ABCB1 p.Leu975Cys
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PMID: 10506575
[PubMed]
Loo TW et al: "The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy."
No.
Sentence
Comment
42
The Cys-less mutant was also modified to contain mutations L332C in TM6 and L975C in TM12 (27).
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ABCB1 p.Leu975Cys 10506575:42:76
status: NEW120 Cys-less P-gp containing the mutations L332C and L975C was expressed with or without MG-132 and cross-linked in the presence or absence of Mg⅐ATP.
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ABCB1 p.Leu975Cys 10506575:120:49
status: NEW121 Figure 5 shows that ATP induces cross-linking in mutant L332C/L975C when expressed in the absence of MG-132.
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ABCB1 p.Leu975Cys 10506575:121:62
status: NEW123 In the presence of MG-132, however, the 150 kDa core-glycosylated mutant L332C/L975C was not cross-linked by oxidant in the absence or presence of ATP (Fig. 5, lanes 6 and 8).
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ABCB1 p.Leu975Cys 10506575:123:79
status: NEW139 HEK 293 cells were transfected with Cys-less P-gp cDNA containing mutations L332C/L975C and then incubated with (ϩMG-132) or without (-MG-132) MG-132 for 24 h. Membranes were then prepared and treated with (ϩ) or without (-) copper phenanthrolene (CuPhen) for 5 min at 37°C in the presence (ϩATP) or absence (-ATP) of 10 mM ATP.
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ABCB1 p.Leu975Cys 10506575:139:82
status: NEW
PMID: 12223492
[PubMed]
Loo TW et al: "Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein."
No.
Sentence
Comment
155
Lower levels of protection were observed with mutants I340C, A841C, L975C, and V982C (Fig. 5).
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ABCB1 p.Leu975Cys 12223492:155:68
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.
Sentence
Comment
81
It has been shown that TM12 (L975C) approaches TM6 (L332C) on the extracellular side during ATP hydrolysis (27).
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ABCB1 p.Leu975Cys 16042402:81:29
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.Leu975Cys 16492138:41:206
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.Leu975Cys 16492138:60:62
status: NEW
No.
Sentence
Comment
165
In 2002, Loo & Clarke found that with hydrolysis of the first molecule of ATP there is a further conformational change such as an -helix rotation between TM6 and TM12, which can be detected by disulfide cross-linking between cysteines (residue L332C in TM6 and L975C in TM12) [66].
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ABCB1 p.Leu975Cys 20088754:165:261
status: NEW
PMID: 8910331
[PubMed]
Loo TW et al: "Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates."
No.
Sentence
Comment
116
B, the above experiment was repeated with Cys-less P-glycoprotein(His)10, mutants containing only one cysteine (L332C or L975C), or a mutant containing both cysteines (L332C ϩ L975C).
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ABCB1 p.Leu975Cys 8910331:116:121
status: NEWX
ABCB1 p.Leu975Cys 8910331:116:182
status: NEW117 C, mutant L332C ϩ L975C was treated with (lane 2) or without (lane 1) copper phenanthroline for 15 min at 37 °C and then solubilized with SDS sample buffer.
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ABCB1 p.Leu975Cys 8910331:117:24
status: NEW151 To test for this possibility, the cDNAs coding for Cys-less P-glycoprotein and mutant L332C ϩ L975C were transfected into NIH 3T3 cells, and drug-resistant colonies were selected in the presence of 45 nM colchicine or 5 nM vinblastine.
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ABCB1 p.Leu975Cys 8910331:151:100
status: NEW156 A, membranes prepared from cells transfected with vector alone (control) or cotransfected with cDNA for mutant L332C in the Cys-less NH2-terminal half-molecule A52 and the cDNA for mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with cDNA for mutant F335C in the NH2-terminal half-molecule A52 and the cDNA for mutant F978C in the COOH-terminal half-molecule A52 were treated with oxidant for various intervals and at different temperatures.
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ABCB1 p.Leu975Cys 8910331:156:188
status: NEW158 B, HEK 293 cells were co-transfected with cDNAs encoding Cys-less NH2-and COOH-terminal half-molecules A52; the cDNAs of mutant L332C in the NH2-terminal half-molecule A52 and the Cys-less COOH-terminal half-molecule A52; the cDNAs of Cys-less NH2-terminal half-molecule-A52 and mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with the cDNAs of the mutant L335C in the NH2-terminal half-molecule A52 and mutant L975C in the COOH-terminal half-molecule A52.
X
ABCB1 p.Leu975Cys 8910331:158:286
status: NEWX
ABCB1 p.Leu975Cys 8910331:158:287
status: NEW167 As shown in Fig. 5, the ATPase activities of both mutants (Cys-less and L332C ϩ L975C) were similar in the presence of 5 mM colchicine, 1 mM verapamil, and 100 M vinblastine.
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ABCB1 p.Leu975Cys 8910331:167:86
status: NEW168 Compared with the wild-type enzyme, both Cys-less and L332C ϩ L975C mutants had lower drug-stimulated ATPase activities.
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ABCB1 p.Leu975Cys 8910331:168:68
status: NEW
PMID: 9261097
[PubMed]
Loo TW et al: "Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12."
No.
Sentence
Comment
82
Effect of Nucleotides and Drug Substrates on Cross-linking-An interesting observation was that the amount of cross-linking seen in mutant L332C/L975C in whole cells (9) varied with the metabolic state of the transfected cells.
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ABCB1 p.Leu975Cys 9261097:82:144
status: NEW84 A possible explanation was that cross-linking of mutant L332C/L975C was promoted by the presence of ATP.
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ABCB1 p.Leu975Cys 9261097:84:62
status: NEW96 pressing mutant L332C/L975C were cross-linked in the presence of nucleotides.
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ABCB1 p.Leu975Cys 9261097:96:22
status: NEW99 These results suggest that cross-linking between L332C and L975C occurred during ATP hydrolysis.
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ABCB1 p.Leu975Cys 9261097:99:59
status: NEW103 To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine.
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ABCB1 p.Leu975Cys 9261097:103:70
status: NEW104 No cross-linked product was observed for mutant L332C/L975C (Fig. 3A).
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ABCB1 p.Leu975Cys 9261097:104:54
status: NEW108 Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein.
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ABCB1 p.Leu975Cys 9261097:108:74
status: NEW109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A).
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ABCB1 p.Leu975Cys 9261097:109:82
status: NEW126 Membranes prepared from HEK 293 cells expressing mutants L332C/L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 5 mM ATP, 5 mM ATP plus 0.2 mM sodium vanadate, 5 mM ADP, 5 mM ADP plus 0.2 mM sodium vanadate, or 5 mM AMP-PNP.
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ABCB1 p.Leu975Cys 9261097:126:63
status: NEW132 Membranes prepared from HEK 293 cells expressing mutants L332C/ L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 1 mM verapamil, 0.1 mM vinblastine, 50 M cyclosporin A, or 5 mM colchicine.
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ABCB1 p.Leu975Cys 9261097:132:64
status: NEW140 ATP hydrolysis rather than nucleotide binding was responsible for cross-linking between L332C/ L975C.
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ABCB1 p.Leu975Cys 9261097:140:95
status: NEW142 The observation that ATP hydrolysis promoted cross-linking between L332C/L975C suggests that inhibition of cross-linking of L332C/L975C in whole cells by verapamil or vinblastine occurred indirectly through depletion of intracellular ATP.
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ABCB1 p.Leu975Cys 9261097:142:73
status: NEWX
ABCB1 p.Leu975Cys 9261097:142:130
status: NEW
PMID: 9405384
[PubMed]
Loo TW et al: "Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate."
No.
Sentence
Comment
21
We show that the drug-stimulated ATPase activities of mutants L339C and A342C (TM6) and L975C, V982C, and A985C (TM12) were particularly sensitive to inhibition by dBBn and that the inhibition was prevented by various drug substrates.
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ABCB1 p.Leu975Cys 9405384:21:88
status: NEW98 In contrast, mutants L339C, A342C, L975C, V982C, and A985C were significantly inhibited by dBBn, because they retained only 10, 40, 13, 25, and 32% of their activities, respectively.
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ABCB1 p.Leu975Cys 9405384:98:35
status: NEW99 The concentration of dBBn required to give 50% inhibition of ATPase activity for mutants L339C, L975C, V982C, A985C, and A342C were 90, 112, 320, 480, and 700 M, respectively.
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ABCB1 p.Leu975Cys 9405384:99:96
status: NEW111 The P-glycoproteins(His)10 of Cys-less and mutants L339C, A342C, L975C, V982C, and A985C were mixed with lipid and then preincubated for 15 min at 4 °C without drug or in the presence of 2 mM verapamil (Ver.
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ABCB1 p.Leu975Cys 9405384:111:65
status: NEW124 Similarly, mutants L339C, L975C, and V982C were also protected from dBBn inactivation by various drug substrates.
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ABCB1 p.Leu975Cys 9405384:124:26
status: NEW127 More modest protection by colchicine was seen for mutants L975C and V982C.
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ABCB1 p.Leu975Cys 9405384:127:58
status: NEW129 It offered little or no protection for mutant V982C and only moderately protected mutants L339C and L975C.
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ABCB1 p.Leu975Cys 9405384:129:100
status: NEW
No.
Sentence
Comment
204
Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules.
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ABCB1 p.Leu975Cys 9841738:204:38
status: NEW209 The cross-linking of the first pair (L332C/L975C) required the presence of ATP, was unaffected by drug substrates, and could not be reversed by treatment with dithiothreitol.
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ABCB1 p.Leu975Cys 9841738:209:43
status: NEW
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.
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ABCB1 p.Leu975Cys 22700974:13:53
status: NEW
PMID: 21991360
[PubMed]
Bikadi Z et al: "Predicting P-glycoprotein-mediated drug transport based on support vector machine and three-dimensional crystal structure of P-glycoprotein."
No.
Sentence
Comment
227
For example, activities of the human P-gp mutants, I340C (in TM6), L975C (in TM12), V981C (in TM12), and V982C (in TM12), were found to be highly protected from inhibition by MTS-rhodamine by pre-treatment with rhodamine B, indicating that these residues likely participate in rhodamine B binding to human P-gp [48].
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ABCB1 p.Leu975Cys 21991360:227:67
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.Leu975Cys 16545467:78:313
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.Leu975Cys 16545467:76:313
status: NEW
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).
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ABCB1 p.Leu975Cys 23733192:82:233
status: NEW83 The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells.
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ABCB1 p.Leu975Cys 23733192:83:27
status: NEW162 Accordingly, cross-linking of mutant T333C/L975C was used to test the effect of F1086A on coupling.
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ABCB1 p.Leu975Cys 23733192:162:43
status: NEW163 The T333C and L975C mutations are located at the extracellular ends of TM segments 6 and 12, respectively (Fig. 3, A and B).
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ABCB1 p.Leu975Cys 23733192:163:14
status: NEW164 Mutant T333C/L975C can be cross-linked when intact cells expressing the mutant are treated with BMOE cross-linker (30).
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ABCB1 p.Leu975Cys 23733192:164:13
status: NEW165 Mutant T333C/L975C, however, does not show cross-linking if membranes containing the mutant are treated only with BMOE (Fig. 4A).
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ABCB1 p.Leu975Cys 23733192:165:13
status: NEW170 To determine whether the F1086A mutation affected NBD/ TMD coupling, it was introduced into mutant T333C/L975C and subjected to cross-linking.
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ABCB1 p.Leu975Cys 23733192:170:105
status: NEW171 It was observed that the F1086A mutation inhibited ATP-dependent cross-linking of the mutant T333C/L975C (Fig. 4B).
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ABCB1 p.Leu975Cys 23733192:171:99
status: NEW187 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).
X
ABCB1 p.Leu975Cys 23733192:187:63
status: NEWX
ABCB1 p.Leu975Cys 23733192:187:82
status: NEW197 Accordingly, the A266F mutation was introduced into mutant F1086A/T333C/L975C to test whether it affected cross-linking between the TMDs.
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ABCB1 p.Leu975Cys 23733192:197:72
status: NEW221 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.
X
ABCB1 p.Leu975Cys 23733192:221:58
status: NEWX
ABCB1 p.Leu975Cys 23733192:221:78
status: NEWX
ABCB1 p.Leu975Cys 23733192:221:101
status: NEW
PMID: 25600711
[PubMed]
Pan L et al: "Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics."
No.
Sentence
Comment
196
Specifically, ATP binding inhibited the crosslink of pairs of human Pgp between TM6 and TM12 at L339C-V982C (mouse L334-V978) and L332C-L975C (mouse L328-L971) but promoted the crosslink of F343C-V982C (mouse F339-V978).
X
ABCB1 p.Leu975Cys 25600711:196:136
status: NEW