ABCMdb

ABCC1 p.Glu507Ala

None has been submitted yet.

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
190 Photolabeling of Mutants E507A and G511I with 8-Azido- [␣-32 P]ATP and Orthovanadate- and BeF-induced Trapping of 8-Azido-[␣-32 P]ADP-The precise role of the CLs in coupling the ATPase activity of ABC transporters to the translocation of their substrates is not yet fully understood (12, 42). Login to comment
192 For this reason, the interactions of the transport-deficient E507A and G511I mutants with ATP were examined. Login to comment
195 Next, the catalytic activity of the E507A and G511I mutants was examined by measuring the amount of azido-[32 P]ADP trapped by orthovanadate under conditions permissive for ATP hydrolysis (26). Login to comment
196 As shown in Fig. 9B, [32 P]ADP trapping by the G511I mutant was reduced by 40%, whereas trapping by the E507A mutant was comparable to wild-type MRP1. 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
240 Azido-[32 P]ATP labeling and vanadate-induced trapping of azido-[32 P]ADP by E507A and G511I mutant MRP1 proteins. 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
267 Although binding of azido-[32 P]ATP by the mutants was not affected, vanadate-induced trapping of azido-[32 P]ADP was moderately reduced for G511I but not for E507A. Login to comment
272 Nevertheless, these authors reported that the LTC4 transport activity of the double mutant was reduced by Ͼ80%, a larger decrease than we observed for either of the single E507A and G511I mutants. Login to comment