ABCMdb

PMID: 24349290

Chufan EE, Kapoor K, Sim HM, Singh S, Talele TT, Durell SR, Ambudkar SV
Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).
PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. Login to comment 55 ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Phe978Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys 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 61 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys However, CsA, tariquidar and valinomycin lose almost completely this ability to inhibit IAAP photo-labeling when residues Y307, Q725 and V982 are mutated to cysteine (i.e., Y307C/Q725C/V982C mutant). Login to comment 69 ABCB1 p.Phe978Cys One mutant, F978C, behaves in a manner similar to that of cysless WT Pgp, with only a change in the binding affinity for CsA, IC50 = 0.54 &#b5;M, compared to 0.05 &#b5;M for cysless WT Pgp (Table 1). Login to comment 71 ABCB1 p.Phe978Cys Similarly, tariquidar inhibits IAAP labeling of F978C to the Figure 1. Login to comment 73 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys 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 74 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys Autoradiograms corresponding to cysless, V982C and Y307C/Q725C/V982C, as representative examples of complete inhibition, partial inhibition and no inhibition of IAAP-labeling, respectively, are shown at the top of the figure. Login to comment 81 ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys The mutants F343C and F343C/V982C also exhibit normal basal activity. Login to comment 82 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Phe978Cys 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 83 ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys The rest of the mutants, Q725C, Y307C (and their corresponding double and triple mutants) and F728 show intermediate levels of basal ATPase activity. Login to comment 90 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Phe978Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys 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 93 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys Valinomycin also stimulated ATP hydrolysis of all mutants except V982C and Q725C/V982C. Login to comment 94 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys It is interesting to observe that the effect of the V982C mutation is not dominant in the rest of the double mutants and even in the triple mutant Y307C/Q725C/V982C, in which case valinomycin does stimulate ATP hydrolysis. Login to comment 95 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys 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 108 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys In Figure 4A, representative histograms show the cell surface expression for single (Y307C), double (Y307C/V982C) and triple (Y307C/ Q725C/V982C) mutants. Login to comment 114 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys Even with the triple (Y307C/Q725C/V982C) mutant the efflux of none of the above-mentioned substrates is completely abolished, although many of these substrates are transported at lower levels when compared to the cysless WT Pgp (Table 2). Login to comment 116 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys Figure 5A shows the transport of rhodamine 123 (Rh123), which is normal for the single (Y307C) and double (Y307C/V982C) mutants but is decreased considerably for the triple (Y307C/Q725C/V982C) mutant. Login to comment 123 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys NBD-CsA loses the ability to inhibit the IAAP labeling of the triple (Y307C/Q725C/V982C) Figure 2. Login to comment 137 ABCB1 p.Val982Cys Interestingly, the only single mutation that has a severe effect on drug transport is V982C. Login to comment 138 ABCB1 p.Val982Cys Cells over-expressing the V982C mutant fail to transport NBD-CsA completely. Login to comment 139 ABCB1 p.Val982Cys However, NBD-CsA is able to partially inhibit IAAP labeling in crude membranes (51%, Table S1), clearly showing that NBD-CsA binds the V982C mutant. Login to comment 141 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys As expected, the corresponding double mutants (Q725C/V982C; F728C/V982C; Figure 3. Login to comment 150 ABCB1 p.Phe343Cys ABCB1 p.Val982Cys doi: 10.1371/journal.pone.0082463.g003 F343C/V982C) show dramatically reduced transport of NBD-CsA. Login to comment 151 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys Nonetheless, Y307C/V982C and the triple mutant Y307C/ Q725C/V982C show some rescue of the NBD-CsA transport. Login to comment 152 ABCB1 p.Tyr307Cys This can most likely be attributed to the presence of the Y307C mutation, as this particular double mutant exhibits about 50-60% transport function with respect to NBD-CsA (Table 2). Login to comment 159 ABCB1 p.Tyr307Cys (A) The left panel shows the cell surface localization of Y307C with human Pgp-specific monoclonal antibody MRK-16 labeling as detected by green fluorescence detector. Login to comment 160 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys The middle panel shows the same for the double mutant Y307C/V982C, and the right panel for the triple mutant Y307C/Q725C/V982C. Login to comment 162 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys The conformation sensitivity towards CsA of single (Y307C), double (Y307C/V982C) and triple (Y307C/Q725C/V982C) mutants was similar to cysless WT Pgp, as shown in the three panels, respectively. Login to comment 175 ABCB1 p.Phe343Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Phe978Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys 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 211 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys The transport functions of single (Y307C), double (Y307/V982C) and triple (Y307C/Q725C/V982C) mutants are differentially affected. Login to comment 213 ABCB1 p.Glu1201Gln ABCB1 p.Glu501Gln The inactive E501Q/E1201Q (EQ) mutant P-gp is used as a control to show completely aborted function as described in Materials and Methods. Login to comment 220 ABCB1 p.Gly251Val The same residues studied in the present work were mutated to arginine by Loo and coworkers [38]; they found these mutations promote or have a neutral effect on maturation of a Pgp processing mutant (G251V) defective in folding, also confirming that these residues are in the drug translocation pathway and/or drug-binding pocket of Pgp. Login to comment 238 ABCB1 p.Gln725Cys Here, we report QZ59-SSS, CsA, tariquidar, valinomycin and FSBA lost the ability to inhibit IAAP-labeling of Q725C to the same extent as cysless WT Pgp. Login to comment 240 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys 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 251 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys In all mutants, even the triple Y307C/Q725C/V982C, ATP hydrolysis is inhibited by both CsA and tariquidar. Login to comment 257 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys In steady-state conditions, calcein-AM and bodipy-placitaxel are transported by the triple (Y307C/Q725C/V982C) mutant to the same extent as cysless WT Pgp. Login to comment 261 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys Therefore, NBD-CsA does not bind to its primary site on Y307C/Q725C/V982C, but to a secondary site, where it is transported at 50-60% of the rate of cysless WT Pgp. Login to comment 290 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The accumulation of substrates was calculated with respect to the accumulation in E556Q/E1201Q mutant Pgp, which is completely inactive [48], and is treated as a negative control for all transport experiments. Login to comment 328 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys Effect of QZ59-SSS-sulfur on the photocrosslinking of cysless WT and mutant Pgpswith IAAP. Inhibition of IAAP-labeling for cysless WT and for triple mutant Y307C/Q725C/V982C at different concentrations of QZ59-SSS-sulfur are shown (graph). Login to comment 333 ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys ABCB1 p.Tyr307Cys Inhibition of IAAP-labeling for single mutants Q725C, Y307C and V982C (upper graph) and for double (Q725C/V982C and Y307C/ V982C) and triple (Y307C/Q725C/V982C) mutants (lower graph) at different concentrations of valinomycin are shown. Inhibition of IAAP-labeling of cysless WT is included in both graphs, as a reference. Login to comment 338 ABCB1 p.Val982Cys ABCB1 p.Gln725Cys ABCB1 p.Tyr307Cys Effect of FSBA on the photocrosslinking of cysless WT and mutant Pgps with IAAP. Inhibition of IAAP-labeling of cysless WT and triple (Y307C/Q725C/V982C) mutant at different concentrations of FSBA are shown (graph). Login to comment