ABCMdb

ABCB1 p.Val982Cys

None has been submitted yet.

Publications

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). Login to comment

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. Login to comment
60 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. Login to comment
160 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. Login to comment
162 (B) Membranes prepared from HEK-293 cells expressing mutants L65C, V982C or L65C/V982C were treated with 0.2 mM M11M for various times at 4◦C. The reactions were stopped by addition of SDS sample buffer containing EDTA and subjected to immunoblot analysis on SDS/7.5% polyacrylamide gels. Login to comment

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. Login to comment
54 In the absence of ATP, mutant L339C(TM6)/V982C- (TM12), but not mutant F343C(TM6)/V982C(TM12), was cross-linked with TMEA. Login to comment
74 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. Login to comment

PMID: 19456124 [PubMed] Crowley E et al: "Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1."

No. Sentence Comment

67 This necessitated the centrifugation (100000g for 30 min) of 500 μL Table 1: Mutagenic Oligonucleotide Primers Used To Generate TM12 Mutationsa mutation primer sequence (50 -30 ) diagnostic restriction digest L976C GAGGATGTTCTAtgtGTATTTTCAGCTGTTG -SpeI F978C GTTCTACTAGTATgTTCtGCaGTTGTCTTTGGTG +PstI A980C CTACTAGTATTTTCAtgcGTTGTCTTTGGTGCCATGGCC -PvuII V982C CTAGTATTTTCAGCgGTTtgCTTTGGTGCCATGGCC -PvuII G984C GCTGTTGTCTTTtGTGCtATGGCCGTGG -NcoI M986C GTATTTGGTGCttgtGCtGTGGGGCAAGTC -NcoI V988C GGTGCCATGGCCtgtGGGCAAGTCAGTTC -BstXI G989C CTTTGGTGCCATGGCCGTGtGcCAAGTCAGTTCATTTGC +BstXI Q990C GGCCGTGGGGtgtGTCtcTTCATTTGCTCC +EarI a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates or removes the indicated restriction site. Login to comment
155 Table 2: Potency and Degree of Drug Stimulation of ATP Hydrolysis by ABCB1a nicardipine vinblastine EC50 (μM) fold stimulation EC50 (μM) fold stimulation Cys-less 4.1 ( 1.1 4.0 ( 0.6 5.91 ( 2.9 2.2 ( 0.2 L976C 5.2 ( 0.2 7.4 ( 1.4 10.0 ( 0.0 3.5 ( 0.6 F978C 24.1 ( 2.3b 9.5 ( 1.4 42.9 ( 4.3b 2.3 ( 0.5 A980C 3.4 ( 0.3 5.1 ( 0.9 12.3 ( 1.8 3.2 ( 0.8 V982C 5.8 ( 0.9 4.2 ( 0.5 2.0 ( 0.7 1.8 ( 0.2 G984C 37.6 ( 11.2b 16.2 ( 6.6b 6.7 ( 1.7 6.2 ( 2.3 M986C 9.2 ( 0.8 4.7 ( 1.1 15.0 ( 2.0b 2.8 ( 0.7 V988C 3.9 ( 0.6 3.1 ( 0.1 7.3 ( 2.3 1.9 ( 0.2 G989C 13.6 ( 1.5 5.1 ( 1.6 4.9 ( 0.9 2.4 ( 0.3 Q990C 6.9 ( 1.1 3.7 ( 1.0 NDc NDc S992C 4.9 ( 0.5 4.2 ( 0.6 7.1 ( 2.6 2.3 ( 0.4 F994C 1.7 ( 0.4 3.2 ( 0.8 5.9 ( 2.5 1.6 ( 0.3 a ATPase activity was plotted as a function of the drug concentration and potency (EC50) and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation. Login to comment

PMID: 20731718 [PubMed] Crowley E et al: "Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1."

No. Sentence Comment

61 The central region of TM12, from V982C to M986C, displayed the highest extent of labelling, with Lext values of 75-100%. Login to comment
79 A similar stretch of TM12 (namely V982C-V988C) displayed the greatest propensity to be labelled with BM, with only isoform M986C being not completely labelled by the probe. Login to comment
118 Conformational changes - amino region of TM12 As shown in Table 2, the amino region of TM12 (L976C-V982C) was not associated with large alterations in topography. Login to comment
128 In complete contrast, V982C did not undergo any alterations of probe accessibility during transition to the nucleotide-bound and posthydrolytic conformational states. Login to comment
139 Mutant CM BM FM Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) L976C 38 ± 5 29 ± 12 66 ± 14 29 ± 18 - - A980C 53 ± 6 34 ± 1 54 ± 8 20 ± 9 - - V982C 98 ± 14 15 ± 6 164 ± 50 27 ± 17 - - G984C 73 ± 14 29 ± 6 84 ± 24 22 ± 7 13 ± 10 ND M986C 89 ± 30 25 ± 10 51 ± 5 3 ± 2 21 ± 2 ND V988C 53 ± 6 37 ± 18 221 ± 63 18 ± 12 - - G989C 64 ± 7 15 ± 6 21 ± 3 9 ± 2 - - S992C 55 ± 4 22 ± 6 51 ± 5 4 ± 1 32 ± 3 25 ± 5 F994C 51 ± 10 11 ± 9 111 ± 35 13 ± 10 129 ± 24 8 ± 3 Conformational changes - central region Two of the residues examined in the central region (G984C and M986C) of TM12 have been shown to accommodate partial labelling with FM, suggestive of aqueous accessibility in the basal state. At M986C, the extent of labelling with the hydrophilic probe was increased following the addition of nonhydrolysable nucleotide. Login to comment
164 ABCB1 isoform Catalytic intermediate CM BM FM L976C Basal ++ +++ ) AMP-PNP +++ ++ ) Vi trapped +++ +++ ) A980C Basal ++ ++ ) AMP-PNP +++ + ) Vi trapped +++ +++ ) V982C Basal +++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G984C Basal +++ +++ + AMP-PNP +++ +++ + Vi trapped +++ ++ ) M986C Basal +++ ++ + AMP-PNP ++ +++ ++ Vi trapped +++ ++ ) V988C Basal ++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G989C Basal ++ + ) AMP-PNP ++ ++ ) Vi trapped ++ + ) S992C Basal ++ ++ + AMP-PNP +++ +++ ++ Vi trapped ++ ++ + F994C Basal ++ +++ +++ AMP-PNP ++ +++ ++ Vi trapped +++ +++ + reflect localization at the membrane-solute interface. Login to comment
183 The homology models predict that both V982C and G984C, located within the centre of the helix, experience little change in molecular environment upon ATP binding, which is in agreement with the biochemical data. Login to comment
228 The structures are shown in the panel as viewed from the translocation pore; the relative environments of V982C (cyan) and G984C (blue) are unaltered by nucleotide binding. Login to comment

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

68 To test these predictions, we introduced pairs of cysteines into a Cys-less mutant of P-glycoprotein to create the mutants F336C/S979C, L339C/V982C, F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
78 No cross-linked product was observed for mutants F336C/S979C and L339C/V982C. Login to comment
107 Mutants S979C/F336C or L339C/V982C did not yield any cross-linked product even in the presence of ATP or drug substrates (data not shown). Login to comment
124 Cross-linking was not observed between F336C/S979C or L339C/V982C, even in the presence of ATP or drug substrates FIG. 2. Login to comment

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. Login to comment
98 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. Login to comment
99 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. Login to comment
111 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. Login to comment
124 Similarly, mutants L339C, L975C, and V982C were also protected from dBBn inactivation by various drug substrates. Login to comment
127 More modest protection by colchicine was seen for mutants L975C and V982C. Login to comment
129 It offered little or no protection for mutant V982C and only moderately protected mutants L339C and L975C. Login to comment

PMID: 9841738 [PubMed] Jones PM et al: "A new structural model for P-glycoprotein."

No. Sentence Comment

211 In contrast, two other potential pairs that lie between the first and second of the four cross-linked pairs within TMs 6 and 12 (Loo & Clarke, 1997), namely F336C/S979C and L339C/V982C, failed to form cross-links. 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

244 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment
237 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment

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]. Login to comment

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]. Login to comment
76 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]. Login to comment

PMID: 24349290 [PubMed] Chufan EE et al: "Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1)."

No. Sentence Comment

7 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 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 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
73 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 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 The mutants F343C and F343C/V982C also exhibit normal basal activity. Login to comment
82 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
90 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 Valinomycin also stimulated ATP hydrolysis of all mutants except V982C and Q725C/V982C. Login to comment
94 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 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 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 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 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 NBD-CsA loses the ability to inhibit the IAAP labeling of the triple (Y307C/Q725C/V982C) Figure 2. Login to comment
137 Interestingly, the only single mutation that has a severe effect on drug transport is V982C. Login to comment
138 Cells over-expressing the V982C mutant fail to transport NBD-CsA completely. Login to comment
139 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 As expected, the corresponding double mutants (Q725C/V982C; F728C/V982C; Figure 3. Login to comment
150 doi: 10.1371/journal.pone.0082463.g003 F343C/V982C) show dramatically reduced transport of NBD-CsA. Login to comment
151 Nonetheless, Y307C/V982C and the triple mutant Y307C/ Q725C/V982C show some rescue of the NBD-CsA transport. Login to comment
160 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 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 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 The transport functions of single (Y307C), double (Y307/V982C) and triple (Y307C/Q725C/V982C) mutants are differentially affected. Login to comment
240 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 In all mutants, even the triple Y307C/Q725C/V982C, ATP hydrolysis is inhibited by both CsA and tariquidar. Login to comment
257 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 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
328 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 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 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

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). Login to comment