ABCB1 p.Ala259Cys
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PMID: 24275649
[PubMed]
Loo TW et al: "Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein."
No.
Sentence
Comment
9
Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol.
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ABCB1 p.Ala259Cys 24275649:9:24
status: NEW128 Cross-linked product was observed in all mutants (data not shown), with the greatest amount present in mutant A259C/W803C (about 75%, Fig. 4A).
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ABCB1 p.Ala259Cys 24275649:128:110
status: NEW129 We then tested whether cross-linking of mutant A259C/ W803C could be inhibited by nucleotides.
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ABCB1 p.Ala259Cys 24275649:129:47
status: NEW137 To test the effect of cross-linking on activity, histidine-tagged mutant A259C/W803C was expressed in HEK 293 cells in the presence of cyclosporine A and P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Ala259Cys 24275649:137:73
status: NEW156 Cross-linking of A259C (IH2) to W803C (IH3) inhibits activity.
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ABCB1 p.Ala259Cys 24275649:156:17
status: NEW157 A, membranes prepared from cells expressing histidine-tagged mutant A259C/ W803C were treated without (afa;) or with (af9;) 0.5 mM CuCl2 for 10 min at 0 &#b0;C in the absence (None) or presence of 5 mM ATP, adenosine 5b18;-(beta,ॹ- imino)triphosphate(AMP.PNP),orADP.Thereactionswerestoppedbyaddition of SDS sample buffer containing no reducing agent, and samples were then subjected to immunoblot analysis.
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ABCB1 p.Ala259Cys 24275649:157:68
status: NEW158 The positions of the cross-linked (X-link) and mature (170-kDa) P-gps are indicated. B, the amount of cross-linked protein relative to total P-gp (170-kDa plus 150-kDa P-gp) was quantitated from three different transfections af9; S.D. C, the histidine-tagged mutant A259C/ W803C was isolated by nickel-chelate chromatography, reconstituted with lipid, and treated without (afa;) or with (af9;) 0.2 mM CuCl2 for 10 min at 20 &#b0;C. The reaction was stopped with 1 mM EDTA.
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ABCB1 p.Ala259Cys 24275649:158:269
status: NEW199 In support of these predictions, we found that clamping IH2 to IH3 by oxidative cross-linking of mutant A259C/W803C or mutations to Tyr1087 inhibited activity.
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ABCB1 p.Ala259Cys 24275649:199:104
status: NEW209 Cross-linking of A259C (blue) in ICL2 to W803C (green) in ICL3 inhibited activity.
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ABCB1 p.Ala259Cys 24275649:209:17
status: NEW
PMID: 24523403
[PubMed]
Loo TW et al: "Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch."
No.
Sentence
Comment
81
P-gp ATPase Activity Is Highly Activated When Residues L175C and N820C Are Cross-linked in Close Proximity-In a previous study, we found that cross-linking the two homologous halves through formation of a direct disulfide bond between A259C in intracellular helix 2 (IH2) and W803C in IH3 inhibited activity (49).
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ABCB1 p.Ala259Cys 24523403:81:235
status: NEW82 It is possible that the A259C/W803C cross-link inhibited activity because these residues are located within IH segments that are predicted to form critical TMD/ NBD contact points.
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ABCB1 p.Ala259Cys 24523403:82:24
status: NEW