ABCMdb

ABCB1 p.Phe343Cys

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Publications

PMID: 12223492 [PubMed] Loo TW et al: "Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein."

No. Sentence Comment

140 The activity of mutant F343C, however, was increased (347%) after treatment with MTS-rhodamine. Login to comment
164 Two mutants, L65C and F343C, showed increased activity after treatment with MTS-rhodamine. Login to comment
167 Mutant F343C showed about 3.5-fold increase in activity after treatment with MTS-rhodamine. Login to comment
168 Because mutant F343C had about 60% of the activity of Cys-less P-gp, reaction with MTS-rhodamine essentially causes a 2-fold increase in activity relative to the Cys-less parent. Login to comment

PMID: 12909621 [PubMed] Loo TW et al: "Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein."

No. Sentence Comment

116 It appears that the presence of verapamil increased the reactivity or proximity of F343C to TMEA. Login to comment
137 Mutant F343C was also treated with various concentrations of TMEA in the absence of verapamil (-Ver). Login to comment

PMID: 14522974 [PubMed] Loo TW et al: "Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites."

No. Sentence Comment

6 Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. Login to comment
7 The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. Login to comment
8 By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. Login to comment
89 The covalent attachment of MTS-rhodamine to Cys-343 seems to mimic the interaction of MTS-rhodamine with Cys-less P-gp, because maximal stimulation of mutant F343C by MTS-rhodamine was 5.8-fold, whereas that of Cys-less P-gp was about 6.2-fold. Login to comment
90 Therefore, it is unlikely that labeling of mutant F343C by MTS-rhodamine is coincidental. Login to comment
93 The rationale is that substrates will not affect the activity of the MTS-rhodamine-treated mutant F343C if they occupy the same binding site as rhodamine but will further stimulate or inhibit the activity if their binding sites are distinct from that of rhodamine. Login to comment
94 Accordingly, mutant F343C was purified by nickel-chelate chromatography after incubation with or without 2 mM MTS-rhodamine, mixed with lipid and assayed for ATPase activity in the presence of different drug substrates. Login to comment
95 Fig. 3 shows that in the absence of substrate, covalent modification of F343C resulted in a 5.8-fold increase in activity over that of the untreated mutant F343C. Login to comment
96 The activity of untreated mutant F343C in the presence of calcein-AM or colchicine was stimulated 12or 9.2-fold, respectively. Login to comment
99 In the presence of verapamil, the MTS-rhodamine-labeled and untreated mutants F343C were stimulated 9.9and 10.3-fold, respectively (Fig. 3). Login to comment
101 Therefore, we tested whether Hoechst 33342 would affect the activity of the MTS-rhodamine-labeled F343C mutant. Login to comment
105 We then tested whether drug substrates could protect mutant F343C from modification by MTS-rhodamine. Login to comment
106 The rationale is that the presence of another drug substrate in the rhodamine-binding site will prevent mutant F343C from being labeled by MTS-rhodamine. Login to comment
107 Similarly, if the binding site of the drug substrate is distinct from that of rhodamine, then mutant F343C should still be labeled by MTS-rhodamine in the presence of these substrates. Login to comment
110 Accordingly, mutant F343C was pre-incubated with 10 mM colchicine, 10 mM verapamil, 3 mM rhodamine B, or no drug substrates, treated with or without 1 mM MTS-rhodamine and then isolated by nickel-chelate chromatography. Login to comment
111 The presence of colchicine or rhodamine B prevented MTS-rhodamine activation of mutant F343C (5.8-fold) by 77% (2-fold) and 60% (2.8-fold), respectively. Login to comment
114 Labeling of mutant F343C by MTS-rhodamine, however, showed little protection with verapamil. Login to comment
117 The presence of the reducing agent inhibited activation of mutant F343C by MTS-rhodamine by more than 80% (Fig. 4). Login to comment
118 FIG. 2. Effect of MTS-rhodamine treatment on the basal activity of Cys-less and F343C mutant P-gps. Login to comment
124 FIG. 3. Effect of drug substrates on the ATPase activity of mutant F343C before and after labeling with MTS-rhodamine. Login to comment
129 The characteristics of the ATPase activity of MTS-rhodamine-labeled mutant F343C were also examined. Login to comment
132 Fig. 4 shows that vanadate trapping inhibits the activity of the activated F343C mutant. Login to comment
133 Labeling of mutant F343C did not appear to affect the affinity of the enzyme for ATP. Login to comment
150 It is interesting that residue F343C is on the same face of the TM6 ␣-helix as residue I340C (Fig. 6A). Login to comment
155 Two observations suggest that MTS-rhodamine attached to F343C mimics the interaction of rhodamine compounds with P-gp. Login to comment
156 First, the ATPase activity of MTS-rhodamine-labeled F343C (5.8-fold) is similar to that observed with MTS-rhodamine and Cys-less P-gp (6.2-fold). Login to comment
157 Second, rhodamine B protected mutant F343C from labeling by MTS-rhodamine. Login to comment
158 Calcein-AM and colchicine did not affect the activity of MTS-rhodamine-treated mutant F343C. Login to comment
162 Verapamil, however, was able to further stimulate the activity of the MTS-rhodamine treated mutant F343C indicating that verapamil could still bind to the mutant protein. Login to comment
163 It is unlikely that verapamil increased activity by binding to unlabeled mutant F343C because labeling of mutant F343C by MTS-rhodamine was saturable (Fig. 2). Login to comment
164 We also found that subjecting MTS-rhodamine treated F343C to a second round of labeling with MTS-rhodamine did not increase ATPase activity (data not shown). Login to comment
166 Inhibition of MTS-rhodamine activation of mutant F343C. Login to comment
200 This may explain why both F343C and I340C can be labeled with MTS-rhodamine, but only F343C is activated upon labeling. Login to comment

PMID: 14670948 [PubMed] Loo TW et al: "Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane."

No. Sentence Comment

92 Residues that react with the MTS derivatives of drug substrates verapamil (I306C) and rhodamine (F343C) to stimulate ATPase activity are shown as closed circles. Login to comment

PMID: 15192095 [PubMed] Rothnie A et al: "The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein."

No. Sentence Comment

130 Values refer to the mean Ϯ S.E. obtained from at least eight independent protein purification preparations. P-gp isoform Substrate affinity , Km Maximal activity, Vmax -Fold stimulationBasal Stimulated Basal Stimulated mM ␮mol Pi min-1 mg protein-1 Cys-less 0.58 Ϯ 0.06 0.38 Ϯ 0.04 0.58 Ϯ 0.15 1.46 Ϯ 0.30 2.9 Ϯ 0.3 V331C 0.50 Ϯ 0.06 0.26 Ϯ 0.02 0.45 Ϯ 0.05 1.54 Ϯ 0.20 3.5 Ϯ 0.3 T333C 0.49 Ϯ 0.05 0.23 Ϯ 0.02 0.35 Ϯ 0.04 1.22 Ϯ 0.15 3.3 Ϯ 0.1 F335C 0.40 Ϯ 0.05 0.24 Ϯ 0.03 0.65 Ϯ 0.15 1.61 Ϯ 0.31 2.2 Ϯ 0.2 S337C 0.53 Ϯ 0.06 0.26 Ϯ 0.04 0.59 Ϯ 0.10 1.67 Ϯ 0.23 3.2 Ϯ 0.4 L339C 0.51 Ϯ 0.07 0.31 Ϯ 0.04 0.57 Ϯ 0.07 1.47 Ϯ 0.15 2.9 Ϯ 0.3 G341C 0.40 Ϯ 0.04 0.24 Ϯ 0.02 0.42 Ϯ 0.03 1.12 Ϯ 0.09 3.1 Ϯ 0.5 F343C 0.41 Ϯ 0.04 0.26 Ϯ 0.03 0.47 Ϯ 0.04 1.17 Ϯ 0.15 2.6 Ϯ 0.3 generate stable covalent bonds with thiol groups under physiological conditions. Login to comment
155 In contrast, F343C in the native protein conformation was accessible to labeling with FM as adjudged by the 81 Ϯ 2% labeling extent (Lext), which was characterized by a half-life (t1/2 ϭ 47 Ϯ 8 min) almost 4 times slower than observed for G324C. Login to comment
166 Values refer to the mean Ϯ S.E. obtained from a minimum of three independent protein purification preparations. P-gp isoform Potency of drug effect Nicardipine, EC50 Vinblastine, EC50 Vanadate, IC50 ␮M ␮M ␮M Cys-less 3.2 Ϯ 0.3 4.2 Ϯ 0.6 4.0 Ϯ 0.4 V331C 3.3 Ϯ 0.4 7.2 Ϯ 1.7 3.2 Ϯ 0.4 T333C 2.3 Ϯ 0.2 4.6 Ϯ 0.4 3.9 Ϯ 0.8 F335C 2.3 Ϯ 0.4 4.2 Ϯ 0.8 5.5 Ϯ 1.1 S337C 2.7 Ϯ 0.5 4.1 Ϯ 1.0 5.8 Ϯ 0.8 L339C 2.1 Ϯ 0.3 5.1 Ϯ 0.8 4.2 Ϯ 0.7 G341C 3.9 Ϯ 0.5 4.0 Ϯ 0.6 6.8 Ϯ 1.3 F343C 2.1 Ϯ 0.3 5.6 Ϯ 2.7 2.7 Ϯ 0.8 FIG. 1. Login to comment
184 Surprisingly, mutant F343C, which was the only isoform labeled by the hydrophilic compound FM, reacted with the amphiphilic probe BM (Lext ϭ 68 Ϯ 11%) with a half-life of only 18 Ϯ 4 min. Login to comment
189 Isoform F343C was the only protein to label with the hydrophilic probe FM, and the labeling was significantly reduced by both AMP-PNP binding (Lext ϭ 29 Ϯ 3%) and vanadate trapping (Lext ϭ 26 Ϯ 4%). Login to comment
191 The residual labeled protein is likely to arise from the proportion of F343C P-gp that was not completely bound with AMP-PNP or vanadate-trapped. Login to comment
192 This was confirmed by similar labeling half-lives compared with nucleotide free F343C P-gp. Login to comment
193 F343C was the only isoform whose labeling with FM was affected by the catalytic cycle because all other mutant P-gp proteins remained inaccessible to labeling with this hydrophilic molecule. Login to comment
200 Isoforms T333C (t1/2 ϭ 16 Ϯ 3 min) and F343C (t1/2 ϭ 18 Ϯ 5 min) labeled with CM with significantly increased rates compared with the values observed in either basal conditions or in the presence of AMP-PNP, reflecting an increased accessibility of the introduced cysteine residues. Login to comment
203 Whereas under basal conditions V331C, L339C, and F343C were accessible to BM, only the latter was labeled (Lext ϭ 90 Ϯ 8%) after AMP-PNP binding to the protein (Fig. 4b). Login to comment
205 This was further confirmed by the observation that the half-life of labeling with BM in F343C was unaffected by the binding of AMP-PNP (t1/2 ϭ 18 Ϯ 1 min) compared with the basal state. Login to comment
206 Vanadate trapping of the F343C isoform also failed to alter either the extent (Lext ϭ 98 Ϯ 3%) or FIG. 4. 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

72 The locations of residues L65C in TM1, I306C in TM5 and F343C in TM6 are shown. Login to comment
127 In this study [41], it was found that the ATPase activity of mutant F343C in TM6 could be permanently activated by covalent attachment of MTS-Rhodamine. Login to comment

PMID: 16813563 [PubMed] Loo TW et al: "Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket."

No. Sentence Comment

161 cysteines facing the drug-binding pocket that may be cross-linked with F728C are L65C(TM1), I306C(TM5) and F343C(TM6) because they were covalently modified with MTS-verapamil (L65C and I306C) or with MTS-Rhodamine (F343C). Login to comment
177 Cross-linking was due to linkage between Cys343 and Cys728 because cross-linked product was not detected on SDS/ PAGE when membranes prepared from HEK-293 cells transfected with the F343C or F728C single cysteine mutant cDNAs, or cotransfected with the F343C and F728C mutant cDNAs, were treated with the homobifunctional cross-linkers (results not shown). 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

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: 18003606 [PubMed] De Rosa MF et al: "Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog."

No. Sentence Comment

213 In contrast, no inhibition with adaGb3 pretreatment was seen for the F343C containing mutants. Login to comment
292 AdaGb3/MDR1 binding may be similar to colchicine, demecolcine, Hoescht 33342, or flupentixol that similarly do not inhibit TM6 F343C disulfide cross-linking (33). Login to comment

PMID: 18303860 [PubMed] Storm J et al: "Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling."

No. Sentence Comment

239 This lack of change is in contrast to the considerable conformational changes previously observed in the segment between V331C to F343C (42). Login to comment
241 This is in good agreement with the data from Rothnie et al. demonstrating similar high accessibility for the adjacent residue, F343C (42). 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
76 Fig. 1D shows that a product with reduced mobility on SDS-PAGE gels was present when mutants F343C/M986C, G346C/G989C, and P350C/ S993C were treated with oxidant. Login to comment
80 We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil. Login to comment
100 The effect of nucleotides on cross-linking was also tested on mutants F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
103 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. Login to comment
105 By contrast, all the drug substrates were effective in blocking cross-linking of mutants F343C/M986C and G346C/G989C (Fig. 3, B and C), but were less effective in preventing cross-linking of mutant P350C/S993C (Fig. 3D). Login to comment
108 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. Login to comment
109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A). Login to comment
126 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. Login to comment
132 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. Login to comment
144 Drug substrates inhibited cross-linking of mutants F343C/ M986C, G346C/G989C, and P350C/S993C. 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

141 Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment

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

No. Sentence Comment

204 Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules. 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: 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

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
81 The mutants F343C and F343C/V982C also exhibit normal basal activity. Login to comment
150 doi: 10.1371/journal.pone.0082463.g003 F343C/V982C) show dramatically reduced transport of NBD-CsA. 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

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