ABCMdb

ABCB1 p.Gln1118Ala

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Publications

PMID: 16487063 [PubMed] Qian F et al: "Molecular model and ATPase activity of carboxyl-terminal nucleotide binding domain from human P-glycoprotein."

No. Sentence Comment

12 The functional role of Gln1118 was investigated by the studying the biochem ical properties of recombinant NBD2 and its mutant Q1118A. Login to comment
45 Construction of the expression plasmid encoding NBD2 and its mutant Q1118A. Login to comment
51 For site directed muta genesis to introduce a Q1118A mutation, nucleotide mutations at positions 3352 (C→G) and 3353 (A→C) were achieved by sequence overlap PCR methodology. Login to comment
54 The sequence of plasmid containing NBD2 Q1118A mutant was confirmed by sequencing. Login to comment
55 Expression, renaturation, and purification of NBD2 and its mutant Q1118A. Login to comment
56 Escherichia coli BL21 cells con taining NBD2 and its mutant Q1118A were cultured in 200 ml of 2YTA medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, and 100 µg/ml ampicillin) at 37°C with vigorous shaking until the absorbance at 600 nm reached 1.2 units. Login to comment
124 Expression and purification of NBD2 and its mutant Q1118A. Login to comment
127 The Q1118A mutation was introduced into the NBD2 sequence by site directed mutagenesis. Login to comment
128 Recombinant proteins encoded by pGEX4T 1 NBD2 and its mutant Q1118A based plas mids were expressed in E. coli cells. Login to comment
129 Expression and purification of the NBD2 and its mutant Q1118A are illustrated in Fig. 6. Login to comment
135 NBD2 protein and mutant Q1118A protein yielded equal purities and in similar amounts. Login to comment
140 Expression and purification of the NBD2 and its mutant Q1118A. Login to comment
142 Expression of mutant Q1118A protein before (lane ) and after (lane 4) IPTG induction. Login to comment
143 Purified NBD2 protein (lane 5) and its mutant Q1118A protein (lane 6). Login to comment
144 S22 QIAN et al. BIOCHEMISTRY (Moscow) Vol. 71 Suppl. 1 2006 ATPase activity of NBD2 and its mutant Q1118A. Login to comment
148 The ATPase activities of the Q1118A mutant and that of the wild type NBD2 were compared. Login to comment
165 The Q1118A mutant was affected in its ATPase activity but this residue might not participate the binding of ATP because the mutation had no influence on the affinity for ATP. Login to comment

PMID: 17897039 [PubMed] Ha SN et al: "Mini review on molecular modeling of P-glycoprotein (Pgp)."

No. Sentence Comment

61 Also, it is reported that mutation of Gln1118 to Ala reduces hydrolysis of ATP. Login to comment

PMID: 25016028 [PubMed] Zolnerciks JK et al: "The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle."

No. Sentence Comment

74 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 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
132 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
142 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 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
152 (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
156 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 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 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
175 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 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 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 Representative plots from cells expressing wild-type (B), NBD1-Q475A (C), NBD2-Q1118A (D), and Q475A/Q1118A (E) ABCB1. Login to comment
197 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 No increase in ECAR in the presence of verapamil was detected in cells expressing the Q-loop double mutant Q475A/Q1118A. Login to comment
212 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 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 (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
216 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 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
253 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