ABCMdb

PMID: 14522974

Loo TW, Bartlett MC, Clarke DM
Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites.
J Biol Chem. 2003 Dec 12;278(50):50136-41. Epub 2003 Oct 1., 2003-12-12 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. Login to comment 8 ABCB1 p.Phe343Cys By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. Login to comment 61 ABCB1 p.Leu531Cys Effect of Rhodamine on Disulfide Cross-linking Analysis-The cDNA for mutant L531C/C1074 was expressed in HEK 293 cells in the absence of cyclosporin A (16). Login to comment 89 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys Therefore, it is unlikely that labeling of mutant F343C by MTS-rhodamine is coincidental. Login to comment 93 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys Therefore, we tested whether Hoechst 33342 would affect the activity of the MTS-rhodamine-labeled F343C mutant. Login to comment 105 ABCB1 p.Phe343Cys We then tested whether drug substrates could protect mutant F343C from modification by MTS-rhodamine. Login to comment 106 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys Labeling of mutant F343C by MTS-rhodamine, however, showed little protection with verapamil. Login to comment 117 ABCB1 p.Phe343Cys The presence of the reducing agent inhibited activation of mutant F343C by MTS-rhodamine by more than 80% (Fig. 4). Login to comment 118 ABCB1 p.Phe343Cys FIG. 2. Effect of MTS-rhodamine treatment on the basal activity of Cys-less and F343C mutant P-gps. Login to comment 124 ABCB1 p.Phe343Cys FIG. 3. Effect of drug substrates on the ATPase activity of mutant F343C before and after labeling with MTS-rhodamine. Login to comment 129 ABCB1 p.Phe343Cys The characteristics of the ATPase activity of MTS-rhodamine-labeled mutant F343C were also examined. Login to comment 132 ABCB1 p.Phe343Cys Fig. 4 shows that vanadate trapping inhibits the activity of the activated F343C mutant. Login to comment 133 ABCB1 p.Phe343Cys Labeling of mutant F343C did not appear to affect the affinity of the enzyme for ATP. Login to comment 136 ABCB1 p.Leu531Cys For example, verapamil-induced conformational change can be monitored by the rate of cross-linking between a cysteine introduced into NBD1 "LSGGQ" site (L531C) and the endogenous cysteine in the NBD2 Walker A site (C1074). Login to comment 139 ABCB1 p.Leu531Cys Rhodamine B, instead of MTS-rhodamine, was used to test the effect on cross-linking of mutant L531C/C1074 because MTS-rhodamine could potentially react with the cysteines in the NBDs. Login to comment 140 ABCB1 p.Leu531Cys Accordingly, membranes were prepared from cells expressing mutant L531C/C1074 and incubated with or without 1 mM rhodamine B at 4 °C in the presence of oxidant (1 mM copper phenanthroline). Login to comment 143 ABCB1 p.Leu531Cys Fig. 5 shows that rhodamine B promoted cross-linking of mutant L531C/C1074. Login to comment 150 ABCB1 p.Ile340Cys ABCB1 p.Phe343Cys It is interesting that residue F343C is on the same face of the TM6 ␣-helix as residue I340C (Fig. 6A). Login to comment 151 ABCB1 p.Ile340Cys Labeling of I340C with MTS-rhodamine, however, resulted in inhibition of ATPase activity (22). Login to comment 152 ABCB1 p.Ile340Cys Rhodamine B also protected I340C from labeling by MTS-rhodamine, indicating that this residue must be close to rhodamine-binding site. Login to comment 154 ABCB1 p.Ile340Cys Covalent modification of I340C may prevent P-gp from undergoing conformational changes during the transport cycle. Login to comment 155 ABCB1 p.Phe343Cys Two observations suggest that MTS-rhodamine attached to F343C mimics the interaction of rhodamine compounds with P-gp. Login to comment 156 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys Second, rhodamine B protected mutant F343C from labeling by MTS-rhodamine. Login to comment 158 ABCB1 p.Phe343Cys Calcein-AM and colchicine did not affect the activity of MTS-rhodamine-treated mutant F343C. Login to comment 162 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys 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 ABCB1 p.Phe343Cys Inhibition of MTS-rhodamine activation of mutant F343C. Login to comment 172 ABCB1 p.Leu531Cys FIG. 5. Effect of rhodamine B on cross-linking of mutant L531C/C1074. Login to comment 173 ABCB1 p.Leu531Cys Membranes were prepared from HEK 293 cells expressing mutant L531C/C1074 and incubated with no drug substrate or with 1 mM rhodamine B (ϩ Rhodamine B) for 15 min at 4 °C. The samples were then treated with 1 mM copper phenanthroline at 4 °C for various intervals. Login to comment 200 ABCB1 p.Ile340Cys ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys This may explain why both F343C and I340C can be labeled with MTS-rhodamine, but only F343C is activated upon labeling. Login to comment