ABCMdb

PMID: 16492138

Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket.
Biochem J. 2006 Jun 15;396(3):537-45., 2006-06-15 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCB1 p.Leu65Cys One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. Login to comment 6 ABCB1 p.Leu65Cys Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. Login to comment 7 ABCB1 p.Leu65Cys The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. Login to comment 41 ABCB1 p.Leu975Cys ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys ABCB1 p.Leu65Cys ABCB1 p.Val982Cys ABCB1 p.Val981Cys ABCB1 p.Gly984Cys ABCB1 p.Ala985Cys 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 44 ABCB1 p.Leu65Cys The mutant L65C was also stably expressed in BHK cells (baby hamster kidney cells). Login to comment 45 ABCB1 p.Leu65Cys Briefly, BHK cells were transfected with the His-tagged mutant L65C cDNA in pMT21 as described previously [35]. Login to comment 47 ABCB1 p.Leu65Cys BHK or HEK-293 cells expressing mutant L65C were also grown in the presence of 10 µM cyclosporin A for 24 h because it acts as a pharmacological/specific chemical chaperone to increase the yield of mature enzyme [37]. Login to comment 49 ABCB1 p.Leu65Cys Reaction with MTS-verapamil and measurement of ATPase activity HEK-293 or BHK cells expressing His-tagged mutant L65C from 20 (10 cm diameter) plates were washed three times with PBS (10 mM sodium phosphate, pH 7.4, and 150 mM NaCl) and then suspended in a total volume of 1.5 ml of TBS (Tris-buffered saline; 10 mM Tris/HCl, pH 8.0, and 150 mM NaCl). Login to comment 60 ABCB1 p.Leu975Cys ABCB1 p.Phe942Cys ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys ABCB1 p.Thr945Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val981Cys ABCB1 p.Val981Cys ABCB1 p.Gly984Cys ABCB1 p.Gly984Cys ABCB1 p.Ala985Cys ABCB1 p.Ala985Cys 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 72 ABCB1 p.Leu65Cys ABCB1 p.Phe343Cys ABCB1 p.Ile306Cys The locations of residues L65C in TM1, I306C in TM5 and F343C in TM6 are shown. Login to comment 86 ABCB1 p.Leu65Cys A potential complicating factor in our earlier study to screen for mutants Figure 2 Reaction and ATPase activity of mutant L65C treated with MTS-verapamil (A) Schematic reaction of a thiol group with MTS-verapamil and attachment of verapamil to the protein via a disulphide bond. Login to comment 87 ABCB1 p.Met51Cys ABCB1 p.Val71Cys (B) His-tagged Cys-less or mutants M51C-V71C 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 1 mM MTS-verapamil. Login to comment 91 ABCB1 p.Leu65Cys (C) His-tagged mutant L65C expressed in HEK-293 cells was solubilized with n-dodecyl-β-D-maltoside, treated with various concentrations of MTS-verapamil and isolated by nickel-chelate chromatography. Login to comment 97 ABCB1 p.His61Cys ABCB1 p.Gly64Cys Table 1 shows that all mutants, except for mutants H61C and G64C, showed characteristics similar to that Table 1 Drug-stimulated ATPase activity of TM1 cysteine mutants ND, not determined because of very low expression. Login to comment 98 ABCB1 p.Gly54Cys ABCB1 p.Ile60Cys ABCB1 p.Val52Cys ABCB1 p.Ala58Cys ABCB1 p.Leu56Cys ABCB1 p.Met51Cys ABCB1 p.Val53Cys ABCB1 p.Ala57Cys ABCB1 p.Met68Cys ABCB1 p.Leu65Cys ABCB1 p.Gly62Cys ABCB1 p.Val71Cys ABCB1 p.His61Cys ABCB1 p.Gly64Cys ABCB1 p.Leu67Cys ABCB1 p.Thr55Cys ABCB1 p.Leu70Cys ABCB1 p.Ala63Cys ABCB1 p.Ile59Cys ABCB1 p.Pro66Cys Verapamil Colchicine Vinblastine Mutant Vmax (%)* S50 (µM)† Vmax (%) S50 (µM) Vmax (%) S50 (µM) M51C 101 11.0 + - 0.6 96 391 + - 36 94 2.4 + - 0.2 V52C ND ND ND ND ND ND V53C 104 12.0 + - 0.2 101 389 + - 30 102 2.2 + - 0.1 G54C ND ND ND ND ND ND T55C 114 10.3 + - 1.1 95 418 + - 22 91 2.2 + - 0.1 L56C 103 12.2 + - 0.3 87 440 + - 41 95 2.5 + - 0.2 A57C 108 11.3 + - 0.3 98 377 + - 34 92 2.4 + - 0.2 A58C 90 12.5 + - 0.2 94 434 + - 20 95 2.6 + - 0.3 I59C 115 11.2 + - 0.8 95 380 + - 33 114 2.5 + - 0.2 I60C 102 11.1 + - 0.7 91 408 + - 18 110 2.5 + - 0.2 H61C 97 54.0 + - 5.0 61 912 + - 86 105 5.4 + - 0.4 G62C ND ND ND ND ND ND A63C 114 10.5 + - 1.2 99 362 + - 42 105 2.0 + - 0.3 G64C 106 45.0 + - 6.0 88 613 + - 55 60 2.4 + - 0.1 L65C 72 9.3 + - 1.1 112 368 + - 32 78 2.0 + - 0.2 P66C 95 13.0 + - 0.5 86 480 + - 39 97 2.8 + - 0.4 L67C 101 12.3 + - 0.3 106 423 + - 21 100 2.3 + - 0.1 M68C 119 9.7 + - 1.1 105 365 + - 32 92 2.3 + - 0.2 M69C 107 11.8 + - 0.6 110 431 + - 25 108 2.2 + - 0.1 L70C 94 11.4 + - 0.7 90 413 + - 18 98 2.3 + - 0.1 V71C 106 11.9 + - 0.3 90 370 + - 27 102 2.5 + - 0.5 Cys-less 100 12.0 + - 1.0 100 412 + - 48 100 2.2 + - 0.3 * Maximum activity relative to that of Cys-less P-gp. Login to comment 101 ABCB1 p.His61Cys ABCB1 p.Gly64Cys Mutants H61C and G64C showed approx. Login to comment 103 ABCB1 p.His61Cys Mutant H61C also exhibited a reduction in the apparent affinity for vinblastine. Login to comment 104 ABCB1 p.Gly54Cys ABCB1 p.Val52Cys ABCB1 p.Gly62Cys The activities of mutants V52C, G54C and G62C were not determined because of very low expression [32]. Login to comment 111 ABCB1 p.Leu65Cys We found that the activity of only one cysteine mutant in TM1 (L65C) was permanently activated after treatment with MTS-verapamil (Figure 2B). Login to comment 112 ABCB1 p.Leu65Cys Mutant L65C showed a 10.7-fold increase in activity after modification with 0.3-1 mM MTS-verapamil compared with an untreated control. Login to comment 115 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys To test whether activation of mutant L65C by MTS-verapamil was due to covalent attachment of verapamil, we treated the modified mutant with 20 mM of the reducing agent DTT (dithiothreitol) and then measured ATPase activity. Figure 3 shows that the presence of DTT almost completely abolished the enhanced ATPase activity of mutant L65C. Login to comment 116 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys After treatment with DTT, the activity of MTS-verapamil-treated mutant L65C Figure 3 Effect of DTT on mutant L65C modified with MTS-verapamil His-tagged mutant L65C was expressed in HEK-293 cells. Login to comment 123 ABCB1 p.Leu65Cys We then tested whether labelling of mutant L65C with MTS-verapamil could be inhibited by the drug substrate verapamil. Login to comment 124 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys The rationale for these experiments was that if residue L65C contributed to binding of MTS-verapamil, then the presence of verapamil should protect mutant L65C from being modified. Login to comment 125 ABCB1 p.Leu65Cys We also tested whether Rhodamine B had any effect on labelling of mutant L65C by MTS-verapamil. Login to comment 127 ABCB1 p.Phe343Cys 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 130 ABCB1 p.Leu65Cys Accordingly, BHK cells stably expressing mutant L65C were used for these studies because they do not show any variation in P-gp expression as observed with transient expression in HEK293 cells. Login to comment 133 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys BHK cells stably expressing His-tagged L65C were harvested, solubilized with n-dodecyl-β-D-maltoside and then incubated with saturating levels of verapamil (2 mM) or Rhodamine B (3 mM) for 10 min at 20◦ C. The samples were then reacted for Figure 4 MTS-verapamil labelling of mutant L65C is inhibited by verapamil BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β- D-maltoside. Insoluble material was removed by centrifugation. Equivalent amounts of supernatant were incubated for 10 min at 20◦C in the presence of no drug (No Drug), 2 mM verapamil (Ver) or 3 mM Rhodamine B (Rhod). Login to comment 136 ABCB1 p.Leu65Cys 10 min at 20◦ C in the absence or presence of 0.1 mM MTS-verapamil, as this was the minimum concentration that gave almost complete modification of mutant L65C (Figure 2C). Login to comment 139 ABCB1 p.Leu65Cys The eluted samples were mixed with lipid, sonicated and assayed for ATPase activity. Figure 4 shows that the presence of verapamil reduced the labelling efficiency of mutant L65C by MTS-verapamil by more than 80%. Login to comment 140 ABCB1 p.Leu65Cys In contrast, the presence of Rhodamine B during labelling with MTS-verapamil caused very little (<10%) reduction in labelling of mutant L65C (Figure 4). Login to comment 142 ABCB1 p.Leu65Cys We then tested whether other drug substrates could still interact with mutant L65C that had been covalently labelled with MTS-verapamil. Login to comment 143 ABCB1 p.Leu65Cys The rationale was that substrates will not affect the ATPase activity of MTS-verapamil-modified mutant L65C if they occupy the same binding site as verapamil or if their binding site significantly overlaps that of verapamil, but will further stimulate or inhibit the activity if their binding sites are different from that of verapamil. Login to comment 145 ABCB1 p.Leu65Cys BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β-D-maltoside and then treated with or without 0.3 mM MTS-verapamil. Login to comment 146 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys His-tagged P-gp was then isolated by nickel-chelate chromatography, mixed with lipids from E. coli, sonicated and assayed for ATPase Figure 5 Effect of drug substrates and inhibitors on the ATPase activity of mutant L65C before and after labelling with MTS-verapamil BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β- D-maltoside and then incubated in the absence (Untreated; A) or presence (+MTS-Verapamil; B) of 0.3 mM MTS-verapamil. Login to comment 152 ABCB1 p.Leu65Cys We used E. coli lipids because the higher basal ATPase activity made it easier for us to detect for inhibition of activity. Figure 5(A) shows that in unmodified mutant L65C, verapamil stimulated the ATPase activity 4.6-fold, whereas calcein-AM and demecolcine stimulated the ATPase activity 7.0and 5.9-fold respectively. Login to comment 154 ABCB1 p.Leu65Cys When mutant L65C was modified with MTS-verapamil however, its basal activity was increased 4.6-fold compared with an untreated sample (Figure 5B). Login to comment 157 ABCB1 p.Leu65Cys These results suggest that modification of mutant L65C by MTS-verapamil blocks interaction of P-gp with drug substrates calcein-AM, demecolcine and trans-(E)-flupentixol, but not its interaction with cyclosporin. Login to comment 158 ABCB1 p.Leu65Cys The characteristics of the mutant L65C after labelling with MTS-verapamil suggested that Cys65 lined the drug-binding pocket. Another method to test for this possibility is to test whether Cys65 can be cross-linked to cysteine residues in the TMs of TMD2 that were previously shown to be involved in drug binding [15,17]. Login to comment 160 ABCB1 p.Thr945Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Val982Cys ABCB1 p.Gly984Cys ABCB1 p.Ala985Cys 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 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys (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 186 ABCB1 p.Leu65Cys Mutant L65C(TM1)/ I306C(TM5) showed only a 2.8-fold increase in activity after treatment with MTS-verapamil (Figure 7), whereas mutant L65C showed >10-fold increase in activity (Figure 2B). Login to comment 189 ABCB1 p.Leu65Cys DISCUSSION Only one mutant in TM1, L65C, was able to adopt a conformation that permanently hydrolysed ATP after modification with MTS-verapamil. Login to comment 191 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys Attachment of MTS-verapamil to Cys65 seems to mimic interaction of P-gp with verapamil because the ATPase activity of the MTS-verapamil-treated mutant L65C was very similar to that of untreated mutant L65C in the presence of saturating levels of verapamil. Login to comment 199 ABCB1 p.His61Cys ABCB1 p.Gly64Cys We also found that mutation of either His61 or Gly64 to cysteine altered the apparent affinity of P-gp for verapamil, colchicine or vinblastine (Table 1). Login to comment 200 ABCB1 p.Leu65Cys ABCB1 p.Leu65Cys ABCB1 p.His61Cys Arrangement of the residues in TM1 in a cylindrical helix (Figure 8A) shows that residues that react with MTS-verapamil (L65C; the present study) show alterations in substrate specificity when mutated (H61C, G644C and L65C; [44,45]) or show ATP-dependent cross-linking (M68 and M69; [30]) and occupy one face of the helix. Login to comment 204 ABCB1 p.Leu65Cys Since the present study has shown that L65C is involved in binding of verapamil, then cross-linking studies between TM1 and TM11 during ATP hydrolysis [30] would indicate that conformational changes in TM1 and TM11 probably contribute to the release of drug substrate during ATP hydrolysis. Login to comment 212 ABCB1 p.Leu65Cys ABCB1 p.Ile306Cys While the ATPase activities of both mutants L65C and I306C modified by MTS-verapamil could not be further stimulated by calcein-AM or demecolcine, the inhibition of their activities were different. Login to comment 213 ABCB1 p.Leu65Cys ABCB1 p.Ile306Cys The ATPase activity of mutant I306C modified by MTS-verapamil could not be inhibited by cyclosporin A or trans-(E)-flupentixol [40], whereas that of MTS-verapamil- modified mutant L65C was inhibited only by cyclosporin A (Figure 5). Login to comment 214 ABCB1 p.Leu65Cys This difference in sensitivity to inhibition by cyclosporin A suggests that both verapamil and cyclosporin A could bind simultaneously to mutant L65C, whereas covalent Figure 8 Arrangement of TM1 residues in a cylindrical helix and MTS-verapamil in the drug-binding pocket (A) The residues of TM1 are arranged in a cylindrical helix. Login to comment 215 ABCB1 p.Leu65Cys ABCB1 p.His61Cys Residues that show alterations in substrate specificity when mutated (H61C, G644C and L65C, circled) or show ATP-dependent cross-linking (Met68 and Met69 , boxed) with residues in TM11 are shown as occupying one face of the helix. Login to comment 227 ABCB1 p.Leu65Cys Labelling of mutant L65C (TM1) (the present study) or Ile306 (TM5) [40] with MTS-verapamil stimulated the ATPase activity of P-gp by more than 8-10-fold but labelling of the double cysteine mutant L65C(TM1)/I306C(TM5) with MTS-verapamil reduced the verapamil-stimulated ATPase activity of the enzyme (Figure 7). Login to comment 229 ABCB1 p.Ile306Cys Therefore the presence of a permanently bound verapamil molecule at I306C may interfere with movement between TM1 and TM11 during ATP hydrolysis (Figure 1B), resulting in inhibition of ATPase activity. Login to comment