ABCMdb

PMID: 25264938

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., [PubMed]

Sentences

No. Mutations Sentence Comment

56 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.Val982Ala 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). Login to comment 154 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.Val982Ala 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. Login to comment 167 ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala NMQ transport activity of P-gp mutants I306A, F343A and F728A was less than 40% of wild type. Login to comment 168 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Phe942Ala ABCB1 p.Ile340Ala ABCB1 p.Phe336Ala ABCB1 p.Val982Ala 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). Login to comment 170 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.Val982Ala 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). Login to comment 172 ABCB1 p.Phe343Ala The data clearly demonstrates that transport activity of F343A for NMQ was abolished due to the mutagenesis, whereas it was partially preserved for convallatoxin. Login to comment 173 ABCB1 p.Val982Ala For the V982A mutant, NMQ transport was similar to that of the Fig. 3. Login to comment 212 ABCB1 p.Phe343Ala ABCB1 p.Val982Ala 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). Login to comment 253 ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe343Ala The P-gp mutants, I306A, F343A and F728A exhibited a NMQ transport activity that was less than 30% of the wild type activity. Login to comment 254 ABCB1 p.Leu975Ala ABCB1 p.Thr945Ala ABCB1 p.Phe942Ala ABCB1 p.Phe336Ala ABCB1 p.Val982Ala 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). Login to comment 255 ABCB1 p.Phe942Ala ABCB1 p.Ile306Ala ABCB1 p.Phe728Ala ABCB1 p.Phe336Ala Convallatoxin transport activity of most mutants (I306A, F336A, I340A, F728A, F942A, T945, and L975) was similar to that of NMQ. Login to comment 259 ABCB1 p.Phe343Ala ABCB1 p.Val982Ala The transport activities of NMQ and convallatoxin were significantly different for the F343A and V982A mutant (Fig. 5D and E). Login to comment 260 ABCB1 p.Phe343Ala NMQ transport activity of F343A was completely diminished, whereas convallatoxin transport activity was conserved. Login to comment 261 ABCB1 p.Phe343Arg Loo et al. (2007) observed that mutation of Phe343 to arginine reduced the affinity of P-gp for rhodamine B and cyclosporine A, but not for vinblastine. Login to comment 263 ABCB1 p.Val982Ala The V982A mutant lost 50% of its convallatoxin transport activity, whereas NMQ transport activity was not changed. Login to comment 265 ABCB1 p.Val982Ala 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). Login to comment