ABCMdb

PMID: 12642584

Maki N, Hafkemeyer P, Dey S
Allosteric modulation of human P-glycoprotein. Inhibition of transport by preventing substrate translocation and dissociation.
J Biol Chem. 2003 May 16;278(20):18132-9. Epub 2003 Mar 17., 2003-05-16 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCB1 p.Phe983Ala A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Login to comment 45 ABCB1 p.Phe983Ala In addition, two cell lines NIHMDR1-WT and NIHMDR1-F983A were generated. Login to comment 46 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala NIHMDR1-WT and NIHMDR1-F983A were created by stepwise selection of NIH3T3 cells transfected with pHaMDR1 and pHaMDR1-F983A plasmids, respectively. Login to comment 48 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala To construct the vector pHaMDR1-F983A, the NsiI-XhoI fragment of the previously described plasmid pTM1MDR1 containing the F983A mutation (25) was cloned into the corresponding sites of pHaMDR1. Login to comment 49 ABCB1 p.Phe983Ala The region corresponding to the NdeI-PstI fragment within pHaMDR1-F983A including the insertion points and the flanking regions was sequenced in its entirety in both directions by automated sequencing (PRISM Ready Reaction DyeDeoxy Terminator Sequencing Kit, PerkinElmer Life Sciences). Login to comment 50 ABCB1 p.Phe983Ala The plasmids pHaMDR1 and pHaMDR1-F983A were calcium phosphate-transfected into NIH3T3 cells and selected with vincristine. Login to comment 52 ABCB1 p.Phe983Ala A stepwise selection was carried out with increasing concentrations of vincristine to generate NIHMDR1-WT and NIHMDR1-F983A cell lines that were able to grow in the presence of 1 ␮M vincristine. Login to comment 164 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala A Single Amino Acid Substitution That Alters the Inhibitory Potential of cis-(Z)-Flupentixol Also Affects cis-(Z)-Flupentixol-induced Formation of Pgp-[125 I]IAAP Complex-To determine the functional significance of the Pgp-[125 I]IAAP complex formation and the elevated level of [125 I]IAAP associated with Pgp-expressing cells in the presence of cis-(Z)-flupentixol, both phenomena were tested in the Pgp mutant F983A, which is largely insensitive to modulation by cis-(Z)-flupentixol (22). Login to comment 165 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala The intracellular accumulation of [125 I]IAAP in cells expressing either the wild-type Pgp (NIHMDR1-WT) or the mutant F983A Pgp (NIHMDR1-F983A) was considerably lower than that of the control NIH3T3 cells, suggesting efficient transport of [125 I]IAAP by both the wild-type and F983A mutant Pgp. Login to comment 166 ABCB1 p.Phe983Ala However, the elevated level of cellular [125 I]IAAP association, in the presence of 5 ␮M cis-(Z)-flupentixol, was selectively abrogated in NIHMDR1-F983A (Fig. 4A). Login to comment 167 ABCB1 p.Phe983Ala Photocross-linking for 5 min at the end of the uptake assay showed no increase in interaction (photocross-linking) between [125 I]IAAP and F983A by cis-(Z)-flupentixol, although under the same conditions increased complex formation was observed between wild-type Pgp and [125 I]IAAP in NIHMDR1-WT cells (Fig. 4B, upper panel). Login to comment 168 ABCB1 p.Phe983Ala In the absence of cis-(Z)-flupentixol, there was no significant difference in the interaction with [125 I]IAAP between the wild-type and the F983A mutant. Login to comment 169 ABCB1 p.Phe983Ala Cyclosporin A (5 ␮M), a competitive inhibitor of Pgp, inhibited both transport and [125 I]IAAP binding by wild-type and F983A with similar efficiency (Fig. 4B, upper panel). Login to comment 170 ABCB1 p.Phe983Ala Similar results were obtained from experiments with membranes isolated from NIHMDR1-WT and NIHMDR1-F983A cells (Fig. 4B, lower panel). Login to comment 176 ABCB1 p.Phe983Ala To investigate the mechanistic significance of this conformational change, UIC2 reactivity was studied in cells expressing the Pgp mutant F983A, which has impaired sensitivity to inhibition by cis-(Z)- flupentixol (Fig. 5B). Login to comment 177 ABCB1 p.Phe983Ala cis-(Z)-Flupentixol was unable to induce any negative effect on UIC2 binding to NIHMDR1-F983A cells FIG. 3. Login to comment 190 ABCB1 p.Phe983Ala Because increased or decreased UIC2 reactivity induced by cyclosporin A or vanadate, respectively, was unaffected in NIHMDR1-F983A (Fig. 5B), compared with that observed in NIHMDR1-WT cells, the possibility of a nonspecific effect could be ruled out. Login to comment 204 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala NIH3T3, NIHMDR1-WT (wild-type Pgp), and NIHMDR1-F983A (mutant F983A) cells were incubated with 1.5 nM [125 I]IAAP in the presence and absence of 5 ␮M cis-(Z)-flupentixol for 60 min at 37 °C. Login to comment 207 ABCB1 p.Phe983Ala Data points represent average value of three identical experiments. B, interaction of [125 I]IAAP with wild-type (WT) Pgp and F983A, in intact cells (upper panel) and in isolated membranes (lower panel). Login to comment 218 ABCB1 p.Phe983Ala B, UIC2 reactivity of Pgp mutant F983A. Login to comment 219 ABCB1 p.Phe983Ala UIC2 binding to NIHMDR1-WT and NIHMDR1-F983A cells was carried out as mentioned above either in the presence of 1 mM sodium orthovanadate (Vi), 5 ␮M cis-(Z)-flupentixol (Cis(Z)), 5 ␮M cyclosporin A (CsA), or in the absence of any modulators. Login to comment 260 ABCB1 p.Phe983Ala Interestingly, formation of the stable Pgp-[125 I]IAAP complex induced by cis-(Z)-flupentixol was remarkably affected in the Pgp mutant F983A (Fig. 4B), drug transport function by which is not inhibited by cis-(Z)-flupentixol (Fig. 4A). Login to comment 262 ABCB1 p.Phe983Ala Because the mutation F983A did not affect [125 I]IAAP transport or the ability of cyclosporin A to block the substrate site (Fig. 4B) and inhibit transport (Fig. 4A), any global effect of the mutation can be ruled out. Login to comment 270 ABCB1 p.Phe983Ala No such conformational change was induced by cis-(Z)- flupentixol in the Pgp mutant F983A, whereas the ability of the competitive inhibitor cyclosporin A to induce its characteristic change in conformation remained unaltered (Fig. 5B), underscoring the functional distinctness of the allosteric site. Login to comment