely abolishes the resistance to colchicine and daunomycin, but the resistance to vinblastine and actinomycin D remains at wild-type levels [17].
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ABCB1 p.Ile306Arg 16545467:53:84
status: NEW54 Residue I306 is at the interface between TMH5 and TMH6, and its replacement by Arg (I306R) results in a 50-fold reduction in affinity for verapamil but the affinity for rhodamine B and colchicines is unaltered [18].
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ABCB1 p.Ile306Arg 16545467:54:84
status: NEW58 Although mutation of only one of these residues (L975A, V981A and F983A) has no effect on the phenotype of the protein [20], double mutations either completely inhibit (V981A/F983A and L975A/V981A) or cause 50% inhibition (L975A/F983A) of Table 1.
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ABCB1 p.Ile306Arg 16545467:58:299
status: NEW59 Published mutations in human and murine P-glycoprotein that alter drug transport in cells Location of mutation Mutation Refs Mutation Refs Mutation Refs Transmembrane helices H61A and others [14] I214L [60] L868W [59] G64R [15] P223A [65] I936A [21] L65R [15] S224P [60] F938A [21] Q139[H/P/R] [60] I306R [18] S939[A/C/T/Y/W/D/F] [21,22] G141V [17] F335A [16] T941A [21] G185V [61,62] V338A [66] Q942A [21] I186N [61] G338A [67,68] A943G [21] G187V [17] A339P [67,68] Y946A [21] G187E [60] G341A [66] S948A [21] A192T [60] S344[A/T/C/Y] [66] Y949A [21] F200L [60] N350I [19] C952A [21] F204S [60] P709A [65] F953A [21] R206L [60] G830V [17] L975A [20] W208G [60] I837L [23] F978A [16] K209E [60] N839I [23] V981A [20] L210I [60] I862F [19] F983A [20] T211P [60] L865F [19] F978A [16] V213A [60] P866A [65] N988D [59] Intracellular domain T169I [60] K177I [60] G288V [17] R170L [60] E180G [60] A931T [19] L171P [60] G181R [60] F934A [21] T172P [60] G183D [60] G935A [21] S176P [60] D184N [60] NBD D555N [63] K1076M [69] E1197Q [64] D558N [64] D1093N [64] D1203N [64] D592N [64] E1125Q [64] D1237N [64] E604Q [64] S1173A [70] E1249Q [64] Review TRENDS in Pharmacological Sciences Vol.27 No.
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ABCB1 p.Ile306Arg 16545467:59:299
status: NEW[hide] Drug rescue distinguishes between different struct... Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2. Loo TW, Clarke DM
Drug rescue distinguishes between different structural models of human P-glycoprotein.
Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2., [PMID:24083983]
Abstract [show]
There is no high-resolution crystal structure of the human P-glycoprotein (P-gp) drug pump. Homology models of human P-gp based on the crystal structures of mouse or Caenorhabditis elegans P-gps show large differences in the orientation of transmembrane segment 5 (TM5). TM5 is one of the most important transmembrane segments involved in drug-substrate interactions. Drug rescue of P-gp processing mutants containing an arginine at each position in TM5 was used to identify positions facing the lipid or internal aqueous chamber. Only the model based on the C. elegans P-gp structure was compatible with the drug rescue results.
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No. Sentence Comment
14 In addition, it was found that the I306R mutation inhibited binding of a subset of P-gp drug substrates.13 These results suggest that residue Ile306 is important for binding of drug substrates and activation of Received: September 12, 2013 Revised: September 29, 2013 Published: October 1, 2013 Figure 1.
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ABCB1 p.Ile306Arg 24083983:14:35
status: NEW[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]
Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
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175 In the N-terminal TMD1 domain, the largest number of arginine mutations predicted to line the drug-binding pocket that inhibited tariquidar rescue were located in TM1 (H61R, G64R, L65R, M68R, M69R, and F72R) and TM5 (F303R, I306R, Y307R, S309R, and Y310R) (Fig. 4, A and E).
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ABCB1 p.Ile306Arg 26507655:175:224
status: NEW193 It was found that 16 of the 28 mutants resembled the G251V/I868R mutant as expression in the presence of 5 òe;M cyclosporine A yielded mature P-gp as the major product in TM1 (H61R, G64R, L65R, M68R, and M69R), TM5 (F303R, I306R, and S309R), TM7 (Q725R and F728R), TM10 (I868R and G872R), TM11 (F942R, T945R, and Q946R), and TM12 (V982R) (Fig. 7).
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ABCB1 p.Ile306Arg 26507655:193:227
status: NEW212 Seventeen of the 30 G251V/arginine mutants (M68R, M69R, and F72R in TM1; I306R, Y307R, S309R, and Y310R in TM5; F336R in TM6; F728R and F732R in TM7; I868R and G872R in TM10; F942R, T945R, M949R, and S952R in TM11; and V982R in TM12) that could not be rescued with tariquidar showed little or no stimulation of ATPase activity with tariquidar (Fig. 8A).
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ABCB1 p.Ile306Arg 26507655:212:73
status: NEW283 We identified 13 additional arginine mutations (H61R, G64R, L65R, and M68R in TM1; A129R in TM2; I306R and S309R in TM5; F343R in TM6; F942R, T945R, Q946R, and Y950R in TM11; and L975R in TM12) (Fig. 11A) that were not predicted to lie within 4.5-&#c5; of the predicted site 3 tariquidar-binding site (9).
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ABCB1 p.Ile306Arg 26507655:283:97
status: NEW