ABCMdb

PMID: 22360349

Mandal D, Moitra K, Ghosh D, Xia D, Dey S
Evidence for modulatory sites at the lipid-protein interface of the human multidrug transporter P-glycoprotein.
Biochemistry. 2012 Apr 3;51(13):2852-66. Epub 2012 Mar 22., [PubMed]

Sentences

No. Mutations Sentence Comment

28 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Thr945Ala ABCB1 p.Met948Ala ABCB1 p.Ile765Ala ABCB1 p.Ile764Ala ABCB1 p.Ala871Phe ABCB1 p.Leu772Ala ABCB1 p.Gly723Ala ABCB1 p.Ile840Ala ABCB1 p.Ile864Ala ABCB1 p.Val715Ala ABCB1 p.Ile768Ala ABCB1 p.Phe716Ala ABCB1 p.Ile719Ala ABCB1 p.Ile867Ala ABCB1 p.Phe770Ala ABCB1 p.Thr837Ala The desired nucleotide replacements were confirmed by Big-Dye version 3.1 (BD Sciences) nucleotide sequencing of the MDR1 open reading frame in recombinant plasmids pKM2-MDR1- V715A, -F716A, -I719A, -G723A, -I764A, -I765A, -I768A, -F770A, -L772A, -T837A, -I840A, -I864A, -I867A, -A871F, -T945A, -M948A, -F983A, -M986A, -V988A, -Q990A, and -V991A. Login to comment 99 ABCB1 p.Ile765Ala ABCB1 p.Ile764Ala ABCB1 p.Leu772Ala ABCB1 p.Gly723Ala ABCB1 p.Val715Ala ABCB1 p.Ile768Ala ABCB1 p.Phe716Ala ABCB1 p.Ile719Ala ABCB1 p.Phe770Ala As one can see in Figure 2A, none of the substitutions in TM7 (G723A, I719A, F716A, and V715A) or TM8 (L772A, F770A, I768A, I765A, and I764A) was critical for stimulation of [125 I]IAAP binding by cis-(Z)-flupentixol. Login to comment 102 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Phe983Ala ABCB1 p.Met948Ala A comparable level of basal binding of [125 I]IAAP to wild-type Pgp and Pgp mutants M948A, F983A, V988A, M986A, and Q990A (Figure 2B) rules out the possibility that the reduced level of stimulation in the mutants is due to loss of [125 I]IAAP binding and not stimulation per se. To investigate whether M948, Q990, F983, M986, and V988 are part of a general module involved in the regulation of substrate binding or are molecular components specific to cis- (Z)-flupentixol, we studied the effect of a structurally unrelated Pgp modulator, cyclosporin A, on binding of [125 I]IAAP to all 21 mutants. Login to comment 110 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Thr945Ala ABCB1 p.Met948Ala ABCB1 p.Ile765Ala ABCB1 p.Ile764Ala ABCB1 p.Ala871Phe ABCB1 p.Leu772Ala ABCB1 p.Gly723Ala ABCB1 p.Ile840Ala ABCB1 p.Ile864Ala ABCB1 p.Val715Ala ABCB1 p.Ile768Ala ABCB1 p.Ile719Ala ABCB1 p.Ile867Ala ABCB1 p.Phe770Ala ABCB1 p.Thr837Ala Stimulation of ATP Hydrolysis by cis-(Z)-Flupentixol and Verapamil cis-(Z)-flupentixol-mediated stimulation verapamil-mediated stimulation maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark WT 7.3 ± 0.4 6.8 ± 1.5 5.6 ± 0.19 6.6 ± 1.04 V715A 6.2 ± 0.2 5.0 ± 0.9 6.4 ± 0.37 16.3 ± 3.3 V716A 5.1 ± 0.2 4.7 ± 1.1 NDb NDb a I719A 4.6 ± 0.08 4.7 ± 0.4 5.8 ± 0.26 6.5 ± 1.31 G723A 5.3 ± 0.3 10.93 ± 2.7 5.4 ± 0.1 11.6 ± 0.88 I764A 4.5 ± 0.4 4.1 ± 2 3.2 ± 0.26 27.9 ± 8.4 I765A 4.96 ± 0.2 2.3 ± 0.47 NDb NDb a I768A 4.8 ± 0.2 1.2 ± 0.43 4.8 ± 0.19 8.5 ± 1.4 F770A 6.6 ± 0.6 10.9 ± 3.9 8.6 ± 0.59 13.2 ± 3.2 L772A 3.6 ± 0.2 2.96 ± 0.9 3.2 ± 0.11 9.5 ± 1.6 T837A 1.1 ± 0.06 NDb a 2.6 ± 0.1 3.1 ± 0.9 I840A 4.0 ± 0.3 25.6 ± 6.5 6.9 ± 0.2 13.5 ± 0.5 I864A 0.6 ± 0.05 NDb a 4.1 ± 0.25 0.9 ± 1.04 I867A 10.2 ± 1.3 72.3 ± 18.5 10.2 ± 0.52 30.7 ± 3.1 A871F NDb NDb NDb NDb a T945A 5.0 ± 0.3 13.7 ± 3.1 6.4 ± 0.15 6.9 ± 0.71 M948A NDb NDb a 6.8 ± 1.5 44.4 ± 12.4 F983A 4.95 ± 0.36 19.59 ± 5.0 6.1 ± 0.38 6.6 ± 1.8 M986A 3.8 ± 0.3 2.6 ± 1.5 7.7 ± 0.76 30.0 ± 8.6 V988A 3.0 ± 0.2 21.4 ± 7.6 6.5 ± 0.31 16.9 ± 2.9 Q990A 1.8 ± 0.4 18.9 ± 2.6 a 7.7 ± 0.58 12.9 ± 3.7 V991A 3.9 ± 0.2 21.1 ± 5.0 5.9 ± 0.59 21.9 ± 7.2 a Mutants with <2-fold maximal stimulation (<25% of the wild-type level). Login to comment 113 ABCB1 p.Val988Ala ABCB1 p.Ala871Phe ABCB1 p.Ile867Ala Although a moderate effect was observed in Pgp mutants I867A, A871F, and V988A, none of the replacements proved to be absolutely critical for inhibition by cyclosporin A (Figure 3). Login to comment 135 ABCB1 p.Ile764Ala ABCB1 p.Leu772Ala Although a moderate loss of stimulation was observed in mutants L772A and I764A, a >2-fold residual activity in each case suggested that neither of the two residues was indispensable. Login to comment 137 ABCB1 p.Ala871Phe This, however, was with the exception of mutant A871F, which replaced a naturally occurring alanine with a phenylalanine. Login to comment 138 ABCB1 p.Ala871Phe The more general effect evident in A871F most likely represented a local structural perturbation caused by replacement of the methyl side chain of alanine with a bulkier benzyl functional group of phenylalanine. Login to comment 200 ABCB1 p.Gln990Ala ABCB1 p.Ile864Ala As one can see in the top panels (middle and right) of Figure 8, the negative synergistic effect of cis-(Z)-flupentixol on verapamil-stimulated ATP hydrolysis was considerably compromised in I864A and Q990A, Pgp mutants defective with respect to ATP-site stimulation by cis-(Z)-flupentixol. Login to comment 201 ABCB1 p.Ile765Ala Reciprocally, the inhibitory effect of verapamil on cis-(Z)-flupentixol-mediated stimulation of the ATP site was obliterated in the verapamil-specific Pgp mutant I765A (Figure 8, bottom right). Login to comment 205 ABCB1 p.Gln990Ala ABCB1 p.Ile765Ala ABCB1 p.Ile864Ala ATP hydrolysis by the wild type and Pgp mutants I864A, Q990A, and I765A, in isolated membrane vesicles, was conducted either after preincubation with 50 μM verapamil (top) or with 50 μM cis-(Z)-flupentixol (bottom) followed by incubation with increasing concentrations of cis-(Z)-flupentixol (top) or verapamil (bottom), using the vanadate-sensitive phosphate release assay. Login to comment 224 ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Phe983Ala Substitutions F983A, M986A, and V988A completely abrogate stimulation of substrate binding (Figure 2) with only modest effects on ATP-site stimulation (Figure 4), which indicates that the two modulatory events are not tightly coupled to each other. Login to comment 225 ABCB1 p.Ile864Ala ABCB1 p.Thr837Ala Similarly, a reciprocal effect was evident in T837A and I864A, where a 34-fold stimulation of substrate binding was retained (Figure 2) even though ATP-site stimulation was almost abrogated (Figure 4). Login to comment