ABCMdb

ABCB4 p.Leu975Cys

None has been submitted yet.

Publications

PMID: 16456713 [PubMed] Loo TW et al: "Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux."

No. Sentence Comment

200 During ATP hydrolysis, conformational changes between TM6 and TM12 were detected by disulfide cross-linking between cysteines introduced at the extracellular ends of these TMs (residue L332C in TM6 and L975C in TM12) (Loo & Clarke, 1996a, 1997b). Login to comment
201 Cross-linking between L332C/L975C prevented conformational changes during ATP hydrolysis, resulting in inhibition of ATPase activity. Login to comment

PMID: 17636884 [PubMed] Loo TW et al: "Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments."

No. Sentence Comment

74 The positions of the catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) and the cysteine mutations in the TM segments used in the disulfide cross-linking studies (L332C, L339C, F343C, F728C, L975C, and V982C) are shown. Login to comment
89 Mutant L332C(TM6)/L975C(TM12) contains one cysteine residue at the extracellular end of TM6 (L332C) and another at the extracellular end of TM12 (L975C) (Figure 1). Login to comment
125 Membranes prepared from HEK 293 cells expressing mutants L332C(TM6)/L975C(TM12), L332C- (TM6)/L975C(TM12)/E556Q(NBD1)/E1201Q(NBD2), L332C- (TM6)/L975C(TM12)/E556Q(NBD1), or L332C(TM6)/L975C- (TM12)/E1201Q(NBD2) were treated with (+) or without (-) 1 mM copper phenanthroline (CuP) for 10 min at 37 °C in the absence (None) or presence of 5 mM ATP (ATP). Login to comment
94 The mutant L332C(TM6)/L975C- (TM12) exhibited extensive cross-linking only in the presence of ATP (Figure 4). Login to comment
200 Mutant L332C(TM6)/L975C- (TM12) normally exhibits cross-linking only in the presence of ATP. Login to comment
234 The cross-linking pattern of mutant L332C(TM6)/L975C- (TM12)/E556Q(NBD1)/E1201Q(NBD2) indicates that ATP hydrolysis induces conformational changes in the TMDs that are distinct from those involving ATP binding. Login to comment

PMID: 22700974 [PubMed] Loo TW et al: "The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together."

No. Sentence Comment

106 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment
201 Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1). Login to comment
202 The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43). Login to comment
203 Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant. Login to comment
206 Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (Ͼ 90% efficiency) when intact cells were treated with BMOE. Login to comment
207 To test for the effect of cross-linking on activity, histidine-tagged T333C/L975C P-gp was isolated by nickel-chelate chromatography before and after cross-linking with BMOE. Login to comment
210 We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C. Login to comment
212 The results suggest that the stimulators may promote the closed conformation where the T333C and L975C residues are far apart. Login to comment
225 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (-) or presence (ϩ) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment
244 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment
104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment
195 Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1). Login to comment
196 The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43). Login to comment
197 Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant. Login to comment
200 Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (b0e; 90% efficiency) when intact cells were treated with BMOE. Login to comment
204 We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C. Login to comment
218 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (afa;) or presence (af9;) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment
237 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment