ABCMdb

PMID: 10350482

Dey S, Hafkemeyer P, Pastan I, Gottesman MM
A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.
Biochemistry. 1999 May 18;38(20):6630-9., 1999-05-18 [PubMed]

Sentences

No. Mutations Sentence Comment

37 ABCB1 p.Phe983Ala In this study we demonstrate that substitution of a single phenylalanine residue with alanine at position 983 in TM 12 of Pgp affects inhibition of Pgp-mediated drug transport by both cis(Z)- and trans(E)-flupentixol, and significantly alters their stereospecific effect on ATP hydrolysis and substrate recognition by Pgp, suggesting a common site of functional interaction for both isomers of flupentixol. Login to comment 126 ABCB1 p.Phe983Ala RESULTS Substitution of Phenylalanine Residue 983 with Alanine SelectiVely Affects Inhibition of Drug Transport by both cis(Z)- and trans(E)-Flupentixol. Login to comment 127 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala In a recent study, seven amino acid residues, L975, V981, F983, M986, V988, Q990, and V991, in the putative TM 12 of human Pgp were substituted individually by alanine (L975A, V981A, F983A, M986A, V988A, Q990A, and V991A). Login to comment 133 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala Consistent with the previous report, all the mutant Pgp`s (L975A, V981A, F983A, M986A, V988A, Q990A, and V991A) were expressed on the cell surface at a comparable level to that of wild-type (WT) Pgp (Figure 1A, upper panel). Login to comment 141 ABCB1 p.Gln990Ala ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Human osteosarcoma (HOS) cells infected with vTF7-3 were transfected with either pTM1 (control) (s), pTM1-MDR1 (wild type) (-), pTM1-MDR1-L975A (L975A) (‚‚‚), pTM1-MDR1-V981A (V981A) (- - -), pTM1-MDR1-F983A (F983A) (- -), pTM1-MDR1-M986A (M986A) (thick dashes), pTM1-MDR1-V988A (V988A) (-‚‚-), pTM1-MDR1-Q990A (Q990A) (-‚-), or pTM1-MDR1-V991A (V991A) (-‚‚‚-) plasmid DNA. Login to comment 143 ABCB1 p.Gln990Ala ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala (A, upper panel) Cells were subjected to FACS analysis after staining with human Pgp external epitope-specific monoclonal antibody MRK-16 (14), as described under Experimental Procedures. (A, lower panel) Total cell lysates were prepared from each cell type, and immunoblot analysis with Pgp-specific monoclonal antibody C219 was performed as described under Experimental Procedures. (B) Similar to section A; cells were infected with vTF7-3, and transfected with either pTM1 (control) (s), pTM1-MDR1 (wild type) (-), pTM1-MDR1-L975A (L975A) (‚‚‚), pTM1-MDR1-V981A (V981A) (- - -), pTM1-MDR1-F983A (F983A) (- -), pTM1-MDR1-M986A (M986A) (thick dashes) pTM1-MDR1-V988A (V988A) (-‚‚-), pTM1-MDR1-Q990A (Q990A) (-‚-), or pTM1-MDR1-V991A (V991A) (-‚‚‚-) plasmid DNAs. Login to comment 155 ABCB1 p.Leu975Ala In the presence of 10 µM cis(Z)-flupentixol or 5 µM trans(E)-flupentixol, the intracellular accumulation of Bodipy FL-verapamil in HOS cells expressing either wild-type Pgp or L975A was increased to approximately the same level as that of the control cells (Figure 2A,B), which had no detectable expression of Pgp, suggesting effective inhibition of Pgp-mediated efflux from the cells. Login to comment 156 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Val991Ala ABCB1 p.Leu975Ala The effects of both cis(Z)- and trans(E)-flupentixol on accumulation of Bodipy FL-verapamil in cells expressing V988A, Q990A, or V991A were identical to that observed in L975A (data not shown), suggesting that none of these substitutions affects inhibition of transport by flupentixol. Login to comment 157 ABCB1 p.Phe983Ala However, the steady-state accumulation of the verapamil derivative in cells expressing F983A was minimally altered in the presence of 10 µM cis(Z)- or 5 µM trans(E)-flupentixol (Figure 2A,B), suggesting an impaired ability of this mutant to respond to inhibition of drug transport by both isomers of flupentixol. Login to comment 158 ABCB1 p.Val981Ala ABCB1 p.Met986Ala In cells expressing mutants V981A (Figure 2A,B) and M986A (data not shown), the level of intracellular accumulation of Bodipy Fl-verapamil was effectively increased by both isomers of flupentixol, but not to the same extent as that of the cells expressing wild-type Pgp. Login to comment 159 ABCB1 p.Val981Ala ABCB1 p.Met986Ala This suggested that inhibition of drug transport by cis(Z)- and trans(E)- flupentixol in V981A and M986A was not complete, indicating a moderate contribution of these residues to the interaction with flupentixol. Login to comment 160 ABCB1 p.Phe983Ala Since substitution of F983 had the most profound effect on the inhibitory potential of flupentixol, the mutant F983A was our obvious choice for further characterization. Login to comment 161 ABCB1 p.Phe983Ala Stimulation of ATP Hydrolysis by cis(Z)-Flupentixol Is Considerably Altered in F983A. Login to comment 163 ABCB1 p.Phe983Ala Since drug transport by F983A was considerably less sensitive to inhibition by cis(Z)- flupentixol, we studied the ability of cis(Z)-flupentixol to modulate the rate of ATP hydrolysis by this mutant. Login to comment 165 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Steady-state accumulation of Bodipy FL-verapamil was measured by FACS in the presence (- - -) and absence (s) of 10 µM cis(Z)-flupentixol (A) or 5 µM trans(E)-flupentixol (B) in HOS cells infected with vTF7-3 and transfected with either pTM1 (pTM1), pTM1-MDR1 (WT), pTM1-MDR1-L975A (L975A), pTM1-MDR1-V981A (V981A), or pTM1-MDR1-F983A (F983A). Login to comment 166 ABCB1 p.Phe983Ala For details, see Experimental Procedures. the wild-type Pgp or F983A, and Pgp-ATPase activity was measured in the presence of varying concentrations of either verapamil or cis(Z)-flupentixol. Login to comment 167 ABCB1 p.Phe983Ala Consistent with their ability to transport Bodipy FL-verapamil, ATP hydrolysis by both wild-type Pgp and F983A in isolated membranes was stimulated by verapamil in a concentration-dependent manner (Figure 3A). Login to comment 168 ABCB1 p.Phe983Ala Although the rate of ATP hydrolysis by F983A in the absence of verapamil was about 70% of that of the wild-type Pgp, the verapamil-stimulated activity was about the same in both Pgp`s. Login to comment 169 ABCB1 p.Phe983Ala A maximum rate of ATP hydrolysis between 25 and 28 nmol mg-1 min-1 was achieved by both wild-type and F983A at a verapamil concentration of 25 µM. Login to comment 173 ABCB1 p.Phe983Ala In contrast, stimulation by cis(Z)-flupentixol of ATP hydrolysis by F983A was significantly impaired, with a maximum of only 12-14 nmol mg-1 min-1 of ATP hydrolyzed at cis(Z)- flupentixol concentrations of 50 µM (Figure 3B). Login to comment 174 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Due to a low basal rate of ATP hydrolysis by F983A, the fold stimulation by cis(Z)-flupentixol (2-fold) was about the same for both wild-type Pgp and F983A; however, in F983A this was achieved at a considerably higher concentration (Figure 3B). Login to comment 175 ABCB1 p.Phe983Ala The Ability of trans(E)-Flupentixol To Inhibit Verapamil-Stimulated ATP Hydrolysis Is Markedly Reduced in F983A. Login to comment 176 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Since Bodipy FL-verapamil transport by F983A was less sensitive to inhibition by trans(E)-flupentixol, we studied its effect on verapamil-stimulated ATP hydrolysis by F983A. Isolated cell membranes from HeLa cells, expressing either wild-type Pgp or F983A, were preincubated with varying concentrations of trans(E)-flupentixol prior to the measurement of vanadate-sensitive ATP hydrolysis in the presence of 25 µM verapamil. Login to comment 177 ABCB1 p.Phe983Ala Consistent with the previous experiment, in the absence of trans(E)-flupentixol the rate of verapamil-stimulated ATP hydrolysis by the wild-type and the mutant (F983A) was around 26 nmol mg-1 min-1. Login to comment 179 ABCB1 p.Phe983Ala In contrast, the ability of trans(E)- flupentixol to inhibit verapamil-stimulated ATP hydrolysis by F983A was significantly reduced (Figure 4A). Login to comment 181 ABCB1 p.Phe983Ala This indicated that residue F983A also plays a crucial role in the inhibition of ATP hydrolysis by trans(E)-flupentixol. Login to comment 182 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala To determine whether the loss of sensitivity to trans(E)- flupentixol in F983A reflected a general defect of the mutant FIGURE 3: Effect of verapamil and cis(Z)-flupentixol on ATP hydrolysis by the wild-type and F983A Pgp`s. Login to comment 183 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Membranes were isolated from HeLa cells infected with vTF7-3 and transfected with either pTM1, pTM1-MDR1 (WT), or pTM1-MDR1-F983A (F983A) plasmid DNAs. Login to comment 184 ABCB1 p.Phe983Ala HeLa cell membranes (20 µg of protein) containing wild-type Pgp (0) or F983A (b) were incubated either with the indicated concentrations of verapamil (A) or with cis(Z)-flupentixol (B) prior to measuring the rate of ATP hydrolysis. Login to comment 188 ABCB1 p.Phe983Ala FIGURE 4: Effect of trans(E)-flupentixol and cyclosporin A on verapamil-stimulated ATP hydrolysis by the wild-type and F983A Pgp`s. Login to comment 189 ABCB1 p.Phe983Ala Vanadate-sensitive ATP hydrolysis by wild-type Pgp (0) and F983A (b) (20 µg of membrane protein) in HeLa cell membranes was measured as described in Figure 3, in the presence of 5 mM ATP, 10 mM MgCl2, 25 µM verapamil, and the indicated concentrations of either trans(E)-flupentixol (A) or cyclosporin A (B). Login to comment 192 ABCB1 p.Phe983Ala in its ability to respond to inhibitors of Pgp-ATPase activity, we studied the effect of cyclosporin A on verapamil-stimulated ATP hydrolysis by wild-type Pgp and F983A. Login to comment 193 ABCB1 p.Phe983Ala Cyclosporin A inhibited verapamil-stimulated ATPase activity with equal potency in both wild type and F983A (Figure 4B). Login to comment 194 ABCB1 p.Phe983Ala The concentrations for half-maximal inhibition were about 0.4 µM for both wild type and F983A. Login to comment 196 ABCB1 p.Phe983Ala Stimulation by cis(Z)-Flupentixol of Photoaffinity Labeling with the Substrate Analogue [125 I]IAAP Is Considerably Affected in F983A. Login to comment 198 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Since cis(Z)-flupentixol was unable to reverse drug transport by F983A, we examined the effect of cis(Z)-flupentixol on [125 I]IAAP labeling of F983A. Isolated membranes from HeLa cells expressing wild-type Pgp or F983A were photoaffinity labeled with 4 nM [125 I]IAAP in the presence of varying (0-100 µM) concentrations of cis(Z)-flupentixol. Login to comment 200 ABCB1 p.Phe983Ala In contrast, stimulation of [125I]IAAP labeling was significantly reduced in F983A (Figure 5B) with a maximal stimulation of only 2-fold (Figure 5C). Login to comment 201 ABCB1 p.Phe983Ala However, stimulation reached its maximum at the same concentration of cis(Z)- flupentixol (10 µM) for both wild-type Pgp and F983A (Figure 5C). Login to comment 205 ABCB1 p.Phe983Ala Photoaffinity Labeling of F983A with [125I]IAAP Is RelatiVely Resistant to Inhibition by trans(E)-Flupentixol. Login to comment 209 ABCB1 p.Phe983Ala Inhibition of [125I]IAAP labeling by trans(E)-flupentixol was considerably altered in F983A, in which case the concentration of trans(E)-flupentixol required for half-maximal inhibition was about 4-fold higher (about 40 µM) than that for the wild-type Pgp. Login to comment 210 ABCB1 p.Phe983Ala Photoaffinity labeling of the wild-type Pgp and F983A with [125 I]IAAP was also carried out in the presence of varying concentrations of cyclosporin A. Login to comment 212 ABCB1 p.Phe983Ala Concentrations of cyclosporin A required for half-maximal and maximal inhibitions were around 0.035 µM for both wild-type Pgp and F983A. Login to comment 213 ABCB1 p.Phe983Ala This excluded the possibility of a general loss of sensitivity to inhibitors of substrate recognition in F983A. Login to comment 218 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala In this study we demonstrate that a single substitution of alanine for phenylalanine at position 983 in the putative TM 12 of Pgp markedly alters the ability of both cis and trans isomers of flupentixol to modulate ATPase activity and [125 I]IAAP labeling of Pgp, and also FIGURE 5: Effect of cis(Z)-flupentixol on [125I]IAAP labeling of the wild-type Pgp and F983A. Login to comment 219 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Membranes were isolated from HeLa cells infected with vTF7-3 and transfected with either (A) pTM1-MDR1 (WT) or (B) pTM1-MDR1-F983A (F983A) plasmid DNAs. Login to comment 221 ABCB1 p.Phe983Ala (C) Quantification of radioactivity associated with the wild-type (0) and F983A (b) Pgp`s bands was done by a STORM 860 phosphorimaging system as described under Experimental Procedures. Login to comment 239 ABCB1 p.Val981Ala ABCB1 p.Met986Ala ABCB1 p.Phe983Ala Although drug transport was inhibited by both cis(Z)- and trans(E)-flupentixol in V981A and M986A (data not shown), inhibition was not complete, FIGURE 6: Effect of trans(E)-flupentixol and cyclosporin A on [125I]IAAP labeling of the wild-type Pgp and F983A. Login to comment 240 ABCB1 p.Phe983Ala HeLa membranes (25 µg of protein) containing either wild-type Pgp (top) or F983A (bottom) were preincubated at room temperature in the presence of the indicated concentrations of either trans(E)-flupentixol (A) or cyclosporin A (B) prior to incubation with 4 nM [125I]IAAP, for 10 min, as described under Experimental Procedures. Following incubation, membranes were photo-cross-linked, and 10 µg of membrane proteins was analyzed by SDS-PAGE and subjected to autoradiography. Login to comment 243 ABCB1 p.Phe983Ala Radioactivity associated with the wild-type (0) and F983A (b) Pgp bands was expressed as percent control using the same method as in Figure 5. Login to comment 246 ABCB1 p.Val981Ala ABCB1 p.Met986Ala ABCB1 p.Phe983Ala It is to be noted that in all three mutants (F983A, V981A, and M986A) the inhibitory effect of both isomers (cis and trans) of flupentixol was affected. Login to comment 255 ABCB1 p.Phe983Ala In F983A the inhibitory effect of cis(Z)- and trans(E)-flupentixol on Pgp-mediated drug transport was considerably reduced without much alteration in the ability of the protein to transport substrates (Figures 1B and 2A,B). Login to comment 257 ABCB1 p.Phe983Ala Therefore, this dissociation of susceptibility to inhibition by flupentixol from drug transport in F983A strongly indicates the allosteric nature of the flupentixol interaction site with Pgp. Login to comment 258 ABCB1 p.Phe983Ala Consistent with this, in F983A the modulatory potency of cis(Z)- and trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered with only a minimal effect on the basal level of ATPase activity and [125 I]IAAP labeling. Login to comment 266 ABCB1 p.Phe983Ala Consistent with the idea of a single site of interaction, both cis(Z)-flupentixol-mediated stimulation (Figure 5C) and trans(E)-flupentixol-mediated inhibition (Figure 6A) of [125I]IAAP labeling were also significantly reduced in F983A. Login to comment 269 ABCB1 p.Phe983Ala In F983A, stimulation of [125I]IAAP labeling was significantly altered with a maximal stimulation of only 2-fold compared to 9-fold stimulation in the wild-type Pgp (Figure 5A-C). Login to comment 270 ABCB1 p.Phe983Ala However, the decrease in stimulation of [125I]IAAP labeling was evident at the same concentration of cis(Z)-flupentixol for both F983A and wild-type Pgp. Login to comment