ABCMdb

PMID: 25016028

Zolnerciks JK, Akkaya BG, Snippe M, Chiba P, Seelig A, Linton KJ
The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle.
FASEB J. 2014 Oct;28(10):4335-46. doi: 10.1096/fj.13-245639. Epub 2014 Jul 11., [PubMed]

Sentences

No. Mutations Sentence Comment

74 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Gln1118Ala ABCB1 p.Gln132Arg ABCB1 p.Gln132Ala ABCB1 p.Gln773Arg ABCB1 p.Gln773Ala ABCB1 p.Gln475Ala Plasmids Mutations were introduced into a plasmid encoding human ABCB1 with a C-terminal hexahistidine tag (pCIneo-wtABCB1-6His; ref. 25) by site-directed mutagenesis (QuikChange XL; Stratagene, La Jolla, CA, USA) using the following oligonucleotides: Q132A, 5=-GGTTGCTGCTTACATCGCGGTTTCATTTTGGTGC- 3=; Q132R, 5=-GGTTGCTGCTTACATTCGAGTTTCATTTTG- GTGC-3=; Q475A, 5=-GGGAAATCATTGGTGTGGTGAGTGCT- GAGCCTGTATTGTTTGCCACCACG-3=; Q773A, 5=-GGAATTA- TTTCTTTTATTACATTTTTCCTTGCGGGTTTCACATTTG- GCAAAGCTGG-3=; Q773R, 5=-GGAATTATTTCTTTTATTA- CATTTTTCCTTCGAGGTTTCACATTTGGCAAAGCTGG-3=; Q1118A, 5=-GGGCATCGTGTCCGCGGAACCCATCCTGTTTG-3=; E556Q, 5=-CCCCAAGATCCTCCTGCTTGATCAGGCCACGT- CAGCCTTGG-3=; and E1201Q, 5=-CAGCCTCATATTTTGCTTCT- TGATCAGGCCACGTCAGCTCTGGATAC-3=. Login to comment 121 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala The Q475A/ Q1118A mutant bound Bodipy-verapamil in the plasma membrane but did not efflux the drug; thus, it accumulated in the plasma membrane of cells expressing the double-Q-loop mutant ABCB1 and also in intracellular compartments (middle panels). Login to comment 122 ABCB1 p.Glu1201Gln In contrast, the catalytically inactive Walker B E1201Q mutant was trapped in the inward-closed conformation, which has a low affinity for drug, and the cells accumulated Bodipy-verapamil intracellularly but not in the plasma membrane (bottom panels). Login to comment 132 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala RESULTS Q loops of the NBDs are essential for drug efflux, but redundancy is built into the molecular mechanism Site-directed mutagenesis was used to introduce glutamine-to-alanine mutations into NBD1 (NBD1-Q475A) and NBD2 (NBD2-Q1118A) and into both NBDs of human ABCB1. Login to comment 133 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln As controls, mutations E556Q and E1201Q in the Walker B motifs of each NBD, which render the transporter catalytically inactive, were also generated. Login to comment 142 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala Cells expressing the single-NBD Q-loop mutants NBD1-Q475A or NBD2-Q1118A, accumulated only low levels of Bodipy-verapamil, similar, but not identical with, those expressing wild-type ABCB1 (Fig. 2C). Login to comment 144 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala In contrast, cells expressing the double mutant Q475A/Q1118A exhibited no efflux activity and, rather surprisingly and reproducibly, accumulated more Bodipy-verapamil than did the mock-transfected Figure 4. Login to comment 151 ABCB1 p.Glu1201Gln cells or the cells expressing the Walker B mutant E1201Q of ABCB1 (Fig. 2C). Login to comment 152 ABCB1 p.Gln1118Ala ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala ABCB1 p.Gln475Ala (The raw dot plot data also show that Bodipy-verapamil accumulation increased linearly with increasing expression of Q475A/ Q1118A; Supplemental Fig. S1.) Double-Q-loop Q475A/Q1118A mutant is trapped in the inward-open conformation The extracellular loops of human ABCB1 form a discontinuous epitope for the antibody UIC2, characterized by Igor Roninson and colleagues (38) and others (39). Login to comment 154 ABCB1 p.Glu1201Gln Consequently, UIC2 binds more readily to wild-type ABCB1 than the Walker B mutant E1201Q (Fig. 2D) in cells that express equal amounts of these proteins, because the Walker B mutant can bind but cannot hydrolyze ATP (40). Login to comment 156 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala UIC2 reproducibly binds most readily to the Q475A/Q1118A mutant, suggesting that it adopts a conformation consistent with the inward-open state that would be expected to have a high affinity for UIC2. Login to comment 161 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala In contrast, cells expressing the Q475A/Q1118A mutant accumulated Bodipy-verapamil in both the intracellular compartments and the plasma membrane, where it colocalized with the 4E3 staining (Fig. 3, middle panels). Login to comment 163 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala The microscopy data are therefore consistent with the UIC2-binding data and suggest that the Q475A/ Q1118A mutant adopts the inward-open conformation, which, in the absence of the 2 Q-loop glutamines, cannot form the NBD-NBD interface and provides additional drug-binding sites in the plasma membrane, which explains the increased accumulation of Bodipy-verapamil in these cells. Login to comment 164 ABCB1 p.Glu1201Gln In contrast, no Bodipy-verapamil was detected bound to the Walker B E1201Q mutant, which is trapped in the inward-closed conformation. Login to comment 175 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala The drug-stimulated ATPase activity of the single-Q-loop mutants was reduced to 9.5 and 8.1% of the wild-type activity for the NBD1-Q475A and NBD2-Q1118A mutants, respectively, and the double mutant was inactive. Login to comment 177 ABCB1 p.Gln1118Ala ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala ABCB1 p.Gln475Ala In vitro ATPase data for NBD1-Q475A, NBD2-Q1118A, and wild-type ABCB1 Mutant af9;Nicardipine (50 òe;M) afa;Nicardipine Relative ATPase activity (%)a Km (mM) Vmax (nmol Pi/min/mg) Km (mM) Vmax (nmol Pi/min/mg) Wild type 0.746 afe; 0.22 1691 afe; 228 0.327 afe; 0.49 151 afe; 54 100 Q475A 2.37 afe; 1.64 162 afe; 70*** 0.284 afe; 0.19 8.0 afe; 1.7* 9.5 Q1118A 2.34 afe; 1.39 138 afe; 51*** 0.573 afe; 0.31 8.2 afe; 1.7* 8.1 Data are averages afe; se of c56;4 independent experiments. Login to comment 190 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala A) ECARs of HEK293T cells expressing wild-type (red), NBD1-Q475A (purple), or NBD2-Q1118A (orange) ABCB1 and mock-transfected cells (blue). Login to comment 193 ABCB1 p.Gln1118Ala ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala ABCB1 p.Gln475Ala Representative plots from cells expressing wild-type (B), NBD1-Q475A (C), NBD2-Q1118A (D), and Q475A/Q1118A (E) ABCB1. Login to comment 197 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala The V1 for cells expressing the single mutants NBD1-Q475A and NBD2-Q1118A was 1.19and 1.13-fold above basal rates, respectively, corresponding to 50 and 35% of the ATPase activity of wild-type ABCB1. Login to comment 198 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala No increase in ECAR in the presence of verapamil was detected in cells expressing the Q-loop double mutant Q475A/Q1118A. Login to comment 207 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg The single mutant TMD1-Q132R effluxed the Bodipy-verapamil with 70% efficiency of the wild-type protein, whereas the TMD2- Q773R was fully active and indistinguishable from wild-type ABCB1 (Fig. 6, dark gray bars, in which transport data are presented as the fold reduction in Bodipy-verapamil accumulation compared to mock-transfected cells). Login to comment 209 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg The double-arginine mutant Q132R/ Q773R retained the ability to export Bodipy-verapamil, but it was significantly reduced to 42% (Pb0d;0.001) of wild-type ABCB1 activity. Login to comment 210 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg This residual activity is most likely due to the partial masking of the positive charge on verapamil by the Bodipy moiety, such that electrostatic repulsion from Q132R or Q773R is incomplete. Login to comment 211 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The catalytically inactive Walker B mutants NBD1-E556Q and NBD2-E1201Q (46) were also tested for comparison and did not efflux Bodipy-verapamil. Login to comment 212 ABCB1 p.Gln1118Ala ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg ABCB1 p.Gln475Ala To test whether each verapamil-binding cavity was coupled to the NBDs via a specific Q loop, we combined the single drug cavity mutants TMD1-Q132R and TMD2- Q773R with the NBD Q-loop mutants NBD1-Q475A and NBD2-Q1118A and compared their transport activity (Fig. 6, striped bars) with that of the drug cavity mutants and also the single-and double-Q-loop mutants (Fig. 6, light gray bars; note that the double-Q-loop mutant has a fold difference that is b0d;1 because it provides additional binding sites for Bodipy-verapamil in the membrane). Login to comment 213 ABCB1 p.Gln1118Ala ABCB1 p.Gln132Arg ABCB1 p.Gln475Ala The drug cavity mutant TMD1-Q132R combined synergistically with NBD1-Q475A to significantly reduce Bodipy-verapamil export activity to 25% of wild-type activity, but in combination with NBD2-Q1118A, the transporter retained the full level of activity of each single mutant. Login to comment 214 ABCB1 p.Gln1118Ala ABCB1 p.Gln132Arg (At 85% of the wild type, this activity was not significantly different from that of the Q132R mutant or the Q1118A mutant.) Login to comment 215 ABCB1 p.Gln132Arg This observation shows that the wild-type Q773- lined verapamil-binding cavity of the Q132R mutant is dedicated to, and only requires, the NBD1 Q loop to couple to the ATP catalytic cycle. Login to comment 216 ABCB1 p.Gln1118Ala ABCB1 p.Gln773Arg ABCB1 p.Gln475Ala In contrast, TMD2- Q773R combined synergistically with both NBD1-Q475A and NBD2-Q1118A to reduce Bodipy-verapamil export activity to 22 and 34% of the wild-type activity, respectively, showing that the wild-type Q132-lined verapamil-binding cavity of these mutants is coupled to and requires the Q loops of both NBDs to trigger efflux. Login to comment 218 ABCB1 p.Gln1118Ala ABCB1 p.Gln1118Ala ABCB1 p.Gln1118Ala ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala ABCB1 p.Gln475Ala ABCB1 p.Gln475Ala ABCB1 p.Gln475Ala Cytosensor data for NBD1-Q475A, NBD2-Q1118A, Q475A/Q1118A, and wild-type ABCB1 Mutant K1 (òe;M) V1 (fold increase) V1 (% wild-type) K2 (òe;M) V2 (fold increase) Wild type 0.56 afe; 0.03 1.38 afe; 0.01 100 23.05 afe; 3.44 1.09 afe; 0.01 Q475A 0.91 afe; 0.10*** 1.19 afe; 0.02*** 50.2 26.54 afe; 4.84 0.94 afe; 0.02*** Q1118A 0.95 afe; 0.17** 1.13 afe; 0.02*** 35.6 84.81 afe; 30.36*** 0.88 afe; 0.06*** Q475A/Q1118A ND ND ND ND ND Data are averages afe; se of c56;3 independent experiments. Login to comment 224 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg The single-Q-loop mutants, combined with the TMD mutants Q132R in TMD1 or Q773R in TMD2 that line the 2 verapamil-binding cavities (12), showed that Bodipy-verapamil bound to the cavity lined by TMD2-Q773 triggers conformation change to the inward-closed state via the conduit of the Q loop in NBD1. Login to comment 225 ABCB1 p.Gln773Arg In reciprocal experiments in which the transporter preferentially engaged drug via the Q132-lined cavity (by introduction of the Q773R mutation), both Q loops were required to efficiently couple efflux of the bound Bodipy-verapamil to the ATP catalytic cycle. Login to comment 251 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Catalytically inactive Walker B mutants NBD1-E556Q and NBD2-E1201Q served as negative controls. Login to comment 253 ABCB1 p.Gln1118Ala ABCB1 p.Gln475Ala ߤ Cells expressing the Q475A/Q1118A mutant ABCB1 accumulated more Bodipy-verapamil than did the nontransfected cells, giving a ratio of b0d;1. ns, not significant. Login to comment