ABCMdb

ABCB1 p.Ala729Cys

Predicted by SNAP2:	C: N (53%), D: D (85%), E: D (80%), F: D (85%), G: D (53%), H: D (80%), I: D (75%), K: D (80%), L: D (80%), M: D (66%), N: D (75%), P: D (80%), Q: D (75%), R: D (80%), S: N (87%), T: D (63%), V: D (80%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	C: N, D: D, E: D, F: D, G: N, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: N, V: D, W: D, Y: D,

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[hide] Transmembrane segment 7 of human P-glycoprotein fo... Biochem J. 2006 Oct 15;399(2):351-9. Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket.
Biochem J. 2006 Oct 15;399(2):351-9., 2006-10-15 [PMID:16813563]

Abstract [show]
P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. 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. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.

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No. Sentence 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
106 Figure 2(B) shows that increasing the level of MTS-verapamil from 0.3 mM to 3 mM did not decrease or increase the basal ATPase activity of mutant A729C. Login to comment
107 In addition, the activity of MTS-verapamil- modified mutant A729C could not be activated further by unmodified verapamil (Figure 2B). Login to comment
108 Therefore MTS-verapamil modification of mutant A729C inhibits its verapamil-stimulated ATPase activity. 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