ABCMdb

ABCB1 p.Ile306Arg

None has been submitted yet.

Publications

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

48 Histidine-tagged wild-type P-gp and mutants I306E and I306R were constructed as described previously (35, 36). Login to comment
77 Stable cell lines expressing wild-type P-gp or mutant I306R were generated using the Flp-In system (Invitrogen, Canada). Login to comment
78 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. Login to comment
81 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. Login to comment
84 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. Login to comment
113 Therefore, an alternative approach to introduce a charged group at position 306 would be to mutate Ile306 to glutamic acid or arginine. Login to comment
123 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. Login to comment
124 Figure 4 shows that mutation of Ile306 to glutamic acid or arginine significantly affected the apparent affinity for verapamil. Login to comment
125 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. Login to comment
126 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. Login to comment
128 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. Login to comment
129 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. Login to comment
130 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. Login to comment
139 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. Login to comment
143 FIGURE 4: Verapamil-stimulated ATPase activity of wild-type and mutant I306R and I306E P-gps. Login to comment
144 Histidine-tagged wild-type, mutant I306R or mutant I306E P-gps were expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment
146 FIGURE 5: Drug-stimulated ATPase activity of wild-type and mutant I306R in the presence of vinblastine, rhodamine B, or colchicine. Login to comment
147 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. Login to comment
151 The mutant I306R, however, showed little activation (<2-fold) even at 300 µM vinblastine. Login to comment
152 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. Login to comment
153 We then determined whether verapamil could interfere with ability of mutant I306R to confer resistance in transfected cells. Login to comment
154 Mutant I306R was selected because it showed the largest decrease in apparent affinity for verapamil (Figure 4). Login to comment
158 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). Login to comment
159 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). Login to comment
162 To test whether mutation I306R affected folding and trafficking of the mutant protein to the cell surface, we performed cell-surface labeling experiments. Login to comment
163 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. Login to comment
165 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). Login to comment
166 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. Login to comment
167 The cells expressing wild-type or mutant I306R P-gps were then incubated with various concentrations of colchicine and verapamil for several days. Login to comment
169 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). Login to comment
170 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). Login to comment
172 By contrast, the relative resistance of mutant I306R to colchicine in the presence of verapamil was only slightly decreased (27.7-fold). Login to comment
174 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. Login to comment
175 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). Login to comment
176 These results show that mutant I306R was still able to interact with hydrophobic substrates. Login to comment
177 FIGURE 6: Cell-surface labeling and effect of verapamil and cyclosporin A on wild-type and mutant I306R P-gp-mediated colchicine resistance. Login to comment
178 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. Login to comment
181 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). Login to comment
270 Mutant I306R affected interaction of P-gp with only some drug substrates (Figures 4 and 5). Login to comment
275 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. Login to comment

PMID: 17848563 [PubMed] Loo TW et al: "Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites."

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). Login to comment
7 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. Login to comment
40 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). Login to comment
69 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. Login to comment
86 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). Login to comment
103 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. Login to comment
165 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). Login to comment
198 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). Login to comment
217 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. Login to comment
276 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). Login to comment
278 The results from the cross-linking protection assay, however, showed that mutant I306R could still interact with vinblastine but with reduced affinity (Fig. 6A). Login to comment

PMID: 21182301 [PubMed] Loo TW et al: "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."

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). Login to comment

PMID: 18328253 [PubMed] Loo TW et al: "Mutational analysis of ABC proteins."

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]. Login to comment
374 The effects of I306R mutation on vinblastine interactions were similar in the ATPase and cross-linking assays. Login to comment
376 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. Login to comment
368 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]. Login to comment
372 The effects of I306R mutation on vinblastine interactions were similar in the ATPase and cross-linking assays. Login to comment

PMID: 16545467 [PubMed] Shilling RA et al: "New light on multidrug binding by an ATP-binding-cassette transporter."

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]. Login to comment
54 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]. Login to comment
58 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. Login to comment
59 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. Login to comment

PMID: 24083983 [PubMed] Loo TW et al: "Drug rescue distinguishes between different structural models of human P-glycoprotein."

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. Login to comment

PMID: 26507655 [PubMed] Loo TW et al: "Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein."

No. Sentence Comment

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). Login to comment
193 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). Login to comment
212 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). Login to comment
283 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). Login to comment