ABCMdb

ABCB1 p.Ala80Cys

None has been submitted yet.

Publications

PMID: 25053414 [PubMed] Loo TW et al: "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."

No. Sentence Comment

51 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 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 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 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 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 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 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 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
112 Cysteines A80C/R741C in a Cys-less background cross-link when the mutant was rescued with a drug substrate. Login to comment
113 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 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 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 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 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 Accordingly, mutations A80C, R741C, and A80C/R741C were introduced into A52-tagged wild-type P-gp. Login to comment
122 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 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 Treatment of the slowly migrating A80C/ R741C protein with 10 mM DTT yielded the 170-kDa protein (Fig. 3). Login to comment
126 It is possible that mutant A80C/R741C showed efficient cross-linking because it was transiently expressed in HEK 293 cells. Login to comment
127 We then tested whether mutant A80C/R741C was also efficiently cross-linked if it was stably expressed in BHK cells. Login to comment
128 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 By contrast, the major products in mutant A80C/R741C were the slowly migrating cross-linked and the 150-kDa proteins. Login to comment
134 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 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 By contrast, the 150-kDa A80C/R741C immature protein, but not the cross-linked product, was sensitive to endoglycosidase H. Login to comment
139 The cross-linked A80C/R741C protein was sensitive only to PNGase F (Fig. 4, right panel). Login to comment
142 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 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 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 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 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 Cysteines A80C/R741C in a wild-type background cross-link in the absence of drug substrates. Login to comment
153 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 Endoglycosidase digestion of wild-type and mutant A80C/ R741C P-gp expressed in BHK cells. Login to comment
158 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 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 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 Cross-linking of A80C and R741C reduced activity severely. Login to comment
167 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 The A80C/R741C Disulfide Bond Inhibits Drug Efflux-We then tested whether mutant A80C/R741C could confer drug resistance. Login to comment
172 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 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 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 These results suggest that the presence of the A80C/R741C disulfide bond inhibited drug efflux. Login to comment
179 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 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 Accordingly, BHK cells expressing A52-tagged mutant A80C/R741C were incubated with medium containing various concentrations of DTT. Login to comment
189 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 Mature A80C/R741C P-gp cross-links after treatment with DTT. Login to comment
192 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 Cross-linking of mutant A80C/R741C inhibits verapamil-stimulated ATPase activity. Login to comment
199 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 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 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 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 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 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 We first tested whether ATP hydrolysis was required for spontaneous cross-linking of A80C/R741C. Login to comment
213 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
216 Both the A80C/R741C and A80C/R741C/E556Q/E1201Q mutants yielded cross-linked P-gp as the major product. Login to comment
218 These results indicate that ATP hydrolysis was not essential for cross-linking mutant A80C/R741C. Login to comment
219 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 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 Cross-linking of A80C/R741C inhibits P-gp-mediated drug resistance. Login to comment
225 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 Expression of cells with DTT enhances the ability of mutant A80C/R741C to confer drug resistance. Login to comment
231 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 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 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 Next, we tested whether ATP binding was required for cross-linking of mutant A80C/R741C. Login to comment
241 This required the use of membranes prepared from cells expressing mutant A80C/R741C in the Cys-less background. Login to comment
243 Accordingly, HEK 293 cells were transfected with the A52-tagged mutant A80C/R741C. Login to comment
251 Therefore, we tested whether vanadate trapping of nucleotides could inhibit cross-linking of mutant A80C/R741C. Login to comment
252 Membranes were prepared from HEK 293 cells expressing A52-tagged A80C/R741C that were treated with DTT. Login to comment
254 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 DISCUSSION We found that linking the P-gp halves by direct cross-linking of extracellular cysteines A80C/R741C inhibited ATPase activity. Login to comment
260 Cross-linking of mutant A80C/R741C does not require ATP hydrolysis and is rapid. Login to comment
261 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 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 ATP is not required for cross-linking mutant A80C/R741C. Login to comment
269 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 Direct linkage of A80C and R741C would favor the open conformation (Fig. 1B). Login to comment
285 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 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

PMID: 25456855 [PubMed] Loo TW et al: "Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation."

No. Sentence Comment

22 Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C). Login to comment
23 Residues A80C/R741C come close enough (<7 A da; ) to spontaneously cross-link in the open conformation (<7 A da; ) but are widely separated (>30 A da; ) in the closed conformation. Login to comment
24 Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SDS-PAGE gels. Login to comment
26 It was found that only tariquidar blocked A80C/ R741C cross-linking. Login to comment
44 P-gp can also be trapped in an open conformation by cross-linking cysteines introduced into extracellular loops (ECL)1 (A80C) and 4 (R741C). Login to comment
45 Residues A80C/R741C come close enough to spontaneously cross-link in the open conformation (6.9 A da; , all distances represent predicted a a carbon distances) but are widely separated in the closed conformation (>30 A da; ). Login to comment
46 The presence of an A80C/R741C disulfide bond inhibits ATPase activity and drug efflux [29]. Login to comment
48 In this study, we determined whether tariquidar blocks formation of the open conformation of P-gp by testing if it inhibits A80C/R741C cross-linking. Login to comment
74 Purification of P-gp and assay of ATPase activity Histidine-tagged A80C/R741C P-gp in the wild-type background was expressed in HEK 293 cells and then isolated by nickel-chelate chromatography as described previously [37]. Login to comment
80 Effect of substrates and inhibitors on A80C/R741C cross-linking BHK cells stably expressing A52-tagged mutant A80C/R741C in the wild-type background [29] were pre-treated for 5 min at 20 8C with PBS containing 10 mM dithiothreitol (to reduce the disulfide bond) in the presence of the following substrate or inhibitor: 500 nM tariquidar, 5 mM cyclosporine A, 10 mM vinblastine, 25 mM verapamil, 25 mM Taxol, 25 mM rhodamine B, 25 mM Hoechst 33342, 25 mM ketoconazole, 10 mM reserpine, 10 mM cis-flupentixol, 10 mM trans-flupentixol or no drug substrate/ inhibitor. Cells were then washed four times with PBS without dithiothreitol but containing the same substrate or inhibitor and then treated with or without 0.1 mM copper phenanthroline in the presence of the same substrate or inhibitor for 3 min at 20 8C. Login to comment
92 Tariquidar inhibits spontaneous cross-linking of A80C/R741C P-gp in intact cells We previously showed that cysteines A80C/R741C spontaneously cross-linked when the mutant was expressed in cells [29]. Login to comment
94 Cysteine A80C is located in ECL1 in the N-half of the protein whereas cysteine R741C is located in ECL4 in the C-half (Fig. 1). Login to comment
98 Therefore, A80C/R741C cross-linking would be expected when P-gp adopts an open conformation during its catalytic cycle since a disulfide bond spans a distance of about 6-7 A da; . Login to comment
99 The structure of tariquidar is shown in Fig. 2A. Our goal was to test if tariquidar would affect A80C/R741C cross-linking. Login to comment
100 The first step was to determine if tariquidar could still interact with P-gp after introduction of the A80C and R741C mutations. Login to comment
102 To test if tariquidar could activate the ATPase activity of A80C/R741C P-gp, the histidine-tagged mutant (in the wild-type background) was transiently expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment
105 Next, we tested the effect of tariquidar on A80C/R741C cross-linking. Login to comment
106 The A52-tagged A80C/R741C mutant (in the wild-type background) was transiently expressed in HEK 293 cells for 18 h in the absence or presence of various concentrations of tariquidar. Login to comment
111 Locations of the A80C and R741C mutations and composition of truncation mutants of P-gp. Login to comment
117 Inhibition of spontaneous A80C/R741C cross-linking in whole cells by tariquidar. Login to comment
119 (B) Isolated histidine-tagged P-gp mutant A80C/R741C (in wild-type background) was mixed with lipid and ATPase activity was determined in the absence or presence of 1 mM tariquidar. Login to comment
122 (C) Whole SDS (no reducing agent) cell extracts of HEK 293 cells expressing mutant A52-tagged A80C/R741C (wild-type background) in the presence of various concentrations of tariquidar (Tariq) were subjected to immunoblot analysis. Login to comment
134 The results show that tariquidar is a potent inhibitor of A80C/ R741C cross-linking. Login to comment
135 By contrast, we previously observed that P-gp substrates such as vinblastine or cyclosporine A did not inhibit spontaneous cross-linking of the A80C/R741C mutant [29]. Login to comment
137 Tariquidar inhibits rapid A80C/R741C cross-linking in the presence of oxidant In a previous study [29], we found that spontaneous cross-linking of mutant A80C/R741C was slow relative to cycling of P-gp through its reaction cycle. Login to comment
138 When cells expressing the A80C/R741C mutant were treated with dithiothreitol to reduce the extracellular disulfide bond, it took about 20 min to achieve 50% cross-linking of the mutant after dithiothreitol was removed. Login to comment
140 The low rate of disulfide bond formation relative to cycling of the enzyme was due to inefficient cross-linking rather than trapping of a rare conformational change as the rate of A80C/ R741 cross-linking increased over 20-fold in the presence of oxidant (copper phenanthroline) [29]. Login to comment
141 Therefore, it was also important to test if tariquidar would also inhibit rapid cross-linking of mutant A80C/R741C in the presence of oxidant. Login to comment
142 We tested if tariquidar would inhibit A80C/R741C cross-linking in the presence of oxidant using BHK cells stably expressing the mutant [29]. Login to comment
143 Stably transfected BHK cells expressing A80C/R741C P-gp (in the wild-type background) were used rather than transiently transfected HEK 293 cells because they remain attached to the plates during the multiple washing steps required for the oxidant cross-linking assays. Login to comment
144 We first treated the cells expressing P-gp A80C/R741C with 10 mM dithiothreitol to reduce the disulfide bond located on the extracellular surface. Login to comment
149 Cross-linked A80C/R741C was the major product (about 80% of total P-gp protein) detected when untreated whole cell extracts of BHK cells expressing the mutant were subjected to immunoblot analysis (Fig. 3A and B, lane A). Login to comment
153 The results show that tariquidar blocked both slow (Fig. 2C) and fast (Fig. 3A and B, lane D) cross-linking of mutant A80C/R741C. Login to comment
154 To test the effects of other drug substrates or modulators on cross-linking of mutant A80C/R741C in the presence of oxidant, the cells were treated for 5 min with 10 mM dithiothreitol in the presence of drug substrates such as vinblastine, verapamil, paclitaxel, rhodamine B, or Hoechst 33342 or inhibitors/ modulators such as cyclosporine A, ketoconazole, reserpine, or the cisor trans-isomers of flupentixol. Login to comment
157 The results show that inhibition of rapid A80C/R741C cross-linking was specific for tariquidar. Login to comment
161 Tariquidar acts as a noncompetitive inhibitor for drug binding and differed from drug substrates because it was the only compound that blocked A80C/R741C cross-linking (Fig. 3). Login to comment
166 Only tariquidar inhibited A80C/R741C cross-linking in the presence of oxidant. Login to comment
167 (A) BHK cells stably expressing A52-tagged mutant A80C/R741C (in wild-type background) were treated without ( ) or with (+) 10 mM dithiothreitol (DTT) to reduce the disulfide bond. Login to comment
208 Does tariquidar also inhibit A80C/R741C cross-linking when A B G268V 0 12.5 25 50 100 200 400 800 [Tar] (nM) 170 kDa 150 kDa GAPDH C D 170 kDa 150 kDa GAPDH None Cyclo Tariq F804D 0 20 40 60 80 100 Percent Mature [Tariq] nM 0 12.5 25 50 100 200 400 800 * * * * * * None Cyclo Tariq 0 20 40 60 80 100 Percent Mature * Fig. 4. Login to comment
223 We previously found that complex carbohydrate was required to cause the cross-linked A80C/R741C full-length protein to migrate slower on SDS-PAGE gels [29]. Login to comment
224 Removal of the carbohydrate from the cross-linked A80C/R741C mutant with endoglycosidase F yielded a product that migrated in the same position as wild-type P-gp lacking carbohydrate. Login to comment
232 The A52-tagged A80C/N-half and R741C/C-half cDNAs (in the Cys-less background) were constructed. Login to comment
233 The A80C/N-half and R741C/C-half proteins were co-expressed in the absence or presence of 10 mM cyclosporine A or 500 nM tariquidar. Login to comment
237 To test for A80C/R741C cross-linking we expressed A52-tagged R741C/C-half P-gp with untagged A80C/N-half P-gp in the absence or presence of 10 mM cyclosporine A or 500 nM tariquidar. Login to comment
238 The presence of an epitope tag on just the C-half protein was used to make it simpler to detect cross-linking between the R741C/C-half protein and A80C/N-half protein. Login to comment
259 (A) Whole cell SDS extracts of HEK 293 cells (containing 10 mM dithiothreitol) expressing A52-tagged A80C/N-half and A52-tagged R741C/C-half Pgp (in Cys-less background) in the absence (None) or presence of 10 mM cyclosporine A (Cyclo) or 500 nM tariquidar (Tariq) were subjected to immunoblot analysis. Login to comment
260 The positions of R741C/C-half or mature or immature forms of A80C/N-half P-gp`s are indicated. Login to comment
264 (C) Whole cell extracts of cells expressing A52-tagged R741C/C-half P-gp together with untagged A80C/N-half P-gp (in Cys-less background) in the absence (None) or presence of 10 m; cyclosporine A (Cyclo) or 500 nM tariquidar (Tariq) before ( ) or after (+) treatment with 10 mM dithiothreitol (DTT) were subjected to immunoblot analysis with monoclonal antibody A52. Login to comment
265 The positions of R741C/C-half cross-linked to mature (Mat) and immature (Immat) forms of A80C/N-half are shown. Login to comment
269 The 170 kDa protein corresponded in size to R741C/C-half cross-linked to mature A80C/ N-half protein. Login to comment
272 The results show that R741C/C-half can spontaneously cross-link to A80C/N-half in the endoplasmic reticulum but cross-linking is inhibited by tariquidar. Login to comment
274 Discussion We found that tariquidar was different from other drug substrates and inhibitors because it blocked A80C/R741C cross-linking. Login to comment
293 How does tariquidar prevent formation of an open conformation in P-gp to block A80C/R741C cross-linking at the cell surface? Login to comment
294 One reason that tariquidar might block A80C/R741C cross-linking is that it was found to bind very tightly to P-gp at the cell surface [20]. Login to comment
297 Tight binding of tariquidar might influence A80C/R741C cross-linking because modeling studies suggest that the tariquidar binding site lies close to residues at the extracellular ends of TM segments 1 and 7 [49]. Login to comment
299 Occupation of the tariquidar-binding site may block movement between ECL1 and ECL4 required for A80C/R741C cross-linking. Login to comment
319 The positions of A80C and R741C are indicated as filled balls. Login to comment