ABCMdb

PMID: 16813563

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 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCB1 p.Phe728Cys Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Login to comment 6 ABCB1 p.Phe728Cys Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. Login to comment 9 ABCB1 p.Phe728Cys 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 ABCB1 p.Phe711Cys ABCB1 p.Ala718Cys ABCB1 p.Val715Cys ABCB1 p.Gly714Cys ABCB1 p.Asn721Cys ABCB1 p.Gly722Cys ABCB1 p.Ile719Cys ABCB1 p.Ile720Cys ABCB1 p.Val713Cys ABCB1 p.Gly723Cys ABCB1 p.Phe716Cys ABCB1 p.Val712Cys ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Leu724Cys ABCB1 p.Ile731Cys ABCB1 p.Ala727Cys ABCB1 p.Gln725Cys ABCB1 p.Pro726Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys 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 90 ABCB1 p.Leu65Cys ABCB1 p.Ile306Cys We have previously shown that modification of specific cysteines in TM1 (L65C) and in TM5 (I306C) with MTS-verapamil caused permanent activation of P-gp ATPase activity (an 8to 11-fold increase in activity compared with untreated P-gp) [27,36]. Login to comment 97 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys ABCB1 p.Pro726Cys 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 98 ABCB1 p.Pro726Cys Mutant P726C, however, showed about a 50% reduction in basal activity after treatment with MTS-verapamil. Login to comment 99 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys We have shown previously that mutants Q725C, F728C and A729C were stimulated more than 10-fold with verapamil [37]. Login to comment 103 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Ala729Cys 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 ABCB1 p.Ala729Cys In addition, the activity of MTS-verapamil- modified mutant A729C could not be activated further by unmodified verapamil (Figure 2B). Login to comment 108 ABCB1 p.Ala729Cys Therefore MTS-verapamil modification of mutant A729C inhibits its verapamil-stimulated ATPase activity. Login to comment 109 ABCB1 p.Phe711Cys ABCB1 p.Phe728Cys ABCB1 p.Ile731Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Ala729Cys ABCB1 p.Gln725Cys (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 ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys We then tested whether drug substrates could protect mutants Q725C and F728C from being labelled by MTS-verapamil. Login to comment 119 ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys Therefore mutant F728C was selected for further analysis because some drug substrates protected it from labelling by MTS-verapamil. Login to comment 127 ABCB1 p.Phe728Cys 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 ABCB1 p.Phe728Cys We then confirmed that activation of mutant F728C was indeed due to covalent attachment of MTS-verapamil to Cys728 . Login to comment 130 ABCB1 p.Phe728Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys We then tested whether other drug substrates could stimulate further the ATPase activity of MTS-verapamil-treated mutant F728C. Login to comment 140 ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Phe728Cys The activity of untreated mutant F728C was stimulated 6.0-fold in the presence of verapamil (Figure 4). Login to comment 151 ABCB1 p.Phe728Cys 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 ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys To distinguish between these possibilities, we compared the properties of mutant Q725C with mutant F728C. Login to comment 155 ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys 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 ABCB1 p.Leu65Cys ABCB1 p.Phe343Cys ABCB1 p.Ile306Cys ABCB1 p.Phe728Cys 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 ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys 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 187 ABCB1 p.Gln725Cys For comparison, we tested the cross-linking characteristics of mutant Q725C with a second cysteine residue introduced at positions Leu65 , Ile306 or Phe343 . Login to comment 224 ABCB1 p.Pro726Cys Labelling of one mutant, P726C, with MTS-verapamil resulted in inhibition of the basal activity of P-gp (Figure 2A). Login to comment 226 ABCB1 p.Pro726Cys Therefore an explanation for inhibition of ATPase activity after MTS-verapamil treatment of mutant P726C is that the bound verapamil molecule interferes with TM conformational changes associated with ATP hydrolysis [42-44]. Login to comment 228 ABCB1 p.Gln725Cys Residue Gln725 did not appear to directly participate in drug binding since the mutant Q725C did not show any changes in apparent affinity for verapamil, vinblastine or colchicine (Table 1). Login to comment 232 ABCB1 p.Phe728Cys 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