ABCMdb

PMID: 16489767

Maki N, Moitra K, Silver C, Ghosh P, Chattopadhyay A, Dey S
Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein.
Biochemistry. 2006 Feb 28;45(8):2739-51., 2006-02-28 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCB1 p.Phe983Ala In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Login to comment 24 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala 1 Abbreviations: Pgp, P-glycoprotein; [125 I]IAAP, [125 I]iodoarylazidoprazosin; [R-32 P]-8N3ATP, [R-32 P]-8-azido-ATP; ATP, adenosine triphosphate; ADP, adenosine diphosphate; TM, transmembrane; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Cis(Z), cis-(Z)-flupentixol; CsA, cyclosporin A; Vi, vanadate; F983A, Pgp F983A mutant; EGTA, ethylene glycol bis( -aminoethyl)-N,N,N',N'- tetraacetic acid. Login to comment 58 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala The recombinant baculovirus containing the Pgp mutant F983A [BV-MDR1-(H6)-F983A] was constructed in the same way as BV-MDR1-(H6) (25) using recombinant plasmid pBac- PakMDR1-(H6)-F983A. Login to comment 59 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala The NdeI-XhoI fragment of pTM1-MDR1-(H6)-F983A (containing the codon for amino acid phenylalanine 983 changed to that of alanine) (47) was cloned into the wild-type MDR1 coding region of plasmid pBacPak9-MDR1-(H6) to generate pBacPakMDR1-(H6)- F983A. Login to comment 60 ABCB1 p.Phe983Ala The recombinant baculovirus BV-MDR1(H6)-F983A was generated according to ref 25. Login to comment 61 ABCB1 p.Phe983Ala For expression of the wild-type Pgp and the mutant F983A, cells were propagated to 80% confluency at 27 °C in the monolayer and infected with the recombinant baculoviruses with a multiplicity of infection of 10 and harvested after 72 h of infection. Login to comment 159 ABCB1 p.Phe983Ala When dissociation of bound [125 I]IAAP in response to vanadate trapping was studied in the Pgp F983A mutant that is impaired in modulation by cis-(Z)-flupentixol (38, 39), no such delay in dissociation was observed even in the presence of 25 µM cis-(Z)-flupentixol (k ) 1.86 ( 0.25/ min, t1/2 ) 0.37 min) (R2 ) 0.99) (Figure 2C,D), suggesting that the inhibitory effect on substrate dissociation is dependent on a functional interaction of cis-(Z)-flupentixol with Pgp. Login to comment 171 ABCB1 p.Phe983Ala Consistent with that, stimulation of ATP hydrolysis by 50 µM prazosin in the Pgp F983A mutant was minimally affected by cis-(Z)- flupentixol (25 µM) (Figure 3B, right), further supporting that the inhibitory effect on substrate-stimulated ATP hydrolysis is mediated through a specific interaction of cis- (Z)-flupentixol with the allosteric site of Pgp. Login to comment 187 ABCB1 p.Phe983Ala Vanadate trapping of wild-type (top and middle panels) and F983A (bottom panel) Pgp was carried out in the presence (middle and lower panels) and absence (top panel) of 25 µM cis-(Z)-flupentixol, similar to the above experiment, except that nonradioactive 8-azido-ADP (1.25 mM) was used for trapping, and trapped samples were incubated and photo-cross-linked with 5 nM [125I]IAAP, a Pgp substrate. Login to comment 190 ABCB1 p.Phe983Ala Key: no modulator, O; 25 µM cis-(Z)-flupentixol, b; 25 µM cis-(Z)-flupentixol in F983A, 0. Login to comment 207 ABCB1 p.Phe983Ala The rate of ATP hydrolysis by the wild-type (left and middle histograms) and F983A (right histogram) Pgp was measured as mentioned in the presence of 50 µM prazosin and varying concentrations (0-50 µM) of the allosteric modulator cis-(Z)- flupentixol (left and right panels) or the competitive modulator verapamil (middle panel). Login to comment 221 ABCB1 p.Phe983Ala Since the stimulatory effect on both substrate interaction and ATP hydrolysis is abrogated in the Pgp F983A mutant (38), which is impaired in modulation by cis-(Z)-flupentixol (38, 39), it suggests that both phenomena are mediated through modulator interaction at the allosteric site. Login to comment 238 ABCB1 p.Phe983Ala The reduced effectiveness of cis-(Z)-flupentixol to inhibit the prazosin-stimulated ATP hydrolysis (Figure 3B, right) in the Pgp F983A mutant further confirms that the inhibitory action of the modulator is mediated through its interaction at the allosteric site of Pgp and not by its nonproductive (nonstimulating) association with the substrate site, blocking access to prazosin. Login to comment 245 ABCB1 p.Phe983Ala The dissociation of bound [125 I]IAAP coupled to vanadate trapping was minimally affected by cis-(Z)-flupentixol in the Pgp F983A mutant (Figure 2C,D), further emphasizing the involvement of the allosteric modulator site in the phenomenon. Login to comment 252 ABCB1 p.Phe983Ala In the Pgp F983A mutant, the stimulatory signal (for ATP hydrolysis) originating from the substrate site is minimally affected in the presence of cis-(Z)-flupentixol (Figure 3B, right); this suggests that the modulator-induced interference in cross talk between the FIGURE 5: Effect of cis-(Z)-flupentixol on dissociation of trapped [R-32P]-8-azido-ADP. Login to comment