ABCMdb

PMID: 15379547

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 [PubMed]

Sentences

No. Mutations Sentence Comment

48 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg Histidine-tagged wild-type P-gp and mutants I306E and I306R were constructed as described previously (35, 36). Login to comment 77 ABCB1 p.Ile306Arg Stable cell lines expressing wild-type P-gp or mutant I306R were generated using the Flp-In system (Invitrogen, Canada). Login to comment 78 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 92 ABCB1 p.Ile306Cys A good candidate is mutant I306C in TM5 (Figure 1A). Login to comment 93 ABCB1 p.Ile306Cys 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). Login to comment 102 ABCB1 p.Ile306Cys Histidine-tagged mutant I306C was expressed in HEK 293 cells, isolated by nickel-chelate chromatography, and mixed with lipid. Login to comment 103 ABCB1 p.Ile306Cys 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. Login to comment 107 ABCB1 p.Ile306Cys Figure 2 shows that treatment of mutant I306C with either MTSET or MTSES profoundly affected the verapamil-stimulated ATPase activity. Login to comment 108 ABCB1 p.Ile306Cys ABCB1 p.Ile306Cys 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. Login to comment 109 ABCB1 p.Ile306Cys 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). Login to comment 113 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg 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 116 ABCB1 p.Ile306Cys Labeling of residue I306C with MTS-verapamil permanently activated P-gp ATPase activity (46). Login to comment 123 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg Figure 4 shows that mutation of Ile306 to glutamic acid or arginine significantly affected the apparent affinity for verapamil. Login to comment 125 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 132 ABCB1 p.Ile306Cys 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. Login to comment 133 ABCB1 p.Ile306Cys Histidine-tagged mutant I306C P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment 139 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg FIGURE 4: Verapamil-stimulated ATPase activity of wild-type and mutant I306R and I306E P-gps. Login to comment 144 ABCB1 p.Ile306Glu ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg The mutant I306R, however, showed little activation (<2-fold) even at 300 µM vinblastine. Login to comment 152 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg We then determined whether verapamil could interfere with ability of mutant I306R to confer resistance in transfected cells. Login to comment 154 ABCB1 p.Ile306Arg Mutant I306R was selected because it showed the largest decrease in apparent affinity for verapamil (Figure 4). Login to comment 158 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg These results show that mutant I306R was still able to interact with hydrophobic substrates. Login to comment 177 ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg 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 ABCB1 p.Ile306Arg 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 196 ABCB1 p.Ile306Cys 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. Login to comment 197 ABCB1 p.Leu531Cys Mutant L531C- (NBD1)/C1074C(NBD2) can be used as a control since the cysteines are in the nucleotide-binding domain (NBD) can be cross-linked (17) and should be accessible to membrane-permeant MTSEA but not to membrane-impermeant MTSES or MTSET (58). Login to comment 201 ABCB1 p.Ile306Cys 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. Login to comment 202 ABCB1 p.Gly872Cys In contrast to mutant L531C(NBD1)/C1074C- (NBD2), cross-linking of mutants I306C(TM5)/I868C(TM10, L339C(TM6)/F942C(TM12), and S222C(TM4)/G872C- (TM10) were significantly inhibited by MTSES and MTSET. Login to comment 209 ABCB1 p.Gly872Cys In panel A, membranes were prepared from HEK 293 cells expressing P-gp mutants L339C(TM6)/ F942C(TM11), I306C(TM5)/I868C(TM10), S222C(TM4)/G872C- (TM10), or G300C(TM5)/F770C(TM8) and were preincubated at 22 °C for 10 min in the presence (+) or absence (-) of various concentrations of MTSES or MTSET. Login to comment 212 ABCB1 p.Leu339Cys ABCB1 p.Ile306Cys 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. Login to comment 229 ABCB1 p.Ile306Cys Modification of mutant I306C by these compounds resulted in a change in affinity for verapamil (Figure 2). Login to comment 231 ABCB1 p.Phe770Cys Not all cysteines in the TM domains were accessible for modification by MTSES or MTSET. Cross-linking of mutants G300C(TM5)/F770C- (TM8) or C137C(TM2)/I935C(TM11), the cysteines of which are located outside the drug-binding pocket, was not affected by MTSES or MTSET. Login to comment 270 ABCB1 p.Ile306Arg Mutant I306R affected interaction of P-gp with only some drug substrates (Figures 4 and 5). Login to comment 272 ABCB1 p.Ile306Cys 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). Login to comment 275 ABCB1 p.Ile306Arg 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