ABCMdb

ABCC1 p.Arg532Ala

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Publications

PMID: 21177244 [PubMed] Iram SH et al: "Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2."

No. Sentence Comment

47 To generate point mutations in CL5, plasmid pBluescriptSK(ϩ)BamHI/SpHI-MRP1 containing a 1.9-kb fragment from pcDNA3.1(-)-MRP1k was used as the template with the following mutagenic primers (substituted nucleotides are underlined): R501A, 5Ј-GAG CAA AGA CAA TGC GAT CAA GCT GAT G-3Ј; K503A, 5Ј-GAC AAT CGG ATC GCG CTG ATG AAC G-3Ј; E507A, 5Ј-GCT GAT GAA CGC AAT TCT CAA TGG G-3Ј; G511I, 5Ј-CTC AAT ATT ATC AAA GTG CTA AAG CTT TAT GCC TGG GAG CTG GC-3Ј; K513A, 5Ј-CTC AAT GGG ATC GCA GTG CTA AAG C-3Ј; K513R, 5Ј-CTC AAT GGG ATC AGA GTG CTA AAG C-3Ј; K516A, 5Ј-GAT CAA AGT GCT AGC GCT TTA TGC CTG-3Ј; K516R, 5Ј-GAT CAA AGT GCT AAG ACT TTA TGC CTG-3Ј; E521A, 5Ј-CTT TAT GCC TGG GCG CTG GCA TTC-3Ј; E521D, 5Ј-CTT TAT GCC TGG GAC CTG GCA TTC-3Ј; R532A, 5Ј-GTG CTG GCC ATT GCG CAG GAG GAG CT-3Ј; E535A, 5Ј-CAT CAG GCA GGA GGC CTT GAA GGT GCT GAA G-3Ј; and E535D, 5Ј- CAG GCA GGA GGA TCT GAA GGT GCT GAA G-3Ј. Login to comment
112 On the other hand, levels of the four remaining CL5 Ala-substituted mutants (R501A, K503A, E507A, and R532A) were comparable with wild-type MRP1 (see Fig. 6A). Login to comment
164 When their transport properties were examined, however, a 50-60% decrease in the [3 H]LTC4 and [3 H]E217betaG transport levels of the R501A, E507A, and R532A mutants was observed (Fig. 6, B and C). Login to comment
166 The reduced E217betaG transport activity of the R501A, E507A, and R532A mutants was analyzed further by determining the kinetic parameters of uptake; the results of these experiments are summarized in Table 1. Login to comment
168 In contrast, the Km (E217betaG) values for the R501A, E507A, and R532A mutants were increased 3-5-fold (18-25 ␮M). Login to comment
186 Photolabeling of MRP1 Mutants R501A, E507A, R532A, and G511I with [3 H]LTC4-To determine whether the decrease in LTC4 uptake by the R501A, E507A, R532A, and G511I mutants was associated with decreased binding of this substrate to MRP1, membrane vesicles enriched for wild-type or mutant MRP1 were photolabeled with [3 H]LTC4. Login to comment
187 As shown in Fig. 8A, [3 H]LTC4 labeling of the R501A and E507A mutants was decreased by 50%, whereas labeling of the R532A mutant was decreased by 70%. Login to comment
199 Levels and organic anion transport activity of MRP1 mutant proteins R501A, K503A, E507A, and R532A. Login to comment
200 A, shown is a representative immunoblot of membrane vesicles (MV) (1 ␮g of protein/lane) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (R501A, K503A, E507A, and R532A) cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 was detected with mAb QCRL-1, and the relative protein expression levels were estimated by densitometry and are shown below the blot. Login to comment
204 TABLE 1 Kinetic parameters of E217betaG uptake by MRP1 mutants R501A, E507A, and R532A Km and Vmax values for E217betaG uptake by membrane vesicles (4 ␮g of protein) prepared from HEK293T cells transfected with wild-type or mutant proteins were determined by measuring ATP-dependent uptake at eight different E217betaG concentrations (0.25-25 ␮M) for 1 min at 37 °C as described under "Experimental Procedures." Login to comment
207 Transfectant Km Vmax a (Vmax/Km) ؋ 10-3 ␮M pmol/mg/min mg/liter/min WT-MRP1 5.4 1088 0.20 R501A 22.9 1274 0.05 E507A 18.1 1150 0.06 R532A 24.5 1031 0.04 a Vmax values have been corrected for differences in protein expression of the mutants relative to WT-MRP1 (Fig. 7A). Login to comment
210 Of the charged amino acid mutants, only K503A exhibited properties comparable with those of wild-type MRP1, whereas the seven remaining Ala-substituted mutants were either poorly expressed (K513A, K516A, E521A, and E535A) or exhibited significantly reduced transport activity (R501A, E507A, and R532A). Login to comment
236 [3 H]LTC4 photolabeling of wild-type and MRP1 mutants R501A, E507A, R532A, and G511I. Login to comment
253 Nevertheless, although the K503A mutant exhibited properties similar to wild-type MRP1, the LTC4 and E217betaG transport activities of R501A, E507A, and R532A were significantly reduced. Login to comment