ABCB1 p.Leu332Cys
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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.Leu332Cys 10506575:42:59
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.Leu332Cys 10506575:120:39
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.Leu332Cys 10506575:121:56
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.Leu332Cys 10506575:123:73
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.Leu332Cys 10506575:139:76
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.Leu332Cys 16042402:81:52
status: NEW171 (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.
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ABCB1 p.Leu332Cys 16042402:171:92
status: NEW
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
39
Construction of mutants containing pairs of cysteines that exhibited ATP-sensitive cross-linking in the presence of copper phenanthroline [L332C- (TM6)/L975C(TM12)] (33), 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate (M14M) [L339C(TM6)/ F728C(TM7)] (34), or tris(2-maleimidoethyl)amine (TMEA) [L339C(TM6)/V982C(TM12) and F343C(TM6)/V982C- (TM12)] (31) were described previously.
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ABCB1 p.Leu332Cys 17636884:39:139
status: NEW74 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.
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ABCB1 p.Leu332Cys 17636884:74:179
status: NEW89 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).
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ABCB1 p.Leu332Cys 17636884:89:93
status: NEW125 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).
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ABCB1 p.Leu332Cys 17636884:125:81
status: NEWX
ABCB1 p.Leu332Cys 17636884:125:132
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.Leu332Cys 20088754:165:244
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.Leu332Cys 8910331:116:112
status: NEWX
ABCB1 p.Leu332Cys 8910331:116:168
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.Leu332Cys 8910331:117:10
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.Leu332Cys 8910331:151:86
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.Leu332Cys 8910331:156:111
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.
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ABCB1 p.Leu332Cys 8910331:158:128
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.Leu332Cys 8910331:167:72
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.Leu332Cys 8910331:168:54
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.Leu332Cys 9261097:82:138
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.Leu332Cys 9261097:84:56
status: NEW96 pressing mutant L332C/L975C were cross-linked in the presence of nucleotides.
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ABCB1 p.Leu332Cys 9261097:96:16
status: NEW99 These results suggest that cross-linking between L332C and L975C occurred during ATP hydrolysis.
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ABCB1 p.Leu332Cys 9261097:99:49
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.Leu332Cys 9261097:103:64
status: NEW104 No cross-linked product was observed for mutant L332C/L975C (Fig. 3A).
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ABCB1 p.Leu332Cys 9261097:104:48
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.Leu332Cys 9261097:108:68
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.Leu332Cys 9261097:109:76
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.Leu332Cys 9261097:126:57
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.Leu332Cys 9261097:132:57
status: NEW140 ATP hydrolysis rather than nucleotide binding was responsible for cross-linking between L332C/ L975C.
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ABCB1 p.Leu332Cys 9261097:140:88
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.Leu332Cys 9261097:142:67
status: NEWX
ABCB1 p.Leu332Cys 9261097:142:124
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.Leu332Cys 9841738:204:32
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.Leu332Cys 9841738:209:37
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).
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ABCB1 p.Leu332Cys 25600711:196:130
status: NEW