ABCMdb

PMID: 23104431

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., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala 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. Login to comment 11 ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillari- dinA, 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. Login to comment 45 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala Removal of the side chain resulted in loss of NMQ transport activity of five human P-gp mutants: I306A, F343A, F728A, T945A, and L975A, which seem to have key role in the transport of NMQ. Login to comment 46 ABCB1 p.Phe942Ala ABCB1 p.Ile306Ala ABCB1 p.Ile340Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala However, transport activity was preserved in L65A, I306A, I340A, F942A, and V982A. Login to comment 62 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Phe942Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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. Login to comment 122 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Phe942Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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). Login to comment 132 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Phe942Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala NMQ transport activity of mutants L65A, F336A, I340A, F942A, andV982A as compared with wild-type P-gp ranged from 60 to 150%, whereas NMQ transport activity of I306A, F343A, F728A, T945A, and L975A varied between 8 and 30%. Login to comment 135 ABCB1 p.Phe942Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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. Login to comment 138 ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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). Login to comment 140 ABCB1 p.Phe942Ala F942A seems to affect the digoxin binding (3.0), although not significantly. Login to comment 180 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Phe942Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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). Login to comment 190 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala The first group of mutants (I306A, F343A, F728A, T945A, and L975A) exhibited a significantly lower NMQ transport activity (8-30% of wild-type P-gp). Login to comment 192 ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala Wild-type, F336A (A), and I340A (B) mutant P-gp membrane vesicles were incubated with 100nM NMQ (containing 10nM [3 H]-NMQ) in the absence or presence of indicated concentrations of cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol. Login to comment 194 ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala The data points have been shown by (ߦ) for wild-type, (a0;) for F336A mutant, and (b2;) for I340A mutant P-gp. Login to comment 202 ABCB1 p.Phe942Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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%). Login to comment 208 ABCB1 p.Phe942Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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. Login to comment 213 ABCB1 p.Phe942Ala ABCB1 p.Leu65Ala ABCB1 p.Val982Ala 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. Login to comment 214 ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala More remarkable were the inhibitory affinities of F336A and I340A, which were significantly different in five cases. Login to comment