ABCB1 p.Val982Ala
| Predicted by SNAP2: | A: N (66%), C: D (53%), D: D (85%), E: D (80%), F: D (75%), G: D (71%), H: D (80%), I: N (87%), K: D (80%), L: N (66%), M: N (78%), N: D (66%), P: D (85%), Q: D (71%), R: D (80%), S: D (59%), T: N (72%), W: D (80%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, W: D, Y: D, |
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[hide] Interaction of digitalis-like compounds with p-gly... Toxicol Sci. 2013 Feb;131(2):502-11. doi: 10.1093/toxsci/kfs307. Epub 2012 Oct 26. Gozalpour E, Wittgen HG, van den Heuvel JJ, Greupink R, Russel FG, Koenderink JB
Interaction of digitalis-like compounds with p-glycoprotein.
Toxicol Sci. 2013 Feb;131(2):502-11. doi: 10.1093/toxsci/kfs307. Epub 2012 Oct 26., [PMID:23104431]
Abstract [show]
Digitalis-like compounds (DLCs), or cardiac glycosides, are produced and sequestered by certain plants and animals as a protective mechanism against herbivores or predators. Currently, the DLCs digoxin and digitoxin are used in the treatment of cardiac congestion and some types of cardiac arrhythmia, despite a very narrow therapeutic index. P-glycoprotein (P-gp; ABCB1) is the only known ATP-dependent efflux transporter that handles digoxin as a substrate. Ten alanine mutants of human P-gp drug-binding amino acids-Leu(65), Ile(306), Phe(336), Ile(340), Phe(343), Phe(728), Phe(942), Thr(945), Leu(975), and Val(982)-were generated and expressed in HEK293 cells with a mammalian baculovirus system. The uptake of [(3)H]-N-methyl-quinidine (NMQ), the P-gp substrate in vesicular transport assays, was determined. The mutations I306A, F343A, F728A, T945A, and L975A abolished NMQ transport activity of P-gp. For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol) were determined. The affinities of digoxin, proscillaridin A, peruvoside, and cymarin for mutants F336A and I340A were decreased two- to fourfold compared with wild type, whereas that of digitoxin and strophanthidol did not change. In addition, the presence of a hydroxyl group at position 12beta seems to reduce the apparent affinity when the side chain of Phe(336) and Phe(942) is absent. Our results showed that a delta-lactone ring and a sugar moiety at 3beta of the steroid body are favorable for DLC binding to P-gp. Moreover, DLC inhibition is increased by hydroxyl groups at positions 5beta and 19, whereas inhibition is decreased by those at positions 1beta, 11alpha, 12beta, and 16beta. The understanding of the P-gp-DLC interaction improves our insight into DLCs toxicity and might enhance the replacement of digoxin with other DLCs that have less adverse drug effects.
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No. Sentence Comment
46 However, transport activity was preserved in L65A, I306A, I340A, F942A, and V982A.
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ABCB1 p.Val982Ala 23104431:46:76
status: NEW62 Ten different P-gp mutants were produced: L65A, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A and all mutations were confirmed by sequencing of full-length P-gp cDNA.
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ABCB1 p.Val982Ala 23104431:62:108
status: NEW122 All the indicated amino acids were replaced by alanine to remove the side chain of the residue (L65A, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A).
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ABCB1 p.Val982Ala 23104431:122:162
status: NEW135 In addition, five P-gp mutants (L65A, F336A, I340A, F942A, and V982A), for which NMQ transport activity was at least 50% of wild-type transport, were selected.
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ABCB1 p.Val982Ala 23104431:135:63
status: NEW138 Comparing the IC50 value of the mutants with those of the wild type (mutant IC50 /wild-type IC50 ), L65A, and V982A showed similar values as wild type (0.6-2.2).
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ABCB1 p.Val982Ala 23104431:138:110
status: NEW180 Fig. 5.ߓ Western blot analysis (A) and NMQ transport activity of wild type and L65A, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A mutant P-gp (B).
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ABCB1 p.Val982Ala 23104431:180:151
status: NEW202 NMQ transport activity of the second group mutants (L65A, F336A, I340A, F942A, and V982A) was not significantly different from that of the wild-type P-gp (60-150%).
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ABCB1 p.Val982Ala 23104431:202:83
status: NEW208 Table 1 The IC50 Values of DLCs Against Wild-Type and Mutant P-gp-Mediated [3 H]-NMQ Transport P-gp Cymarin Digitoxin Digoxin Peruvoside Proscillaridin A Strophanthidol IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 Wild type 432ߙ&#b1;ߙ90 9ߙ&#b1;ߙ2.1 188ߙ&#b1;ߙ24 214ߙ&#b1;ߙ41 25ߙ&#b1;ߙ3.5 242ߙ&#b1;ߙ61 L65A 800ߙ&#b1;ߙ99 1.9 17ߙ&#b1;ߙ3.4 1.9 217ߙ&#b1;ߙ33 1.2 469ߙ&#b1;ߙ73* 2.2 48ߙ&#b1;ߙ7.2 1.9 186ߙ&#b1;ߙ18 0.8 F336A 979ߙ&#b1;ߙ48 2.3 14ߙ&#b1;ߙ1.9 1.6 524ߙ&#b1;ߙ114 2.8 528ߙ&#b1;ߙ92** 2.5 111ߙ&#b1;ߙ19** 4.4 291ߙ&#b1;ߙ45 1.2 I340A 1181ߙ&#b1;ߙ103** 2.7 19ߙ&#b1;ߙ4.3 2.0 439ߙ&#b1;ߙ138 2.3 527ߙ&#b1;ߙ37** 2.5 79ߙ&#b1;ߙ21* 3.1 156ߙ&#b1;ߙ14 0.6 F942A 821ߙ&#b1;ߙ256 1.9 8ߙ&#b1;ߙ1.0 0.9 558ߙ&#b1;ߙ145 3.0 273ߙ&#b1;ߙ29 1.3 18ߙ&#b1;ߙ1 0.7 187ߙ&#b1;ߙ24 0.8 V982A 620ߙ&#b1;ߙ106 1.4 12ߙ&#b1;ߙ2.1 1.3 345ߙ&#b1;ߙ73 1.8 291ߙ&#b1;ߙ10 1.4 22ߙ&#b1;ߙ3.4 0.9 144ߙ&#b1;ߙ9 0.6 Note.
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ABCB1 p.Val982Ala 23104431:208:1179
status: NEW213 Inhibition of NMQ transport by L65A, F942A, and V982A with six DLCs showed that in only one case the mutation caused a significant difference in the IC50 value (ratio of 2.2) of the DLCs compared with wild-type P-gp.
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ABCB1 p.Val982Ala 23104431:213:48
status: NEW[hide] Convallatoxin: a new P-glycoprotein substrate. Eur J Pharmacol. 2014 Dec 5;744:18-27. doi: 10.1016/j.ejphar.2014.09.031. Epub 2014 Sep 28. Gozalpour E, Greupink R, Bilos A, Verweij V, van den Heuvel JJ, Masereeuw R, Russel FG, Koenderink JB
Convallatoxin: a new P-glycoprotein substrate.
Eur J Pharmacol. 2014 Dec 5;744:18-27. doi: 10.1016/j.ejphar.2014.09.031. Epub 2014 Sep 28., [PMID:25264938]
Abstract [show]
Digitalis-like compounds (DLCs), such as digoxin and digitoxin that are derived from digitalis species, are currently used to treat heart failure and atrial fibrillation, but have a narrow therapeutic index. Drug-drug interactions at the transporter level are frequent causes of DLCs toxicity. P-glycoprotein (P-gp, ABCB1) is the primary transporter of digoxin and its inhibitors influence pharmacokinetics and disposition of digoxin in the human body; however, the involvement of P-gp in the disposition of other DLCs is currently unknown. In present study, the transport of fourteen DLCs by human P-gp was studied using membrane vesicles originating from human embryonic kidney (HEK293) cells overexpressing P-gp. DLCs were quantified by liquid chromatography-mass spectrometry (LC-MS). The Lily of the Valley toxin, convallatoxin, was identified as a P-gp substrate (Km: 1.1+/-0.2 mM) in the vesicular assay. Transport of convallatoxin by P-gp was confirmed in rat in vivo, in which co-administration with the P-gp inhibitor elacridar, resulted in increased concentrations in brain and kidney cortex. To address the interaction of convallatoxin with P-gp on a molecular level, the effect of nine alanine mutations was compared with the substrate N-methyl quinidine (NMQ). Phe343 appeared to be more important for transport of NMQ than convallatoxin, while Val982 was particularly relevant for convallatoxin transport. We identified convallatoxin as a new P-gp substrate and recognized Val982 as an important amino acid involved in its transport. These results contribute to a better understanding of the interaction of DLCs with P-gp.
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No. Sentence Comment
56 Nine different P-gp mutants, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A, were produced and sequencing of full-length P-gp cDNA was used to confirm all mutations (Gozalpour et al., 2013).
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ABCB1 p.Val982Ala 25264938:56:89
status: NEW154 Nine amino acids were replaced by alanine (I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A) and P-gp mutants were expressed in HEK293 cells to produce membrane vesicles.
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ABCB1 p.Val982Ala 25264938:154:103
status: NEW168 The transport activity of F336A, F942A, T945A and L975A for NMQ ranged from 49% to 57%, whereas I340A showed increased activity of 120% and V982A had about the same activity as wild type (Fig. 5B).
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ABCB1 p.Val982Ala 25264938:168:140
status: NEW170 Convallatoxin and NMQ transport activity were not significantly different for I306A, F336A, I340A, F728A, F942A, T945A, and L975A (Fig. 5C), whereas they differed significantly for F343A and V982A (Fig. 5D and E).
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ABCB1 p.Val982Ala 25264938:170:191
status: NEW173 For the V982A mutant, NMQ transport was similar to that of the Fig. 3.
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ABCB1 p.Val982Ala 25264938:173:8
status: NEW212 The transport activity of F343A and V982A mutants for NMQ and convallatoxin were compared using an unpaired Student's t-test: nnn Po0.0001 (D and E).
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ABCB1 p.Val982Ala 25264938:212:36
status: NEW254 The transport activity of F336A, F942A, T945A, L975A and V982A, were conserved (45-100% of wild type) (Fig. 5B) similar to our previous results (Gozalpour et al., 2013).
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ABCB1 p.Val982Ala 25264938:254:57
status: NEW259 The transport activities of NMQ and convallatoxin were significantly different for the F343A and V982A mutant (Fig. 5D and E).
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ABCB1 p.Val982Ala 25264938:259:97
status: NEW263 The V982A mutant lost 50% of its convallatoxin transport activity, whereas NMQ transport activity was not changed.
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ABCB1 p.Val982Ala 25264938:263:4
status: NEW265 We previously showed that V982A did not influence the affinity of DLCs such as cymarin, digitoxin, digoxin, peruvoside, proscillaridin A and strophanthidol (Gozalpour et al., 2013).
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ABCB1 p.Val982Ala 25264938:265:26
status: NEW