ABCMdb

ABCB1 p.Thr333Cys

None has been submitted yet.

Publications

PMID: 15192095 [PubMed] Rothnie A et al: "The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein."

No. Sentence Comment

130 Values refer to the mean Ϯ S.E. obtained from at least eight independent protein purification preparations. P-gp isoform Substrate affinity , Km Maximal activity, Vmax -Fold stimulationBasal Stimulated Basal Stimulated mM ␮mol Pi min-1 mg protein-1 Cys-less 0.58 Ϯ 0.06 0.38 Ϯ 0.04 0.58 Ϯ 0.15 1.46 Ϯ 0.30 2.9 Ϯ 0.3 V331C 0.50 Ϯ 0.06 0.26 Ϯ 0.02 0.45 Ϯ 0.05 1.54 Ϯ 0.20 3.5 Ϯ 0.3 T333C 0.49 Ϯ 0.05 0.23 Ϯ 0.02 0.35 Ϯ 0.04 1.22 Ϯ 0.15 3.3 Ϯ 0.1 F335C 0.40 Ϯ 0.05 0.24 Ϯ 0.03 0.65 Ϯ 0.15 1.61 Ϯ 0.31 2.2 Ϯ 0.2 S337C 0.53 Ϯ 0.06 0.26 Ϯ 0.04 0.59 Ϯ 0.10 1.67 Ϯ 0.23 3.2 Ϯ 0.4 L339C 0.51 Ϯ 0.07 0.31 Ϯ 0.04 0.57 Ϯ 0.07 1.47 Ϯ 0.15 2.9 Ϯ 0.3 G341C 0.40 Ϯ 0.04 0.24 Ϯ 0.02 0.42 Ϯ 0.03 1.12 Ϯ 0.09 3.1 Ϯ 0.5 F343C 0.41 Ϯ 0.04 0.26 Ϯ 0.03 0.47 Ϯ 0.04 1.17 Ϯ 0.15 2.6 Ϯ 0.3 generate stable covalent bonds with thiol groups under physiological conditions. Login to comment
141 Typical time courses for labeling of T333C with FM and G341C with CM are shown in Fig 1, b and c, respectively. Login to comment
142 Panel b shows a time-dependent increase in the labeling of T333C P-gp (lanes i-vii). Login to comment
154 Isoforms V331C, T333C, F335C, S337C, L339C, and G341C displayed labeling extents in the range 7-12%, and none was significantly different from the Cys-less isoform (ANOVA). Login to comment
163 Isoforms T333C, F335C, S337C, and G341C did not label with BM since the Lext values (19-27%) were not signif- TABLE II Potency of drugs that affect the ATPase activity of purified reconstituted single cysteine mutants of P-gp Pure, reconstituted P-gp (0.3 ␮g) was incubated in the presence of ATP (2 mM) and varying concentrations of nicardipine, vinblastine, or vanadate. Login to comment
166 Values refer to the mean Ϯ S.E. obtained from a minimum of three independent protein purification preparations. P-gp isoform Potency of drug effect Nicardipine, EC50 Vinblastine, EC50 Vanadate, IC50 ␮M ␮M ␮M Cys-less 3.2 Ϯ 0.3 4.2 Ϯ 0.6 4.0 Ϯ 0.4 V331C 3.3 Ϯ 0.4 7.2 Ϯ 1.7 3.2 Ϯ 0.4 T333C 2.3 Ϯ 0.2 4.6 Ϯ 0.4 3.9 Ϯ 0.8 F335C 2.3 Ϯ 0.4 4.2 Ϯ 0.8 5.5 Ϯ 1.1 S337C 2.7 Ϯ 0.5 4.1 Ϯ 1.0 5.8 Ϯ 0.8 L339C 2.1 Ϯ 0.3 5.1 Ϯ 0.8 4.2 Ϯ 0.7 G341C 3.9 Ϯ 0.5 4.0 Ϯ 0.6 6.8 Ϯ 1.3 F343C 2.1 Ϯ 0.3 5.6 Ϯ 2.7 2.7 Ϯ 0.8 FIG. 1. Login to comment
168 Structures of maleimide-containing probes (a) and the fluorescence profiles of SDS-PAGE gels obtained for time courses of fluorescein maleimide labeling of T333C (b) and coumarin maleimide labeling of G341C (c). Login to comment
200 Isoforms T333C (t1/2 ϭ 16 Ϯ 3 min) and F343C (t1/2 ϭ 18 Ϯ 5 min) labeled with CM with significantly increased rates compared with the values observed in either basal conditions or in the presence of AMP-PNP, reflecting an increased accessibility of the introduced cysteine residues. Login to comment
212 Isoform T333C, which was inaccessible to BM under basal conditions, remained so after nucleotide binding and only labeled to a significant extent in the vanadate-trapped state (Lext ϭ 85 Ϯ 2%). 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

13 Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. Login to 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
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

PMID: 23733192 [PubMed] Loo TW et al: "Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface."

No. Sentence Comment

82 IH2 Mediates TMD/NBD Coupling in the P-gp Drug Pump JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20327 Effect of Mutations on ATP-dependent Cross-linking of P-gp Extracellular Segments-Mutant T333C/L975C contains cysteines predicted to reside at the extracellular ends of TM segments 6 and 12, respectively (30). Login to comment
83 The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells. Login to comment
162 Accordingly, cross-linking of mutant T333C/L975C was used to test the effect of F1086A on coupling. Login to comment
163 The T333C and L975C mutations are located at the extracellular ends of TM segments 6 and 12, respectively (Fig. 3, A and B). Login to comment
164 Mutant T333C/L975C can be cross-linked when intact cells expressing the mutant are treated with BMOE cross-linker (30). Login to comment
165 Mutant T333C/L975C, however, does not show cross-linking if membranes containing the mutant are treated only with BMOE (Fig. 4A). Login to comment
170 To determine whether the F1086A mutation affected NBD/ TMD coupling, it was introduced into mutant T333C/L975C and subjected to cross-linking. Login to comment
171 It was observed that the F1086A mutation inhibited ATP-dependent cross-linking of the mutant T333C/L975C (Fig. 4B). Login to comment
187 A and B, membranes prepared from cells expressing mutant T333C/L975C (A) or T333C/L975C/F1086A (B) were treated with BMOE in the absence (None) or presence of nucleotides (ATP, AMP-PNP, or ADP). Login to comment
197 Accordingly, the A266F mutation was introduced into mutant F1086A/T333C/L975C to test whether it affected cross-linking between the TMDs. Login to comment
221 D, membranes prepared from cells expressing mutants T333C/L975C (None), T333C/L975C/F1086A, or T333C/L975C/F1086A/A266F were treated with BMOE in the presence (af9;) or absence (afa;) of ATP. Login to comment

PMID: 26507655 [PubMed] Loo TW et al: "Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein."

No. Sentence Comment

66 Mutant T333C/L975C was cross-linked in whole cells at 20 &#b0;C with 0.5 mM BMOE (bismaleimidoethane) cross-linker (Thermo Fisher Scientific, Burlington, ON) in the absence or presence of 0.25 òe;M tariquidar (30). Login to comment
118 The conditions for cross-linking mutants T333C/L975C and P517C/I1050C, however, were different. Login to comment
119 Mutant T333C/L975C was cross-linked with BMOE using intact cells because ATP hydrolysis is required for cross-linking (41). Login to comment