ABCMdb

PMID: 12421806

Loo TW, Bartlett MC, Clarke DM
Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains.
J Biol Chem. 2003 Jan 17;278(3):1575-8. Epub 2002 Nov 5., 2003-01-17 [PubMed]

Sentences

No. Mutations Sentence Comment

27 ABCB1 p.Leu531Cys One mutant (L531C/Cys1074 ) contained a cysteine in the NH2-terminal 531 LSGGQ535 site and an endogenous cysteine (Cys1074 ) in the COOH-terminal Walker A site (1070 GSSGCGKC1077 ). Login to comment 28 ABCB1 p.Leu1176Cys The other mutant (Cys431 / L1176C) contained the endogenous Cys431 in the NH2-terminal Walker A site (427 GNSGCGKS434 ) and another cysteine in the COOH-terminal 1176 LSGGQ1180 site. Login to comment 56 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys Fig. 1 shows that that cross-linking was almost complete in mutants L531C/Cys1074 and Cys431 /L1176C when treated with 0.5 mM copper phenanthroline for 15 min at 21 °C. Login to comment 63 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys Fig. 1 shows that inhibition of cross-linking in mutants L531C/Cys1074 and Cys431 /L1176C was observed only after treatment with vanadate plus Mg-ATP. Login to comment 70 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys Therefore, it is possible that the conformational changes in the TMDs may be monitored by changes in the cross-linking patterns in mutants L531C/Cys1074 and Cys431 /L1176C. Login to comment 73 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys These drug substrates were then tested on mutants L531C/Cys1074 and Cys431 / L1176C. Login to comment 74 ABCB1 p.Leu531Cys Fig. 2 shows that the ATPase activity of mutant L531C/Cys1074 was stimulated by calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil (6.4-, 6.8-, 3.5-, and 4-fold, respectively). Login to comment 76 ABCB1 p.Leu531Cys Cyclosporin A, Hoechst 33342, and trans(E)-flupentixol inhibited the activity of mutant L531C/Cys1074 with 50% inhibition occurring at concentrations of about 0.12, 0.67, and 1.1 mM, respectively. Login to comment 77 ABCB1 p.Leu1176Cys Similar results were obtained with mutant Cys431 / L1176C (data not shown). Login to comment 79 ABCB1 p.Leu531Cys Accordingly, membranes from mutant L531C/ Cys1074 were preincubated for 10 min at 21 °C with 1 mM calcein-AM, 2 mM demecolcine, 1 mM cis(Z)-flupentixol, 0.2 mM verapamil, 0.5 mM cyclosporin A, 0.5 mM Hoechst 33342, or 1 mM trans(E)-flupentixol. Login to comment 83 ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys ABCB1 p.Leu1176Cys Membranes were prepared from HEK 293 cells expressing mutants L531C/Cys1074 (L531C/C1074) or Cys431 /L1176C (C431/L1176C). Login to comment 90 ABCB1 p.Leu531Cys Effect of drug substrates on the ATPase activity of P-gp mutant L531C/Cys1074 . Login to comment 91 ABCB1 p.Leu531Cys Histidine-tagged mutant L531C/Cys1074 was isolated by nickel-chelate chromatography, mixed with E. coli lipids, and sonicated. Login to comment 96 ABCB1 p.Leu531Cys Fig. 3 shows the effects of substrates on cross-linking of mutant L531C/Cys1074 . Login to comment 101 ABCB1 p.Leu1176Cys We then tested the effect of drug substrates on cross-linking of mutant Cys431 /L1176C. Login to comment 106 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys The effect of substrates on mutants L531C/Cys1074 and Cys431 /L1176C were very similar. Login to comment 117 ABCB1 p.Leu531Cys Effect of drug substrate on cross-linking of mutant L531C/Cys1074 . Login to comment 118 ABCB1 p.Leu531Cys Membranes containing P-gp mutant L531C/Cys1074 were pre-incubated with no drug, calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, or trans(E)-flupentixol and then treated with oxidant at 4 °C for the indicated times. Login to comment 122 ABCB1 p.Leu1176Cys Effect of drug substrates on cross-linking of mutant Cys431 /L1176C. Login to comment 123 ABCB1 p.Leu1176Cys Cross-linking on membranes containing P-gp mutant Cys431 /L1176C were done as described in the legend to Fig. 3. Login to comment