PMID: 24349290

Chufan EE, Kapoor K, Sim HM, Singh S, Talele TT, Durell SR, Ambudkar SV
Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).
PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013., [PubMed]
Sentences
No. Mutations Sentence Comment
7 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:7:109
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:7:103
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:7:97
status: NEW
view ABCB1 p.Tyr307Cys details
In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. Login to comment
55 ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:55:154
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:55:217
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:55:182
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:55:210
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:55:223
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:55:236
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:55:249
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:55:288
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:55:168
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:55:243
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:55:161
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:55:230
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:55:282
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 24349290:55:175
status: NEW
view ABCB1 p.Phe978Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:55:147
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:55:204
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:55:276
status: NEW
view ABCB1 p.Tyr307Cys details
For biochemical studies, crude membranes were prepared from High-Five insect cells infected with baculovirus coding for cysless WT, single mutants Y307C, F343C, Q725C, F728C, F978C, V982C, double mutants Y307C/V982C, F343C/V982C, Q725C/V982C, F728C/V982C, and a triple mutant Y307C/Q725C/V982C. Login to comment
61 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:61:185
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:61:179
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:61:173
status: NEW
view ABCB1 p.Tyr307Cys details
However, CsA, tariquidar and valinomycin lose almost completely this ability to inhibit IAAP photo-labeling when residues Y307, Q725 and V982 are mutated to cysteine (i.e., Y307C/Q725C/V982C mutant). Login to comment
69 ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 24349290:69:12
status: NEW
view ABCB1 p.Phe978Cys details
One mutant, F978C, behaves in a manner similar to that of cysless WT Pgp, with only a change in the binding affinity for CsA, IC50 = 0.54 &#b5;M, compared to 0.05 &#b5;M for cysless WT Pgp (Table 1). Login to comment
71 ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 24349290:71:48
status: NEW
view ABCB1 p.Phe978Cys details
Similarly, tariquidar inhibits IAAP labeling of F978C to the Figure 1. Login to comment
73 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:73:71
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:73:113
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:73:126
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:73:139
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:73:169
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:73:61
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:73:133
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:73:47
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:73:107
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:73:163
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:73:54
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:73:120
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:73:157
status: NEW
view ABCB1 p.Tyr307Cys details
Inhibition of IAAP labeling for single mutants Q725C, Y307C, F728C and V982C (upper graphs) and for double Q725C/V982C, Y307C/V982C, F728C/V982C, and triple Y307C/Q725C/V982C (lower graphs) mutants at different concentrations of (A) CsA and (B) tariquidar, are shown. Inhibition of IAAP labeling for cysless WT is included in all graphs, as a reference. Login to comment
74 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:74:41
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:74:63
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:74:57
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:74:51
status: NEW
view ABCB1 p.Tyr307Cys details
Autoradiograms corresponding to cysless, V982C and Y307C/Q725C/V982C, as representative examples of complete inhibition, partial inhibition and no inhibition of IAAP-labeling, respectively, are shown at the top of the figure. Login to comment
81 ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:81:12
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:81:22
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:81:28
status: NEW
view ABCB1 p.Val982Cys details
The mutants F343C and F343C/V982C also exhibit normal basal activity. Login to comment
82 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:82:65
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:82:130
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:82:59
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 24349290:82:140
status: NEW
view ABCB1 p.Phe978Cys details
However a majority of the mutants show low basal activity; F728C/V982C shows the lowest (4 &#b1;1.6 nmol Pi/min/mg protein) while V982C and F978C shows fairly low activity (11 &#b1; 2.2 and 13 &#b1; 0.6, respectively). Login to comment
83 ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:83:25
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:83:32
status: NEW
view ABCB1 p.Tyr307Cys details
The rest of the mutants, Q725C, Y307C (and their corresponding double and triple mutants) and F728 show intermediate levels of basal ATPase activity. Login to comment
90 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:90:190
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:90:240
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:90:282
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:90:300
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:90:335
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:90:258
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:90:276
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:90:158
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:90:184
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:90:328
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 24349290:90:353
status: NEW
view ABCB1 p.Phe978Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:90:208
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:90:234
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:90:322
status: NEW
view ABCB1 p.Tyr307Cys details
Mutation(s) CsA Tariquidar Max Inhibition (%) IC50 (&#b5;M) Max Inhibition (%) IC50 (&#b5;M) Cysless WT 86 &#b1; 3 0.05 &#b1; 0.01 97 &#b1; 4 0.14 &#b1; 0.03 Q725C 24 &#b1; 4 -- 37 -- Q725C/V982C 11 -- 22 -- Y307C 35 &#b1; 2 -- ND -- Y307C/V982C 46 -- ND -- F728C 48 -- 40 -- F728C/V982C 49 -- ND -- V982C 56 0.40 64 0.70 Y307C/Q725C/ V982C 12 -- 23 -- F978C 86 0.54 73 3.6 Mean values with standard errors are reported when more than two experiments were carried out; otherwise only average values are reported. Login to comment
93 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:93:65
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:93:81
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:93:75
status: NEW
view ABCB1 p.Gln725Cys details
Valinomycin also stimulated ATP hydrolysis of all mutants except V982C and Q725C/V982C. Login to comment
94 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:94:52
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:94:159
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:94:153
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:94:147
status: NEW
view ABCB1 p.Tyr307Cys details
It is interesting to observe that the effect of the V982C mutation is not dominant in the rest of the double mutants and even in the triple mutant Y307C/Q725C/V982C, in which case valinomycin does stimulate ATP hydrolysis. Login to comment
95 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:95:103
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:95:136
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:95:97
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:95:130
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:95:124
status: NEW
view ABCB1 p.Tyr307Cys details
FSBA also stimulates the ATP hydrolysis of most of the mutants, with the exception of the double F728C/V982C and the triple Y307C/Q725C/V982C mutant. Login to comment
108 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:108:107
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:108:139
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:108:133
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:108:85
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:108:101
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:108:126
status: NEW
view ABCB1 p.Tyr307Cys details
In Figure 4A, representative histograms show the cell surface expression for single (Y307C), double (Y307C/V982C) and triple (Y307C/ Q725C/V982C) mutants. Login to comment
114 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:114:34
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:114:28
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:114:22
status: NEW
view ABCB1 p.Tyr307Cys details
Even with the triple (Y307C/Q725C/V982C) mutant the efflux of none of the above-mentioned substrates is completely abolished, although many of these substrates are transported at lower levels when compared to the cysless WT Pgp (Table 2). Login to comment
116 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:116:113
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:116:186
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:116:180
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:116:88
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:116:107
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:116:174
status: NEW
view ABCB1 p.Tyr307Cys details
Figure 5A shows the transport of rhodamine 123 (Rh123), which is normal for the single (Y307C) and double (Y307C/V982C) mutants but is decreased considerably for the triple (Y307C/Q725C/V982C) mutant. Login to comment
123 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:123:82
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:123:76
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:123:70
status: NEW
view ABCB1 p.Tyr307Cys details
NBD-CsA loses the ability to inhibit the IAAP labeling of the triple (Y307C/Q725C/V982C) Figure 2. Login to comment
137 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:137:86
status: NEW
view ABCB1 p.Val982Cys details
Interestingly, the only single mutation that has a severe effect on drug transport is V982C. Login to comment
138 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:138:26
status: NEW
view ABCB1 p.Val982Cys details
Cells over-expressing the V982C mutant fail to transport NBD-CsA completely. Login to comment
139 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:139:135
status: NEW
view ABCB1 p.Val982Cys details
However, NBD-CsA is able to partially inhibit IAAP labeling in crude membranes (51%, Table S1), clearly showing that NBD-CsA binds the V982C mutant. Login to comment
141 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:141:53
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:141:66
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:141:60
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:141:47
status: NEW
view ABCB1 p.Gln725Cys details
As expected, the corresponding double mutants (Q725C/V982C; F728C/V982C; Figure 3. Login to comment
150 ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:150:40
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:150:46
status: NEW
view ABCB1 p.Val982Cys details
doi: 10.1371/journal.pone.0082463.g003 F343C/V982C) show dramatically reduced transport of NBD-CsA. Login to comment
151 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:151:19
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:151:60
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:151:54
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:151:13
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:151:47
status: NEW
view ABCB1 p.Tyr307Cys details
Nonetheless, Y307C/V982C and the triple mutant Y307C/ Q725C/V982C show some rescue of the NBD-CsA transport. Login to comment
152 ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:152:58
status: NEW
view ABCB1 p.Tyr307Cys details
This can most likely be attributed to the presence of the Y307C mutation, as this particular double mutant exhibits about 50-60% transport function with respect to NBD-CsA (Table 2). Login to comment
159 ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:159:58
status: NEW
view ABCB1 p.Tyr307Cys details
(A) The left panel shows the cell surface localization of Y307C with human Pgp-specific monoclonal antibody MRK-16 labeling as detected by green fluorescence detector. Login to comment
160 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:160:60
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:160:121
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:160:115
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:160:54
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:160:109
status: NEW
view ABCB1 p.Tyr307Cys details
The middle panel shows the same for the double mutant Y307C/V982C, and the right panel for the triple mutant Y307C/Q725C/V982C. Login to comment
162 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:162:74
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:162:105
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:162:99
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:162:52
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:162:68
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:162:93
status: NEW
view ABCB1 p.Tyr307Cys details
The conformation sensitivity towards CsA of single (Y307C), double (Y307C/V982C) and triple (Y307C/Q725C/V982C) mutants was similar to cysless WT Pgp, as shown in the three panels, respectively. Login to comment
175 ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:175:298
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 24349290:175:570
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:175:249
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:175:411
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:175:468
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:175:523
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:175:577
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:175:640
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:175:198
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:175:516
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:175:146
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:175:461
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:175:633
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 24349290:175:352
status: NEW
view ABCB1 p.Phe978Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:175:95
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:175:404
status: NEW
view ABCB1 p.Tyr307Cys details
Mutation(s) Cell surface expression Transport function CalAM BD-PRA NBD-CsA Rh123 Dauno BD-PAC Y307C 100 90-100 80-90 90-100 90-100 90-100 90-100 Q725C 100 90-100 90-100 90-100 90-100 90-100 90-100 F728C 100 90-100 80-90 90-100 90-100 90-100 90-100 V982C 100 90-100 80-100 <10 90-100 90-100 90-100 F343C 50-60 90-100 80-100 90-100 90-100 90-100 90-100 F978C 100 90-100 90-100 90-100 90-100 90-100 90-100 Y307C/ V982C 100 90-100 50-60 50-60 90-100 90-100 90-100 Q725C/ V982C 100 90-100 80-90 <20 90-100 90-100 90-100 F728C/ V982C 30-40 55-65 30-40 <20 70-80 50-60 90-100 F343C/ V982C 70-80 90-100 80-90 <20 90-100 90-100 90-100 Y307/ Q725C/ V982C 100 90-100 30-40 50-60 70-80 60-70 90-100 For cell surface expression, the cells were incubated with MRK-16 antibody for 30 min followed by FITC-labeled anti-mouse secondary antibody for 30 min. Login to comment
211 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:211:56
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:211:87
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:211:81
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:211:35
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:211:75
status: NEW
view ABCB1 p.Tyr307Cys details
The transport functions of single (Y307C), double (Y307/V982C) and triple (Y307C/Q725C/V982C) mutants are differentially affected. Login to comment
213 ABCB1 p.Glu1201Gln
X
ABCB1 p.Glu1201Gln 24349290:213:19
status: NEW
view ABCB1 p.Glu1201Gln details
ABCB1 p.Glu501Gln
X
ABCB1 p.Glu501Gln 24349290:213:13
status: NEW
view ABCB1 p.Glu501Gln details
The inactive E501Q/E1201Q (EQ) mutant P-gp is used as a control to show completely aborted function as described in Materials and Methods. Login to comment
220 ABCB1 p.Gly251Val
X
ABCB1 p.Gly251Val 24349290:220:200
status: NEW
view ABCB1 p.Gly251Val details
The same residues studied in the present work were mutated to arginine by Loo and coworkers [38]; they found these mutations promote or have a neutral effect on maturation of a Pgp processing mutant (G251V) defective in folding, also confirming that these residues are in the drug translocation pathway and/or drug-binding pocket of Pgp. Login to comment
238 ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:238:109
status: NEW
view ABCB1 p.Gln725Cys details
Here, we report QZ59-SSS, CsA, tariquidar, valinomycin and FSBA lost the ability to inhibit IAAP-labeling of Q725C to the same extent as cysless WT Pgp. Login to comment
240 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:240:94
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:240:132
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:240:145
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:240:158
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:240:83
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Phe728Cys
X
ABCB1 p.Phe728Cys 24349290:240:152
status: NEW
view ABCB1 p.Phe728Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:240:76
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:240:139
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:240:69
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:240:126
status: NEW
view ABCB1 p.Tyr307Cys details
The partial inhibition of IAAP labeling observed for single mutants (Y307C, Q725C, F728C, and V982C) and even double mutants (Y307C/V982C, Q725C/V982C, F728C/V982C) is indicative of some drug interaction with Pgp (Figure 1). Login to comment
251 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:251:44
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:251:38
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:251:32
status: NEW
view ABCB1 p.Tyr307Cys details
In all mutants, even the triple Y307C/Q725C/V982C, ATP hydrolysis is inhibited by both CsA and tariquidar. Login to comment
257 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:257:104
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:257:98
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:257:92
status: NEW
view ABCB1 p.Tyr307Cys details
In steady-state conditions, calcein-AM and bodipy-placitaxel are transported by the triple (Y307C/Q725C/V982C) mutant to the same extent as cysless WT Pgp. Login to comment
261 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:261:68
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:261:62
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:261:56
status: NEW
view ABCB1 p.Tyr307Cys details
Therefore, NBD-CsA does not bind to its primary site on Y307C/Q725C/V982C, but to a secondary site, where it is transported at 50-60% of the rate of cysless WT Pgp. Login to comment
290 ABCB1 p.Glu1201Gln
X
ABCB1 p.Glu1201Gln 24349290:290:88
status: NEW
view ABCB1 p.Glu1201Gln details
ABCB1 p.Glu556Gln
X
ABCB1 p.Glu556Gln 24349290:290:82
status: NEW
view ABCB1 p.Glu556Gln details
The accumulation of substrates was calculated with respect to the accumulation in E556Q/E1201Q mutant Pgp, which is completely inactive [48], and is treated as a negative control for all transport experiments. Login to comment
328 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:328:168
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:328:162
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:328:156
status: NEW
view ABCB1 p.Tyr307Cys details
Effect of QZ59-SSS-sulfur on the photocrosslinking of cysless WT and mutant Pgpswith IAAP. Inhibition of IAAP-labeling for cysless WT and for triple mutant Y307C/Q725C/V982C at different concentrations of QZ59-SSS-sulfur are shown (graph). Login to comment
333 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:333:64
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:333:106
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:333:123
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:333:154
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:333:47
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:333:100
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:333:148
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:333:54
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:333:116
status: NEW
view ABCB1 p.Tyr307Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:333:142
status: NEW
view ABCB1 p.Tyr307Cys details
Inhibition of IAAP-labeling for single mutants Q725C, Y307C and V982C (upper graph) and for double (Q725C/V982C and Y307C/ V982C) and triple (Y307C/Q725C/V982C) mutants (lower graph) at different concentrations of valinomycin are shown. Inhibition of IAAP-labeling of cysless WT is included in both graphs, as a reference. Login to comment
338 ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 24349290:338:147
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Gln725Cys
X
ABCB1 p.Gln725Cys 24349290:338:141
status: NEW
view ABCB1 p.Gln725Cys details
ABCB1 p.Tyr307Cys
X
ABCB1 p.Tyr307Cys 24349290:338:135
status: NEW
view ABCB1 p.Tyr307Cys details
Effect of FSBA on the photocrosslinking of cysless WT and mutant Pgps with IAAP. Inhibition of IAAP-labeling of cysless WT and triple (Y307C/Q725C/V982C) mutant at different concentrations of FSBA are shown (graph). Login to comment