ABCMdb

PMID: 25456855

Loo TW, Clarke DM
Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation.
Biochem Pharmacol. 2014 Dec 15;92(4):558-66. doi: 10.1016/j.bcp.2014.10.006. Epub 2014 Oct 22., [PubMed]

Sentences

No. Mutations Sentence Comment

22 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys It was found that only tariquidar blocked A80C/ R741C cross-linking. Login to comment 28 ABCB1 p.Phe804Asp Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp. Login to comment 44 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The presence of an A80C/R741C disulfide bond inhibits ATPase activity and drug efflux [29]. Login to comment 48 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 68 ABCB1 p.Phe804Asp After 5 h at 37 8C, the medium was replaced with fresh medium with or without 5 mM cyclosporine A or 0.5 mM tariquidar for rescue of mutant F804D and 500 nM tariquidar, 10 mM cyclosporine A, 50 mM verapamil, 20 mM vinblastine or 100 mM capsaicin for rescue of TMD1+2. Login to comment 74 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Next, we tested the effect of tariquidar on A80C/R741C cross-linking. Login to comment 106 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Locations of the A80C and R741C mutations and composition of truncation mutants of P-gp. Login to comment 117 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Inhibition of spontaneous A80C/R741C cross-linking in whole cells by tariquidar. Login to comment 119 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys (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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys (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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The results show that tariquidar is a potent inhibitor of A80C/ R741C cross-linking. Login to comment 135 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The results show that inhibition of rapid A80C/R741C cross-linking was specific for tariquidar. Login to comment 161 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 163 ABCB1 p.Gly268Val First, we tested if tariquidar could rescue the G268V processing mutant. Login to comment 164 ABCB1 p.Gly268Val The G268V mutant was selected because we previously found that it could be rescued with vinblastine, verapamil, or cyclosporine A [44]. Login to comment 166 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Only tariquidar inhibited A80C/R741C cross-linking in the presence of oxidant. Login to comment 167 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys (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 174 ABCB1 p.Gly268Val processing mutations like G268V inhibit P-gp maturation because they trap P-gp in the endoplasmic reticulum as a partially folded immature 150 kDa core-glycosylated protein. Login to comment 177 ABCB1 p.Gly268Val Mutant G268V containing an A52 epitope tag was transiently expressed in HEK 293 cells in the presence of various concentrations of tariquidar for 18 h. Login to comment 183 ABCB1 p.Gly268Val The results show that tariquidar acted as a potent pharmacological chaperone to promote maturation of G268V P-gp. Login to comment 184 ABCB1 p.Gly268Val By comparison, 20-fold higher concentrations of high affinity drug substrates/modulators such as verapamil, vinblastine or cyclosporine A were required to promote maturation of G268V P-gp [44]. Login to comment 185 ABCB1 p.Phe804Asp We previously found that some P-gp mutants such as F804D could not be rescued by any drug substrate [45]. Login to comment 186 ABCB1 p.Phe804Asp The F804D mutation is located in intracellular helix 3 that mediates contact between the third intracellular loop (ICL3) in TMD2 and NBD1. Login to comment 187 ABCB1 p.Phe804Asp To test if tariquidar could promote maturation of F804D, the A52-tagged mutant was transiently expressed in the presence of 300 nM of the compound for 18 h. Login to comment 188 ABCB1 p.Phe804Asp Immunoblot analysis of whole cell SDS extracts showed that tariquidar could promote maturation of F804D to yield mature P-gp as about 50% of the steady-state product (Fig. 4C and D). Login to comment 199 ABCB1 p.Gly268Val These concentrations of drug substrates were chosen because they maximally promoted maturation of the G268V mutant [44]. Login to comment 208 ABCB1 p.Gly268Val ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Phe804Asp 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 215 ABCB1 p.Phe804Asp (C) Whole cell extracts of HEK 293 cells expressing A52-tagged P-gp processing mutant F804D in the absence (None) or presence of 10 mM cyclosporine A (Cyclo) or 300 nM tariquidar (Tariq) were subjected to immunoblot analysis. Login to comment 223 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The A52-tagged A80C/N-half and R741C/C-half cDNAs (in the Cys-less background) were constructed. Login to comment 233 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys (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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The positions of R741C/C-half or mature or immature forms of A80C/N-half P-gp`s are indicated. Login to comment 264 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys (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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The positions of R741C/C-half cross-linked to mature (Mat) and immature (Immat) forms of A80C/N-half are shown. Login to comment 266 ABCB1 p.Arg741Cys ABCB1 p.Arg741Cys (D) The amount of cross-linked (X-linked) R741C/C-half relative to total R741C/C-half P-gp was determined before ( ) or after (+) treatment with dithiothreitol (DTT). Login to comment 269 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The 170 kDa protein corresponded in size to R741C/C-half cross-linked to mature A80C/ N-half protein. Login to comment 272 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Discussion We found that tariquidar was different from other drug substrates and inhibitors because it blocked A80C/R741C cross-linking. Login to comment 293 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys 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 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys Occupation of the tariquidar-binding site may block movement between ECL1 and ECL4 required for A80C/R741C cross-linking. Login to comment 306 ABCB1 p.Phe804Asp Perhaps tariquidar acts as a particularly effective pharmacological chaperone to rescue P-gp processing mutants such as F804D (Fig. 4C) because it interacts at both the predicted Rand H-sites to have an additive effect on maturation. Login to comment 319 ABCB1 p.Ala80Cys ABCB1 p.Arg741Cys The positions of A80C and R741C are indicated as filled balls. Login to comment