ABCMdb

PMID: 25053414

Loo TW, Clarke DM
Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.
J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22., [PubMed]

Sentences

No. Mutations Sentence Comment

51 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Mutations A80C, R741C, or A80C plus R741C were introduced into A52-tagged Cys-less P-gp or A52- or 10-histidine-tagged wild-type P-gps as described previously (25). Login to comment 65 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Drug Resistance Assays-Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutant A80C/R741C were generated as described previously (33). Login to comment 73 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Cross-linking of Mutant A80C/R741C in the Presence or Absence of ATP-HEK 293 cells were transiently transfected with the A52-tagged mutant A80C/R741C (in a Cys-less background) and then grown for 48 h at 30 &#b0;C to promote maturation of P-gp. Login to comment 82 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys P-gp Is Inhibited by Extracellular Loop Cross-linking 24750 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER RESULTS Cysteines Introduced into ECL 1 (A80C) and ECL 4 (R741C) of Cys-less P-gp Cross-link Spontaneously-The major goal of the study was to use disulfide cross-linking to test whether we could detect the open conformation of P-gp in intact cells and whether locking P-gp in an open conformation would have an effect on its activity. Login to comment 89 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys To test whether cysteines introduced at positions 80 and 741 would cross-link, mutants A80C, R741C, and A80C/R741C were constructed in a Cys-less P-gp background. Login to comment 97 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Mutants A80C, R741C, and A80C/R741C resembled the Cys-less parent because the 150-kDa immature protein was the major product when expressed in the absence of cyclosporine A (Fig. 2A). Login to comment 98 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cyclosporine A promoted maturation of the Cys-less, A80C, and R741C mutants to yield mature 170-kDa P-gp as the major product (Fig. 2A). Login to comment 99 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys By contrast, mutant A80C/R741C in the presence of cyclosporine A yielded a protein that migrated with slower mobility than the mature 170-kDa P-gp (Fig. 2A). Login to comment 109 ABCB1 p.Leu175Asn ABCB1 p.Ala80Arg ABCB1 p.Pro517Ile The distances (in &#c5;ngstrom) are between the Cॷ carbons of amino acids Ala-80/Arg-741, Leu-175/Asn-820, and Pro-517/Ile- 1050. Login to comment 112 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cysteines A80C/R741C in a Cys-less background cross-link when the mutant was rescued with a drug substrate. Login to comment 113 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys A, A52-tagged Cys-less P-gp (C-less) and mutants A80C, R741C, or A80C/R741C were transiently expressed in HEK 293 cells in the absence (afa;) or presence (af9;) of 5 òe;M cyclosporine A (Cyclo). Login to comment 114 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Whole cell SDS extracts (containing no thiol-reducing agent) were subjected to immunoblot analysis. B, whole cells SDS extracts of mutant A80C/R741C grown in the presence of cyclosporine A were treated without (afa;) or with (af9;) 10 mM DTT prior to immunoblot analysis. Login to comment 115 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The posi- tionsofcross-linked(X-link),mature(170-kDa),andimmature(150-kDa)P-gps are indicated. P-gp Is Inhibited by Extracellular Loop Cross-linking SEPTEMBER 5, 2014ߦVOLUME 289ߦNUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 24751 at SEMMELWEIS UNIV OF MEDICINE on December 12, was supported by the observation that treatment of mutant A80C/R741C grown in cyclosporine A with 10 mM DTT converted the slow migrating P-gp protein into the 170-kDa protein (Fig. 2B). Login to comment 116 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The results suggest that mature 170-kDa A80C/ R741C protein efficiently cross-links when expressed in the presence of cyclosporine A. Login to comment 117 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Cysteines A80C (ECL1) and R741C (ECL4) Can Cross-link Spontaneously in the Absence of Drug Substrates-Because maturation of mutant A80C/R741C in the Cys-less background required the presence of cyclosporine A, we wanted to determine whether this mutant, when introduced into a wild-type background, would cross-link spontaneously in the absence of cyclosporine A. Login to comment 119 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Accordingly, mutations A80C, R741C, and A80C/R741C were introduced into A52-tagged wild-type P-gp. Login to comment 122 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The expression of mutants A80C and R741C also resembled wild-type P-gp in that the major product was the 170-kDa protein. Login to comment 123 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The major product in mutant A80C/R741C, however, was a protein that migrated with slower mobility than the mature 170-kDa slower-migrating protein (Fig. 3). Login to comment 124 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Treatment of the slowly migrating A80C/ R741C protein with 10 mM DTT yielded the 170-kDa protein (Fig. 3). Login to comment 126 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys It is possible that mutant A80C/R741C showed efficient cross-linking because it was transiently expressed in HEK 293 cells. Login to comment 127 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys We then tested whether mutant A80C/R741C was also efficiently cross-linked if it was stably expressed in BHK cells. Login to comment 128 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Accordingly, BHK cells were cotransfected with A52-tagged wild-type P-gp or mutant A80C/R741C (in a wild-type background) cDNAs and a plasmid containing a neomycin marker (pWL-neo). Login to comment 133 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys By contrast, the major products in mutant A80C/R741C were the slowly migrating cross-linked and the 150-kDa proteins. Login to comment 134 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys It appeared that the mature 170-kDa A80C/R741C protein was cross-linked efficiently because little (b0d;5% of total P-gp) of the mature 170-kDa protein was present (Fig. 4, right panel). Login to comment 136 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Whole cell SDS extracts from cells expressing wild-type P-gp or mutant A80C/ R741C were treated with endoglycosidases that cleaved only core sugars (endoglycosidase H) or all carbohydrate (PNGase F). Login to comment 138 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys By contrast, the 150-kDa A80C/R741C immature protein, but not the cross-linked product, was sensitive to endoglycosidase H. Login to comment 139 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The cross-linked A80C/R741C protein was sensitive only to PNGase F (Fig. 4, right panel). Login to comment 142 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cysteines A80C (ECL1) and R741C (ECL4) Can Cross-link Spontaneously After Synthesis and Maturation of P-gp-Disulfide bonds are formed in the endoplasmic reticulum of eukaryotic cells (reviewed in Ref. 39). Login to comment 143 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys It is possible that A80C/R741C may form a disulfide bond during synthesis in the endoplasmic reticulum but that the cross-linked protein did not run with altered mobility in SDS-PAGE gels so that it was not detected. Login to comment 144 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys A way to overcome this problem was to test whether the disulfide bond in mutant A80C/R741C could reform if synthesis of new P-gp was blocked and cross-linked P-gp at the cell surface was reduced. Login to comment 145 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Accordingly, BHK cells expressing A52-tagged A80C/ R741C P-gp were pretreated for 1 h with cycloheximide to inhibit protein synthesis (40). Login to comment 146 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The cells were then treated briefly (1 min) with 5 mM DTT to reduce the A80C/R741C disulfide bond at the cell surface. Login to comment 152 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cysteines A80C/R741C in a wild-type background cross-link in the absence of drug substrates. Login to comment 153 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys A52-tagged WT P-gp and mutants A80C, R741C, or A80C/R741C were transiently expressed in HEK 293 cells in the absence of drug substrates. Login to comment 157 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Endoglycosidase digestion of wild-type and mutant A80C/ R741C P-gp expressed in BHK cells. Login to comment 158 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Whole cell SDS extracts of BHK cells expressing A52-tagged wild-type P-gp or mutant A80C/R741C (wild-type background) that were grown without drug substrates were treated without (afa;)orwith(af9;)endoglycosidaseHf (H)orPNGaseF(F)andsubjectedtoimmu- noblot analysis. Login to comment 159 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The positions of cross-linked (X-link), mature (170-kDa), immature (150-kDa), and unglycosylated (140-kDa) P-gps are indicated. P-gp Is Inhibited by Extracellular Loop Cross-linking 24752 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER About 50% of mutant A80C/R741C was cross-linked by 18 min and, at 54 min, the amount of cross-linked P-gp resembled that of the untreated control (Fig. 5). Login to comment 161 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys The A80C/R741C Disulfide Bond Reduces P-gp Drug-stimulated ATPase Activity-To test whether the A80C/R741C mutant was active, histidine-tagged wild-type P-gp or mutant A80C/R741C were transiently expressed in HEK 293 cells. Login to comment 165 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cross-linking of A80C and R741C reduced activity severely. Login to comment 167 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys It was found however, that the verapamil-stimulated ATPase activity of mutant A80C/R741C was not significantly different from wild-type P-gp when assayed in the presence of DTT (Fig. 6). Login to comment 169 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys The A80C/R741C Disulfide Bond Inhibits Drug Efflux-We then tested whether mutant A80C/R741C could confer drug resistance. Login to comment 172 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys We first tested whether the A80C/R741C disulfide bond would affect the ability of P-gp to confer drug resistance on BHK cells. Login to comment 173 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys BHK control cells and cells expressing equivalent levels of wild-type or mutant A80C/R741C P-gp were incubated with various concentrations of vinblastine or colchicine to determine the concentration needed to inhibit cell growth. Login to comment 176 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys By contrast, for cells expressing mutant A80C/R741C, the LD50s occurred at 2.3 and 0.6 nM (3-fold-increase in resistance) and 1.8 and 0.2 òe;M (about 3-fold increase in resistance) for vinblastine and colchicine, respectively. Login to comment 178 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys These results suggest that the presence of the A80C/R741C disulfide bond inhibited drug efflux. Login to comment 179 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Reduction of the A80C/R741C Disulfide Bond Promotes Drug Efflux-Would reduction of the A80C/R741C disulfide bond increase the ability of P-gp to confer drug resistance? Login to comment 182 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys We also found that significant reduction of the A80C/R741C bond required 100-fold (30 mM) higher levels of beta-mercaptoethanol.3 An alternative approach to reduce the A80C/R741C disulfide bond was be to use DTT. Login to comment 184 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Accordingly, BHK cells expressing A52-tagged mutant A80C/R741C were incubated with medium containing various concentrations of DTT. Login to comment 189 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys We then tested whether mutant A80C/R741C was able to confer resistance to vinblastine when incubated in the presence 3 T. W. Loo and D. M. Clarke, unpublished observations. Login to comment 191 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Mature A80C/R741C P-gp cross-links after treatment with DTT. Login to comment 192 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys BHK cells expressing A52-tagged mutant A80C/R741C (wild-type background) were incubated for 1 h in the presence of 0.5 mg/ml cycloheximide. Login to comment 198 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cross-linking of mutant A80C/R741C inhibits verapamil-stimulated ATPase activity. Login to comment 199 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Histidine-tagged P-gp (WT) or mutant A80C/R741C (in a wild-type background) was transiently expressed in HEK 293 cells. Login to comment 203 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Accordingly, control BHK cells and BHK cells expressing equivalent levels of wild-type or mutant A80C/R741C P-gp were incubated in the presence of various concentrations of vinblastine and 1.1 mM DTT. Login to comment 204 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Because this assay required about 6 days, it was necessary to change the medium daily because the half-life of DTT is only several hours (47) and because the reduced A80C/R741C mutant could readily reform the cross-link (Fig. 5). Login to comment 205 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys It was found that wild-type P-gp conferred a similar level of resistance to vinblastine in the presence of DTT (about 60-fold) (Fig. 8B), whereas mutant A80C/R741C showed an approximately 25-fold increase in the ability to confer resistance (10-fold increase compared with the absence of DTT). Login to comment 206 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The results indicate that mutant A80C/R741C can confer relatively high levels of drug resistance when the disulfide bond is reduced. Login to comment 207 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Cysteines A80C/R741C Cross-link rapidly in the Absence or Presence of ATP-Spontaneous cross-linking of mutant A80C/ R741C after treatment of the cells with 5 mM DTT was relatively slow because about 50% cross-linking occurred after 18 min at 37 &#b0;C (Fig. 5). Login to comment 210 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys We first tested whether ATP hydrolysis was required for spontaneous cross-linking of A80C/R741C. Login to comment 212 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln These catalytic carboxylates have been shown previously to be essential for ATP hydrolysis because mutating both Glu-556 and Glu-1201 to glutamine completely abolished ATPase activity (48, 49). Login to comment 213 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys HEK 293 cells were then transfected with A52-tagged wild-type P-gp, mutant A80C/R741C (in a wild-type background), or A80C/R741C/E556Q/E1201Q (in a wild-type background). Login to comment 215 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln It was observed that the presence of E556Q/E1201Q mutations did not inhibit cross-linking (Fig. 9A). Login to comment 216 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Both the A80C/R741C and A80C/R741C/E556Q/E1201Q mutants yielded cross-linked P-gp as the major product. Login to comment 218 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys These results indicate that ATP hydrolysis was not essential for cross-linking mutant A80C/R741C. Login to comment 219 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys We then determined whether spontaneous cross-linking of A80C/R741C and A80C/R741C/E556Q/E1201Q mutants was inefficient by testing the effect of an oxidant (copper phenanthroline) to catalyze formation of the disulfide bond at a reduced temperature. Login to comment 220 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys HEK 293 cells were transfected with the A52-tagged mutants A80C/R741C and A80C/R741C/E556Q/ E1201Q (in a wild-type background). Login to comment 224 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cross-linking of A80C/R741C inhibits P-gp-mediated drug resistance. Login to comment 225 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys BHK cells expressing no P-gp, A52-tagged wild-type P-gp (WT) or mutant A80C/R741C (in a wild-type background) were incubated in the presence of various concentrations of vinblastine or colchicine to determine the LD50 concentration. Login to comment 230 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Expression of cells with DTT enhances the ability of mutant A80C/R741C to confer drug resistance. Login to comment 231 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys A, BHK cells expressing A52-tagged mutant A80C/R741C (in a wild-type background) were incubated for 18 h in the presence of various concentrations of DTT. Login to comment 234 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys B, BHK cells expressing A52-tagged wild-type P-gp (WT), mutant A80C/R741C, or no P-gp were incubated in the presence of various concentrations of vinblastine and 1.1 mM DTT to determine the LD50 concentration. Login to comment 238 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys P-gp Is Inhibited by Extracellular Loop Cross-linking 24754 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER stopped by addition of SDS sample buffer containing 50 mM EDTA and no reducing agent. Samples were then subjected to immunoblot analysis. Fig. 9B shows that treatment of DTT-treated cells with oxidant almost completely cross-linked the mature 170-kDa protein in both mutants, A80C/R741C and A80C/R741C/E556Q/E1201Q, within 1 min. Login to comment 240 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Next, we tested whether ATP binding was required for cross-linking of mutant A80C/R741C. Login to comment 241 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys This required the use of membranes prepared from cells expressing mutant A80C/R741C in the Cys-less background. Login to comment 243 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Accordingly, HEK 293 cells were transfected with the A52-tagged mutant A80C/R741C. Login to comment 251 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Therefore, we tested whether vanadate trapping of nucleotides could inhibit cross-linking of mutant A80C/R741C. Login to comment 252 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Membranes were prepared from HEK 293 cells expressing A52-tagged A80C/R741C that were treated with DTT. Login to comment 254 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The reactions were stopped by addition of 2afb; SDS sample buffer containing no thiol-reducing agent. Samples were then subjected to immunoblot analysis. Fig. 10B shows that vanadate trapping of nucleotides inhibits cross-linking of mutant A80C/R741C. Login to comment 255 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys DISCUSSION We found that linking the P-gp halves by direct cross-linking of extracellular cysteines A80C/R741C inhibited ATPase activity. Login to comment 257 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys Direct cross-linking of the cysteines L175C and N820C (23) or cross-linking of mutant P571C/I1050C with the short 1,4-butanediyl bismethanethiosulfonate cross-linker (7.8 &#c5;) (24) highly activated P-gp ATPase activity over 10-fold in the absence of drug substrates. Login to comment 258 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Cysteines L175C/N820C are located in the intracellular loops (Fig. 1, C and D), whereas cysteines P517C/I1050C are located in the NBDs (Fig. 1, C and D). Login to comment 259 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys An explanation for the difference is that linkage of the homologous halves at the L175C/N820C or P517C/I1050C sites would tend to favor the closed conformation (Fig. 1C) to FIGURE 9. Login to comment 260 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Cross-linking of mutant A80C/R741C does not require ATP hydrolysis and is rapid. Login to comment 261 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys A, HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants A80C/R741C (in a wild-type background), or A80C/ R741C/E556Q/E1201Q. Login to comment 263 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys Samples were then subjected to immunoblot analysis. B, HEK 293 cells expressing A52-tagged mutants A80C/R741C or A80C/R741C/E556Q/E1201Q (in a wild-type background) were treated with 10 mM DTT for 3 min. Login to comment 268 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ATP is not required for cross-linking mutant A80C/R741C. Login to comment 269 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys A, HEK 293 cells expressing A52-tagged mutant A80C/R741C (in a Cys-less background) were treated with 10 mM DTT for 3 min. Login to comment 280 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Direct linkage of A80C and R741C would favor the open conformation (Fig. 1B). Login to comment 285 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys In agreement with the mouse P-gp studies, we found that ATP did not cause any detectable changes in A80C/R741C cross-linking efficiency (Fig. 10). Login to comment 303 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Inhibition of P-gp by cross-linking of A80C/R741C was different from antibody inhibition because both ATPase activity and drug efflux were inhibited. Login to comment