ABCMdb

PMID: 16042402

Loo TW, Bartlett MC, Clarke DM
ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein.
Biochemistry. 2005 Aug 2;44(30):10250-8., 2005-08-02 [PubMed]

Sentences

No. Mutations Sentence Comment

81 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys It has been shown that TM12 (L975C) approaches TM6 (L332C) on the extracellular side during ATP hydrolysis (27). Login to comment 97 ABCB1 p.Met68Cys ABCB1 p.Met69Cys Figure 2A shows that the slower migrating product in mutants M68C(TM1)/Y950C(TM11), M68C- (TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C- (TM1)/A954C(TM11), and M69C(TM1)/F957C(TM11) was greatly reduced upon exposure to 10 mM dithiothreitol. Login to comment 105 ABCB1 p.Met69Cys Mutants M68C(TM1)/ Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/ A954C(TM11), M69C(TM1)/A954C(TM11), and M69C- (TM1)/F957C(TM11) all showed a time-dependent increase FIGURE 1: Model of packing of the TM segments of P-gp and location of the introduced cysteines. Login to comment 134 ABCB1 p.Met68Cys ABCB1 p.Met69Cys Membranes were prepared from HEK 293 cells (A), expressing mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C- (TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), M69C(TM1)/ F957C(TM11), or the negative mutant F72C(TM1)/F957C(TM11); (B) the single cysteine mutants M68C(TM1), M69C(TM1), Y950C(TM11), Y953C(TM11), A954C(TM11), or F957C(TM11); or (C) coexpressing the single cysteine mutants M68C(TM1) plus Y950C(TM11), M68C(TM1) plus Y953C(TM11), M68C(TM1) plus A954C(TM11), M69C(TM1) plus A954C(TM11), or M69C- (TM1) plus F957C(TM11). Login to comment 158 ABCB1 p.Ala954Cys ABCB1 p.Tyr950Cys Finally, we tested whether mutants M68C(TM1)/Y950C- (TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C- (TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) that showed ATP-dependent cross-linking were still active. Login to comment 163 ABCB1 p.Met69Cys Accordingly, the histidine-tagged P-gp mutants M68C(TM1)/ Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/ A954C(TM11), M69C(TM1)/A954C(TM11), and M69C- (TM1)/F957C(TM11) and the Cys-less P-gp were expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment 171 ABCB1 p.Leu332Cys (B) Membranes prepared from HEK 293 cells expressing mutant M69C(TM1)/A954C(TM11) or mutant L332C- (TM6)/L975C(TM12) were preincubated with 10 mM colchicine (Colch), 0.05 mM cyclosporin A (Cyclo), 1 mM verapamil (Ver), 0.1 mM vinblastine (Vin), or no drug substrate (None) for 15 min at 20 °C. The samples were then incubated with buffer containing 5 mM ATP and with (+) or without (-) oxidant for 10 min at 37 °C. The reactions were stopped by addition of SDS sample buffer containing EDTA and subjected to immunoblot analysis. Login to comment 173 ABCB1 p.Met68Cys Histidine-tagged wild-type, Cys-less, and P-gp mutants M68C- (TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/ A954C(TM11), M69C(TM1)/A954C(TM11), or M69C(TM1)/ F957C(TM11) were expressed in HEK 293 cells. Login to comment 187 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys Labeling of residues F942C and T945C by these thiol-reactive drug substrate analogues was inhibited by the presence of drug substrate. Login to comment