ABCMdb

PMID: 20394729

Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.
Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Only mutant L175C/N820C was cross-linked. Login to comment 58 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys The majority of mutant L175C/N820C was cross-linked after treatment with M1M cross-linker (Fig. 2A). Login to comment 59 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linked product was not observed in the other 11 mutants (Fig. 2A) or in the single cysteine mutants L175C and N820C (data not shown). Login to comment 60 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys When mutant L175C/N820C was treated with various concentrations of M1M, it was found that the highest amount of cross-linked product was obtained in the presence of about 0.01-0.02 mM cross-linker and decreased with higher concentrations of cross-linker (Fig. 2B). Login to comment 62 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys To test whether mutant L175C/N820C was still active, the histidine-tagged version of mutant L175C/N820C was isolated by nickel-chelate chromatography and assayed for drug-stimulated ATPase activity. Login to comment 64 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys It was found that activation and apparent affinity of mutant L175C/N820C for the drug substrates verapamil, vinblastine, rhodamine B, and colchicine were not significantly different from the Cys-less parent (data not shown). Login to comment 65 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys For example, mutant L175C/N820C showed 50% stimulation of ATPase activity (S50) in the presence of 12.6 ± 1.0 lM verapamil, 44 ± 7 lM rhodamine B, 450 ± 40 lM colchicine, and 2.2 ± 0.4 lM vinblastine. Login to comment 67 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Therefore, the mutant was selected for further analysis. Cross-linking of mutant L175C/N820C with M1M (Fig. 2A) suggested that these residues were in close proximity. Login to comment 78 ABCB1 p.Asn820Cys Mutant L175C/N820C was cross-linked with 0.02 mM M1M at 0 °C for various intervals in the presence of 0.3 mM verapamil or 5 mM Mg.ATP. Login to comment 83 ABCB1 p.Asn820Cys Since mutant L175C/N820C was active, we tested the effect of cross-linking with M1M on drug-stimulated ATPase activity. Login to comment 84 ABCB1 p.Asn820Cys Membranes prepared from cells expressing mutant L175C/N820C were first treated for 15 min at 0 °C in the presence or absence of M1M cross-linker. Login to comment 86 ABCB1 p.Asn820Cys Fig. 4A shows that untreated mutant L175C/N820C exhibited about a 12-fold increase in verapamil-stimulated ATPase activity with basal and verapamil-stimulated ATPase activities of 0.14 and 1.75 lmol Pi/min/mg P-gp, respectively. Login to comment 92 ABCB1 p.Asn820Cys We compared the effects of treating mutant L175C/N820C with low (0.02 mM; promotes cross-linking) or high (3 mM; inhibits cross-linking) concentrations of M1M on ATPase activity. Login to comment 93 ABCB1 p.Asn820Cys Treatment of the mutant L175C/N820C with 0.02 mM M1M elevated the basal ATPase activity (Fig. 4A) while treatment with 3 mM cross-linker yielded ATPase activities that were similar to those of untreated control (data not shown). Login to comment 94 ABCB1 p.Asn820Cys It was also found that the activity of mutants containing only L175C or N820C mutation was not affected by treatment with 0.02 mM M1M. Login to comment 97 ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys ABCB1 p.Asp821Cys ABCB1 p.Asp821Cys ABCB1 p.Ala822Cys ABCB1 p.Ala822Cys ABCB1 p.Thr176Cys ABCB1 p.Thr176Cys ABCB1 p.Thr176Cys ABCB1 p.Thr176Cys ABCB1 p.Thr176Cys ABCB1 p.Thr176Cys ABCB1 p.Asp178Cys ABCB1 p.Asp178Cys ABCB1 p.Asp178Cys ABCB1 p.Asp178Cys ABCB1 p.Asp178Cys ABCB1 p.Asp178Cys Verapamil and rhodamine B were selected for study because thiol-reactive derivatives of these drug substrates have been used extensively in cysteine mutagenesis studies to characterize the X-link 170 kDa + + + _ + ++ + + + T176C D177C D178C L175C T176C D177C D178C L175C N820C + D821C + M1M A822C + ++ + T176C D177C D178C L175C B 0.0003 0.001 0.003 0.009 0.027 0.08 0.24 0.73 [M1M] (mM) X-link 170 kDa 0 2.2 C 0 20 40 60 80 100 PercentCross-linked 0.0003 0.001 0.003 0.009 0.027 0.08 0.24 0.73 [M1M] (mM) 0 2.2 A Fig. 2. Login to comment 100 ABCB1 p.Asn820Cys (B) Mutant L175C/N820C was treated with various concentrations of M1M cross-linker for 15 min at 0 °C. Login to comment 104 ABCB1 p.Asn820Cys Effect of ATP and verapamil on cross-linking of mutant L175C/N820C. Login to comment 105 ABCB1 p.Asn820Cys (A) Membranes from cells expressing mutant L175C/N820C were incubated in the presence of no drug (None), 0.3 mM verapamil (+ verapamil) or 5 mM ATP plus 10 mM MgCl2 (+ Mg.ATP) for 15 min at 0 °C. Login to comment 111 ABCB1 p.Asn820Cys Fig. 4B shows that untreated mutant L175C/N820C showed 50% stimulation (S50) of ATPase activity with 12.6 ± 1.0 lM verapamil and 44 ± 7 lM rhodamine B. Login to comment 113 ABCB1 p.Asn820Cys Mutant L175C/N820C cross-linked with M1M showed an S50 of 4.3 ± 0.4 lM and 7.8 ± 0.8 lM for verapamil and rhodamine B, respectively. Login to comment 114 ABCB1 p.Asn820Cys We also tested the activity of untreated and M1M-treated mutant L175C/N820C in the presence of various concentrations of ATP. Login to comment 121 ABCB1 p.Asn820Cys The cross-linking results, however, suggest that L175C/N820C were already in close proximity in the absence of drug substrate or ATP (Figs. Login to comment 138 ABCB1 p.Asn820Cys M1M-cross-linked mutant L175C/N820C still showed drug-stimulated ATPase activity with drug substrates verapamil and rhodamine B (Fig. 4B). Login to comment