ABCMdb

ABCB1 p.Phe728Cys

None has been submitted yet.

Publications

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

No. Sentence Comment

5 Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Login to comment
6 Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. Login to comment
9 Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Login to comment
74 Mutant Verapamil S50 (µM) Vinblastine S50 (µM) Colchicine S50 (µM) F711C 9.8 + - 1.1 2.3 + - 0.2 410 + - 20 V712C 10.0 + - 0.8 1.9 + - 0.2 320 + - 40 V713C 9.9 + - 1.3 1.8 + - 0.2 340 + - 40 G714C 14.1 + - 2.1 1.9 + - 0.3 410 + - 30 V715C 9.8 + - 1.3 1.8 + - 0.3 330 + - 40 F716C 10.1 + - 1.0 2.1 + - 0.1 340 + - 30 C717C 10.0 + - 1.5 2.3 + - 0.3 390 + - 20 A718C 10.5 + - 1.3 2.0 + - 0.1 330 + - 30 I719C 10.5 + - 1.5 1.9 + - 0.2 370 + - 30 I720C 12.4 + - 0.7 2.4 + - 0.2 390 + - 10 N721C 13.0 + - 1.2 1.7 + - 0.2 380 + - 40 G722C ND ND ND G723C 11.1 + - 0.4 2.0 + - 0.3 410 + - 20 L724C 12.0 + - 1.5 2.4 + - 0.3 340 + - 30 Q725C 11.7 + - 1.1 2.0 + - 0.1 390 + - 20 P726C 11.9 + - 1.0 1.9 + - 0.2 450 + - 30 A727C 21.2 + - 3.1 1.9 + - 0.2 480 + - 40 F728C 48.7 + - 2.2 2.7 + - 0.3 830 + - 90 A729C 34.3 + - 3.0 2.3 + - 0.2 760 + - 80 I730C 12.0 + - 1.1 2.0 + - 0.1 420 + - 30 I731C 13.5 + - 1.9 1.9 + - 0.2 410 + - 30 Cys-less 11.6 + - 1.3 2.1 + - 0.3 400 + - 40 into the endoplasmic reticulum during synthesis of the protein. Login to comment
85 Table 1 shows that two mutants, F728C and A729C, showed about 4.2and 3-fold decreases respectively, in the apparent affinity for verapamil when compared with Cys-less P-gp. Login to comment
97 Samples of the isolated P-gp were mixed with lipid and assayed for ATPase activity and compared with untreated P-gp. All of the mutants, except Q725C, P726C, F728C and A729C, were unaffected by treatment with MTS-verapamil (Figure 2A). Login to comment
99 By contrast, the ATPase activity of mutants Q725C, F728C and A729C increased 2.6-, 4.7and 4.5-fold respectively, compared with untreated mutant P-gp. Login to comment
100 Although mutants Q725C, F728C and A729C showed increased ATPase activity after treatment with MTS-verapamil, the activities were less than 50% of the verapamil-stimulated ATPase activity of Cys-less P-gp (maximum 11.1-fold stimulation; Figure 2B). Login to comment
101 We have shown previously that mutants Q725C, F728C and A729C were stimulated more than 10-fold with verapamil [37]. Login to comment
103 Therefore it was possible that reaction of MTS-verapamil with mutants Q725C, F728C and A729C was incomplete or that they were completely modified but the bound verapamil was in a sub-optimal conformation for activation of ATPase activity. Login to comment
104 To distinguish between these two possibilities, mutants Q725C, F728C and A729C (activated by 0.3 mM MTS-verapamil) were treated with or without 3 mM MTS-verapamil. Login to comment
109 In contrast, labelling of mutants Q725C or F728C with 3 mM MTS-verapamil increased their ATPase activities further Figure 2 Effect of MTS-verapamil on basal ATPase activity of TM7 cysteine mutants (A) His-tagged Cys-less or mutants F711C/I731C P-gps were expressed in HEK-293 cells and solubilized with n-dodecyl-β-D-maltoside. Insoluble material was removed by centrifugation and the supernatants were treated with or without 0.3 mM MTS-verapamil. Login to comment
112 (B) Cys-less, Q725C, F728C and A729C P-gp mutants were treated with or without 3 mM MTS-verapamil and His-tagged P-gp isolated by nickel-chelate chromatography. Equivalent amounts of P-gp were mixed with lipid and ATPase activity determined in the presence (+) or absence (-) of 1 mM verapamil (Ver). Login to comment
117 Therefore mutants Q725C and F728C were chosen for further investigation because their labelling with MTS-verapamil appeared to mimic the interaction of P-gp with unmodified verapamil. Login to comment
118 We then tested whether drug substrates could protect mutants Q725C and F728C from being labelled by MTS-verapamil. Login to comment
119 HEK-293 cells expressing mutants Q725C or F728C were solubilized with n-dodecyl-β-D-maltoside and then treated with or without saturating levels of verapamil (3 mM), cyclosporin A (0.2 mM), colchicine (10 mM) or trans-(E)-flupentixol (0.6 mM). Login to comment
123 Figure 3 shows that the substrates verapamil and cyclosporin A protected mutant F728C, but not mutant Q725C, from labelling with MTS-verapamil. Login to comment
126 Therefore mutant F728C was selected for further analysis because some drug substrates protected it from labelling by MTS-verapamil. Login to comment
127 The reaction of mutant F728C with higher concentrations of MTS-verapamil showed that maximum stimulation (11.5-fold) occurred in the presence of 3-10 mM MTS-verapamil (see Figure 1 of supplementary data at http://www.BiochemJ.org/ bj/399/bj3990351add.htm). Login to comment
129 We then confirmed that activation of mutant F728C was indeed due to covalent attachment of MTS-verapamil to Cys728 . Login to comment
130 HEK-293 cells expressing mutant F728C were treated with 3 mM MTS-verapamil for 10 min at 20◦ C and His-tagged P-gp isolated by nickel-chelate chromatography. Login to comment
132 Exposure of the MTS-verapamil labelled F728C mutant to DTT caused a 75% decrease in ATPase activity (see Figure 3 Inhibition of MTS-verapamil labelling of mutants Q725C and F728C by substrates HEK-293 cells expressing mutants Q725C or F728C were solubilized with n-dodecyl- β-D-maltoside. Insoluble material was removed by centrifugation. Login to comment
138 We then tested whether other drug substrates could stimulate further the ATPase activity of MTS-verapamil-treated mutant F728C. Login to comment
140 Mutant F728C was treated with or without Figure 4 Effect of drug substrates and inhibitors on the ATPase activity of mutants F728C and Q725C before and after labelling with MTS-verapamil HEK-293 cells expressing His-tagged mutants F728C or Q725C were solubilized with n-dodecyl-β-D-maltoside and then incubated in the absence (A and C) or presence (B and D) of 3 mM MTS-verapamil. Login to comment
148 The activity of untreated mutant F728C was stimulated 6.0-fold in the presence of verapamil (Figure 4). Login to comment
151 When mutant F728C was treated with MTS-verapamil, its activity could not be increased or decreased further with calcein-AM, demecolcine or trans-(E)-flupentixol (Figure 4B). Login to comment
154 To distinguish between these possibilities, we compared the properties of mutant Q725C with mutant F728C. Login to comment
155 The activity of mutant Q725C was very similar to that of mutant F728C when assayed in the presence of calcein-AM, demecolcine, verapamil, cyclosporin A or trans-(E)-flupentixol before and after labelling with MTS-verapamil (Figures 4C and 4D). Login to 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
232 We found that the Km of mutant F728C for ATP remained at about 1 mM before and after modification with MTS-verapamil (results not shown). Login to comment

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

No. Sentence Comment

270 C, panel i, membranes prepared from HEK 293 cells expressing mutant L339C/F728C were preincubated for 15 min at 22 °C with different concentrations of adaGb3 (0-500 ␮M) (lanes 2-6). Login to comment

PMID: 18596043 [PubMed] Loo TW et al: "Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11."

No. Sentence Comment

194 TABLE 2 Concentrations of drug substrates required to inhibit cross-linking by 50% Mutant Vinblastine Cyclosporin A Rhodamine B ␮M ␮M ␮M L339C/F728C 0.5 Ϯ 0.2a 0.8 Ϯ 0.3 52 Ϯ 14 G64R/L339C/F728C 53 Ϯ 15b 1.5 Ϯ 0.4 61 Ϯ 10 M68R/L339C/F728C 266 Ϯ 62b 1.3 Ϯ 0.3 57 Ϯ 7 V71R/L339C/F728C 0.6 Ϯ 0.2 0.8 Ϯ 0.2 57 Ϯ 10 a Each value is the mean Ϯ S.D. (n ϭ 3-4 separate cross-linking experiments). 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
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
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
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
141 As expected, the corresponding double mutants (Q725C/V982C; F728C/V982C; Figure 3. 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
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