ABCMdb

ABCC1 p.Pro1088Ala

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Publications

PMID: 14722114 [PubMed] Koike K et al: "Identification of proline residues in the core cytoplasmic and transmembrane regions of multidrug resistance protein 1 (MRP1/ABCC1) important for transport function, substrate specificity, and nucleotide interactions."

No. Sentence Comment

139 The locations of the MSD3 Pro residues mutated in this study are highlighted, and the approximate boundaries of the TM helices are indicated by dashed lines. B, shown is a representative immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (P1003A, P1060A, P1068A, P1088A, P1113A, P1120A, P1121A, P1150A, and P1191A) MRP1 cDNAs. MRP1 proteins were detected with mAb QCRL-1, and relative levels of expression estimated by densitometry are indicated; equal protein loading was confirmed by Amido Black staining of the polyvinylidene difluoride membrane and is shown below the blot. Login to comment
147 As shown in Fig. 4A, [3 H]LTC4 uptake levels of the P1003A, P1068A, P1088A, and P1150A mutants were ϳ40-50% less than those of wild-type MRP1, whereas LTC4 uptake by the other five MSD3 Pro mutants was similar to wild-type MRP1. Login to comment
148 Levels of [3 H]E217betaG uptake by six of the nine MSD3 mutants (P1003A, P1060A, P1068A, P1088A, P1120A, and P1121A) were moderately reduced (by 30-55%), whereas uptake of this substrate by the P1113A and P1191A mutants was comparable with wild-type MRP1. Login to comment
150 [3 H]MTX uptake by the P1150A mutant was also dramatically increased (ϳ6-fold) whereas uptake of this antifolate by the remaining eight MSD3 Pro mutants was either moderately reduced (by 50-60% for P1068A, P1088A, and P1120A) or comparable with wild-type MRP1 (P1003A, P1060A, P1113A, P1121A, and P1191A) (Fig. 4C). Login to comment
154 Relative to wild-type MRP1, apigenin-stimulated GSH uptake by the P1068A, P1088A, and P1150A mutants was significantly reduced (by 50-70%), whereas uptake by the other six MSD3 Pro FIG. 4. Login to comment
159 TABLE III Summary of effects of MSD3 Pro substitutions on MRP1 vesicular transport activities Substrate % wild-type MRP1 activitya ECL6 P1003A CL6 TM14 P1088A ECL7 P1113A TM15 CL7 P1060A P1068A P1120A P1121A P1150A P1191A LTC4 60 90 60 50 100 100 110 50 90 E217betaG 65 50 55 45 90 60 70 220 90 MTX 80 80 40 45 100 50 85 620 100 GSH 80 95 50 30 120 140 80 35 100 E13SO4 75 75 90 50 120 90 80 65 60 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in 2-3 independent experiments (for details, see legend to Fig. 4 and text). Login to comment
162 GSH-stimulated E13SO4 uptake by the P1003A, P1060A, P1088A, P1150A, and P1191A mutants was moderately reduced (by 25-50%), whereas uptake of this sulfated estrogen by the other four MSD3 Pro mutants (P1068A, P1113A, P1120A, and P1121A) was comparable with wild-type MRP1. Login to comment
185 The horizontal white scale bar in the image represents 20 ␮m. Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Uptake by MRP1 Pro Mutants-MSD2 TM mutants P343A, P448A, P478A, P557A, and P595A, MSD3 CL7 mutant P1150A, and TM14 mutant P1088A whose [3 H]LTC4 or [3 H]E217betaG transport properties were substantially altered relative to wild-type MRP1 were further characterized by kinetic analyses (Table IV). Login to comment
193 The apparent Km(LTC4) values for the MSD3 mutants P1088A and P1150A were also somewhat decreased (55 and 40 nM, respectively) compared with wild-type MRP1 (72 nM), as were their Vmax(LTC4) values (by ϳ50%), yielding overall LTC4 transport efficiencies (Vmax/Km) for these two mutants of 4.15 and 4.55, respectively, compared with a value of 5.86 for wild-type MRP1. Login to comment
195 In contrast, the apparent Km(E217betaG) value for P1088A was increased 1.4-fold (1.39 ␮M). Login to comment
196 On the other hand, the Vmax(E217betaG) values of P1150A and P1088A (160 and 165 pmol mg-1 min-1 , respectively) were comparable with wild-type MRP1 (176 pmol mg-1 min-1 ). Login to comment
197 Thus, the overall E217betaG transport efficiency of P1150A was enhanced ϳ4-fold whereas that of P1088A was reduced by 35%, largely because of changes in the apparent uptake affinity of the mutant proteins for this conjugated estrogen. Login to comment
198 Photolabeling of Wild-type and Pro Mutant MRP1 Proteins with [3 H]LTC4-To investigate further whether the reduced [3 H]LTC4 transport activity of the TM mutants P343A, P448A, P478A, P557A, P595A, and P1088A and the CL7 mutant P1150A was associated with a decrease in substrate binding, photolabeling experiments were carried out with this intrinsically photoactivable arachidonic acid derivative. Login to comment
204 Radiolabeled vesicles enriched for wild-type MRP1 (WT-MRP1) and MRP1 mutants P343A, P448A, P1088A, P1150A, and empty vector control (pcDNA3.1(-)) are shown in the upper panel, and radiolabeled vesicles enriched for WT-MRP1 and mutants P478A, P557A, P595A, P1150A, and pcDNA3.1(-) are shown in the lower panel. Login to comment
205 TABLE IV Kinetic parameters of vesicular LTC4 and E217beta G uptake by selected Pro mutants of MRP1 Km Vmax Vmax/ Km Mutant:WT nM pmol mg-1 min-1 ϫ10-3 Vmax/Km LTC4 WT-MRP1 72 422 5.86 1.0 P343A 50 203 4.06 0.7 P448A 39 155 3.97 0.7 P1088A 55 229 4.15 0.7 P1150A 40 182 4.55 0.8 WT-MRP1 115 674 5.85 1.0 P478A 49 160 3.24 0.6 P557A 63 191 3.02 0.5 P595A 485 239 0.49 0.1 E217betaG WT-MRP1 1017 176 0.17 1.0 P1088A 1390 165 0.12 0.7 P1150A 243 160 0.66 3.9 take of this conjugated organic anion at different ATP concentrations (Fig. 8A). Login to comment
264 In contrast to the MSD2 mutants, none of the nine MSD3 Pro mutants examined showed a comparable global decrease in transport activity, although Ala substitution of Pro1088 (at the membrane cytosol interface of TM14) and to a lesser extent Pro1003 (in ECL6) did cause a significant reduction in the transport of all five organic anion substrates tested. Login to comment
265 The significantly reduced transport activity of the P1088A mutant, together with our previous findings that non-conservative mutations of polar residues in the same region of TM14 alter MRP1 transport activity, confirm that the TM14-cytosol interface is a particularly important region for MRP1 activity (25, 29).3 Other MSD3 Pro mutants showed more substrate-selective changes in transport activity. Login to comment