ABCMdb

PMID: 24083983

Loo TW, Clarke DM
Drug rescue distinguishes between different structural models of human P-glycoprotein.
Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCB1 p.Ile306Cys It was found that labeling of the I306C mutant with a thiol-reactive derivative of the substrate verapamil activated ATPase activity ~8-fold12 and labeling was blocked by verapamil. Login to comment 14 ABCB1 p.Ile306Arg In addition, it was found that the I306R mutation inhibited binding of a subset of P-gp drug substrates.13 These results suggest that residue Ile306 is important for binding of drug substrates and activation of Received: September 12, 2013 Revised: September 29, 2013 Published: October 1, 2013 Figure 1. Login to comment 15 ABCB1 p.Gly251Val Drug rescue of TM5 and TM9 G251V P-gp arginine mutants. Login to comment 18 ABCB1 p.Gly251Val An asterisk indicates a significant difference from the amount of the mature form observed when the G251V parent was expressed without cyclosporine A (~5% mature). Login to comment 23 ABCB1 p.Gly251Val Accordingly, the ability of drug substrates to promote maturation of a processing mutant (G251V) containing an arginine at each position in TM5 was used to map the locations of residues that faced the lipid bilayer (would prevent rescue) or the aqueous channel (would be rescued). Login to comment 24 ABCB1 p.Gly251Val ABCB1 p.Gly251Val The rationale for using this assay was that drug substrates such as cyclosporine A can promote maturation of a P-gp processing mutant (G251V).14 The G251V mutation is located in the second intracellular loop (ICL2) (Figure 1A) and appears to trap P-gp in a partially folded conformation as a 150 kDa core-glycosylated protein. Login to comment 25 ABCB1 p.Gly251Val Expression in the presence of a drug substrate induces G251V to complete the folding process to yield an active mature 170 kDa protein.15 Introduction of an arginine onto the lipid face of TM5 would inhibit drug rescue. Login to comment 27 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Ser298Arg ABCB1 p.Ile297Arg Examples of drug rescue of G251V and TM5 mutants G251V/I297R and G251V/S298R are shown in Figure 1B. When processing mutant G251V is expressed in the absence of cyclosporine A, the major product was the immature 150 kDa protein (~95% of total P-gp). Login to comment 29 ABCB1 p.Ser298Arg ABCB1 p.Ile297Arg Mutant S298R but not I297R could be rescued by cyclosporine A. Login to comment 30 ABCB1 p.Gly251Val Arginine mutations were then introduced into each position of TM5 or TM9 in the G251V background. Login to comment