ABCMdb

PMID: 16729976

Maki N, Dey S
Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1).
Biochem Pharmacol. 2006 Jul 14;72(2):145-55. Epub 2006 Apr 25., [PubMed]

Sentences

No. Mutations Sentence Comment

47 ABCB1 p.Phe983Ala Replacement of the F983 with alanine (F983A) abrogates inhibition of [125 I]IAAP transport as well as stimulation of Pgp-[125 I]IAAP interaction by cis-(Z)-flupentixol, without any effect on the basal level of [125 I]IAAP binding and transport by Pgp [25]. Login to comment 59 ABCB1 p.Phe983Ala Previously characterized [25] mouse NIHMDR1-WT and NIHMDR1-F983A cells were used for the studies with intact cells. Login to comment 118 ABCB1 p.Phe983Ala Replacement of F983 by an alanine (F983A) abrogates both stimulation of [125 I]IAAP binding to Pgp as well as inhibition of transport by cis-(Z)- flupentixol [25]. Login to comment 119 ABCB1 p.Phe983Ala Replacement of F983 by an alanine (F983A) abrogates both stimulation of [125 I]IAAP binding to Pgp as well as inhibition of transport by cis-(Z)- flupentixol [25]. Login to comment 120 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala We studied [125 I]IAAP binding to the wild type Pgp as well as to the Pgp F983A mutant in NIHMDR1-WT and NIHMDR1-F983A cells, respectively, in the presence of several Pgp modulators, both structurally related (Fig. 1A and B) and unrelated (Fig. 1C) to cis-(Z)-flupentixol. Login to comment 121 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala A detectable level of [125 I]IAAP binding to the wild type and the Pgp F983A mutant was observed (Fig. 2A-C, autoradiogram, none) in the absence of any modulator. Login to comment 122 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala The relatively lower level of [125 I]IAAP signal in the Pgp F983A mutant was due to a reduced level of the expression of the mutant Pgp in NIHMDR1-F983A cells, as determined by immunoblotting with a Pgp specific antibody PEPG13 (see immunoblots). Login to comment 123 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala As evident in Fig. 2A, thioxanthene derivatives cis-(Z)-clopentixol, trans-(E)-clopentixol, cis-(Z)- 753, compound 789, cis-(Z)-768, and trans-(E)-768, all stimulated [125 I]IAAP binding to the wild type Pgp (autoradiogram, upper panel), and the stimulatory effect was abrogated in the Pgp F983A mutant (autoradiogram, lower panel). Login to comment 124 ABCB1 p.Phe983Ala As evident in Fig. 2A, thioxanthene derivatives cis-(Z)-clopentixol, trans-(E)-clopentixol, cis-(Z)- 753, compound 789, cis-(Z)-768, and trans-(E)-768, all stimulated [125 I]IAAP binding to the wild type Pgp (autoradiogram, upper panel), and the stimulatory effect was abrogated in the Pgp F983A mutant (autoradiogram, lower panel). Login to comment 127 ABCB1 p.Phe983Ala The inhibitory potential of these compounds on [125 I]IAAP binding remained unaltered in the Pgp F983A mutant (Fig. 2C, autoradiogram, lower panel), suggesting no involvement of F983 in the modulation of Pgp function by those compounds. Login to comment 128 ABCB1 p.Phe983Ala The inhibitory potential of these compounds on [125 I]IAAP binding remained unaltered in the Pgp F983A mutant (Fig. 2C, autoradiogram, lower panel), suggesting no involvement of F983 in the modulation of Pgp function by those compounds. Login to comment 159 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 4 5 - 1 5 5150 Fig. 2 - (A) Effect of thioxanthenes on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Login to comment 160 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 4 5 - 1 5 5 150 Fig. 2 - (A) Effect of thioxanthenes on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Login to comment 161 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Following incubation, cells were exposed to UV irradiation for 5 min, lysed, and resolved by SDS-PAGE (80,000 cells per well) as indicated in Section 2; (B) effect of phenothiazines on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done as described for (A), except that the modulators added were 5 mM cyclosporin A (CsA) (lane 2), 10 mM promethazine (lane 3), 10 mM chlorpromazine (lane 4), 10 mM trifluperazine (lane 5), 10 mM thioridazine (lane 6), or 1% DMSO (none) (lane 1); (C) effect of other modulators on Pgp- [125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done exactly the same way as described in (A), except modulators used were 5 mM cyclosporin A (CsA) (lane 2), 5 mM rapamycin (lane 3), 10 mM nicardipine (lane 4), 10 mM niguldipine (lane 5), or in the absence of any modulator (none) (lane 1). Login to comment 162 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Following incubation, cells were exposed to UV irradiation for 5 min, lysed, and resolved by SDS-PAGE (80,000 cells per well) as indicated in Section 2; (B) effect of phenothiazines on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done as described for (A), except that the modulators added were 5 mM cyclosporin A (CsA) (lane 2), 10 mM promethazine (lane 3), 10 mM chlorpromazine (lane 4), 10 mM trifluperazine (lane 5), 10 mM thioridazine (lane 6), or 1% DMSO (none) (lane 1); (C) effect of other modulators on Pgp- [125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done exactly the same way as described in (A), except modulators used were 5 mM cyclosporin A (CsA) (lane 2), 5 mM rapamycin (lane 3), 10 mM nicardipine (lane 4), 10 mM niguldipine (lane 5), or in the absence of any modulator (none) (lane 1). Login to comment 209 ABCB1 p.Phe983Ala The stimulatory effect on [125 I]IAAP binding, and its abrogation in the Pgp F983A mutant, provided us with a convenient assay to study the specificity of the allosteric site. Login to comment 210 ABCB1 p.Phe983Ala The stimulatory effect on [125 I]IAAP binding, and its abrogation in the Pgp F983A mutant, provided us with a convenient assay to study the specificity of the allosteric site. Login to comment