ABCB1 p.Thr945Cys
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PMID: 10585407
[PubMed]
Loo TW et al: "Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane."
No.
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Comment
120
Fig. 5 shows that the majority of the mutants, except for G939C, F942C, T945C, and Y953C, were not affected by treatment with dBBn.
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ABCB1 p.Thr945Cys 10585407:120:72
status: NEW122 In contrast, the activities of mutants F942C and T945C, were almost completely inhibited by dBBn (80 and 85%, respectively).
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ABCB1 p.Thr945Cys 10585407:122:49
status: NEW123 The activities of mutants F942C and T945C were inhibited to FIG. 3.
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ABCB1 p.Thr945Cys 10585407:123:36
status: NEW138 By contrast, the verapamil-, vinblastine-, or colchicine-stimulated ATPase activities of mutants F942C or T945C were inhibited by more than 70% when pretreated with dBBn.
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ABCB1 p.Thr945Cys 10585407:138:106
status: NEW139 We then tested whether the presence of verapamil, vinblastine, or colchicine could protect mutants F942C or T945C from inactivation by dBBn.
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ABCB1 p.Thr945Cys 10585407:139:108
status: NEW143 The activities of mutants F942C and T945C, however, were protected by pretreatment with substrate (Fig. 6B).
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ABCB1 p.Thr945Cys 10585407:143:36
status: NEW157 Protection of mutant T945C from dBBn inhibition by drug substrates.
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ABCB1 p.Thr945Cys 10585407:157:21
status: NEW159 Histidine-tagged Cys-less and mutants F942C and T945C P-gp were transiently expressed in HEK 293 cells in the presence of 10 M cyclosporin A and isolated by nickel-chelate chromatography. Equivalent amounts of each P-gp were mixed with lipid and incubated with 1 mM dBBn for 5 min at 37 °C, quenched with cysteine, and then assayed for verapamil (1 mM)-, vinblastine (0.1 mM)-, or colchicine (5 mM)-stimulated ATPase activity.
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ABCB1 p.Thr945Cys 10585407:159:48
status: NEW161 Equivalent amounts of histidine-tagged Cys-less or mutants F942C or T945C P-gp were preincubated for 15 min at 4 °C without or with 2 mM verapamil (Ver.
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ABCB1 p.Thr945Cys 10585407:161:68
status: NEW183 Reaction of dBBn with two mutants, F942C and T945C, significantly inhibited substrate-stimulated ATPase activity.
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ABCB1 p.Thr945Cys 10585407:183:45
status: NEW184 The presence of verapamil, vinblastine, or colchicine protected mutants F942C and T945C from inhibition by dBBn.
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ABCB1 p.Thr945Cys 10585407:184:82
status: NEW
PMID: 10681495
[PubMed]
Loo TW et al: "The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis."
No.
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Comment
77
In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Thr945Cys 10681495:77:1321
status: NEW
PMID: 16042402
[PubMed]
Loo TW et al: "ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein."
No.
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Comment
187
Labeling of residues F942C and T945C by these thiol-reactive drug substrate analogues was inhibited by the presence of drug substrate.
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ABCB1 p.Thr945Cys 16042402:187:31
status: NEW
PMID: 16492138
[PubMed]
Loo TW et al: "Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket."
No.
Sentence
Comment
41
A series of double cysteine mutants containing L65C in TM1 with another cysteine in TMD2 (C-terminal TMD containing TM7-TM12) predicted to line the drug-binding pocket [34] (i.e. F942C or T945C in TM11 and L975C, V981C, V982C, G984C or A985C in TM12) were also constructed for cross-linking analysis.
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ABCB1 p.Thr945Cys 16492138:41:188
status: NEW60 Disulphide cross-linking analysis Mutants L65C, F942C, T945C, L975C, V981C, V982C, G984C, A985C, L65C/F942C, L65C/T945C, L65C/975C, L65C/V981C, L65C/V982C, L65C/G984C and L65C/A985C were transiently expressed in HEK-293 cells.
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ABCB1 p.Thr945Cys 16492138:60:55
status: NEWX
ABCB1 p.Thr945Cys 16492138:60:114
status: NEW160 Accordingly, Figure 6 Disulphide cross-linking of P-gp mutants (A) Membranes were prepared from HEK-293 cells (A) expressing mutants L65C, L65C/T945C, L65C/V982C, L65C/G984C or L65C/A985C.
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ABCB1 p.Thr945Cys 16492138:160:146
status: NEW
PMID: 21182301
[PubMed]
Loo TW et al: "The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants."
No.
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Comment
309
It was not surprising that the mutations affected rhodamine B-stimulated ATPase activity because it was previously shown that treatment of mutants W232C, N296C, and T945C (E875C was not tested) with a thiol-reactive analogue of rhodamine inhibited activity (15).
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ABCB1 p.Thr945Cys 21182301:309:165
status: NEW368 These results are consistent with those from cysteine mutagenesis studies where modification of mutant T945C by thiol-reactive analogues of verapamil or rhodamine or mutants W232C and N296C by a thiol reactive analogue of rhodamine inhibited their activity (13, 15).
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ABCB1 p.Thr945Cys 21182301:368:103
status: NEW
PMID: 16545467
[PubMed]
Shilling RA et al: "New light on multidrug binding by an ATP-binding-cassette transporter."
No.
Sentence
Comment
78
Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,2].
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ABCB1 p.Thr945Cys 16545467:78:550
status: NEW76 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,25].
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ABCB1 p.Thr945Cys 16545467:76:550
status: NEW