ABCMdb

ABCC1 p.Trp445Ala

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PMID: 12388549 [PubMed] Koike K et al: "Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1."

No. Sentence Comment

48 Tryptophan substitutions were generated in the pGEM-3Z and pBluescriptSK(ϩ) plasmids above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): MSD1 Trp mutants W40A (5Ј-G GTC CTC GTG GCC GTG CCT TG-3Ј, W47A (5Ј-GT TTT TAC CTC GCC GCC TGT TTC CCC-3Ј), W82A (5Ј-C TTG GGA TTT TTG CTG GCG ATC GTC TGC TGG GC-3Ј), W86A (5Ј-G CTG TGG ATC GTC TGC GCT GCA GAC CTC TTC TAC TC-3Ј), W94A (5Ј-C CTC TTC TAC TCT TTC GCG GAA AGA AGT CGG GGC-3Ј), W142A (5Ј-GGG ATC ATG CTC ACT TTC GCA CTG GTA GCC CTA ATG TG-3Ј), W142F (5Ј-G CTC ACT TTT TTC CTG GTA GCC C-3Ј); MSD2 Trp mutants W361A (5Ј-C ACG AAG GCC CCA GAT GCG CAG GGC TAC TTC TAC-3Ј), W445A (5Ј-G TAC ATT AAC ATG ATC GCG TCA GCC CCC CTG CAA G-3Ј), W445F (5Ј-CG TAC ATT AAC ATG ATC TTC TCA GCC CCC CTG CAA GTC-3Ј), W445Y (5Ј-CC ACG TAC ATT AAC ATG ATC TAC TCA GCG CCC CTG CAA GTC-3Ј), W459A (5Ј-GCT CTC TAC CTC CTG GCG CTG AAT CTG GGC CC-3Ј), W553A (5Ј-G GGC ACC TTC ACC GCG GTC TGC ACG CCC-3Ј), W553F (5Ј-G GGC ACC TTC ACC TTC GTC TGC ACG CCC-3Ј), W553Y (5Ј-GCC CTG GGC ACC TTC ACA TAT GTC TGC ACG CCC-3Ј; and MSD3 Trp mutants W1198A (5Ј-C GTG GCC AAC AGG GCG CTG GCC GTG CGG C-3Ј), W1198F (5Ј-GTG GCC AAC AGG TTC CTG GCC GTG CGG C-3Ј), W1198Y (5Ј-GTG GCC AAC AGG TAC CTG GCC GTG CGG C-3Ј). Login to comment
50 Construction of MRP1-GFP Fusion Proteins-Constructs encoding GFP fusion proteins of selected MRP1 Trp mutations were generated by exchanging the 1.3-kb ClaI/AflII fragment of a pcDNA3.1(-)-MRP1-GFP construct with the comparable fragments containing the W445A, W553A, and W1198A mutations generated above and designated pcDNA3.1-W445A/MRP1-GFP, pcDNA3.1-W553A/MRP1-GFP, and pcDNA3.1-W1198A/MRP1-GFP, respectively (39). Login to comment
108 All four mutants generated (W361A, W445A, W459A, and W553A) were expressed at levels 60-90% those of wild-type MRP1, indicating that none of the mutations had a major effect on the expression levels of the protein. Login to comment
110 After 1 min, ATP-dependent [3 H]LTC4 uptake by the W445A and W553A MRP1 mutants was reduced by ϳ75 and 50%, respectively, whereas uptake by the W361A and W459A mutants was comparable with wild-type MRP1 (Fig. 3B). Login to comment
113 In contrast, after 1 min, [3 H]E217betaG uptake by the W361A, W445A, and W553A MRP1 mutants was ϳ50, 25, and 10%, respectively, of wild-type MRP1 (Fig. 3D). Login to comment
117 As shown in Fig. 4A, Ala substitution of Trp445 and Trp553 essentially eliminated apigenin-stimulated [3 H]GSH uptake by MRP1. Login to comment
119 Similarly, GSH-stimulated E13SO4 uptake levels by the W445A and W553A mutants were just 30 and Ͻ10% of wild-type MRP1 levels, respectively, whereas uptake by the W361A mutant was similar to wild-type MRP1, and uptake by W459A MRP1 was reduced by just 25% (Fig. 4B). Login to comment
121 A, ATP-dependent uptake of [3 H]LTC4 was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector pcDNA3.1(-) (E) and vectors containing wild-type MRP1 (f) and MSD2 Trp-Ala mutant MRP1 cDNAs (W361A, Œ; W445A, ; W459A, ࡗ; and W553A, q). Login to comment
124 C, the time course of ATP-dependent uptake of [3 H]E217betaG by wild-type MRP1 and MSD2 mutants W361A, W445A, W459A, and W553A was measured as described for A. D, relative levels of [3 H]E217betaG uptake at 1 min are shown and were determined from the time course shown in C as described for B. Login to comment
126 Finally, [3 H]MTX uptake by the W445A and W553A MRP1 mutants, as observed for GSH and E13SO4 uptake, was dramatically reduced by more than 80% (Fig. 4C). Login to comment
139 One mutant, W445Y, was reproducibly expressed at lower levels (approximately half) than the corresponding W445A and W445F mutants for reasons that are presently unclear. Login to comment
140 The most conservatively substituted MRP1-Trp445 mutant, W445Y, showed significant transport activity with respect to all five MRP1 substrates compared with the W445A mutant after correcting for differences in MRP1 expression levels. Login to comment
142 The Phe-substituted Trp445 mutant, W445F, showed levels of transport intermediate between those of the W445A and W445Y mutants. Login to comment
148 In contrast to the W445A and W1198A mutants, more conservative substitutions of the Ala-substituted Trp553 mutant W553A were much less effective in restoring MRP1 transport activity. Login to comment
153 Relative uptake levels of 3 H-labeled organic anions by membrane vesicles enriched for wild-type MRP1 (solid bar) and W361A, W445A, W459A, and W553A mutant MRP1 proteins (shaded bars) were determined as described under "Experimental Procedures." Login to comment
182 Thus, Ala substitution of Trp445 essentially eliminated the transport of all five MRP1 substrates tested. Login to comment
189 Ala substitution of Trp1198 in MSD3 also resulted in a broad and profound decrease in MRP1 transport activity except for FIG. 6. ATP-dependent organic anion transport activity of wild-type and mutant MRP1 containing conservative Phe and Tyr substitutions of Trp1198 in TM16 of MSD3. A, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (W445A, W445F, and W445Y; W553A, W553F and W553Y; W1198A, W1198F, and W1198Y) MRP1 cDNAs. MRP1 proteins were detected with monoclonal antibody QCRL-1, and relative levels of expression shown under the blot were estimated by densitometry. Login to comment
191 Ala-substituted mutants (W445A, W553A, and W1198A; shaded bars) were included for comparison. Login to comment
224 HEK293T cells were transfected with pcDNA3.1(-)MRP1K-GFP (WT-MRP1-GFP) (A), pcDNA3.1-W445A/MRP1-GFP (B), pcDNA3.1-W553A/MRP1-GFP (C), and pcDNA3.1-W1198A/MRP1-GFP (D), and 48 h later, cells were processed for confocal fluorescence microscopy. Login to comment

PMID: 14965249 [PubMed] Haimeur A et al: "The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation."

No. Sentence Comment

264 Thus, Ala substitution of Trp445 (TM8), Trp553 (TM10) and Trp1198 (TM16) eliminated or substantially reduced transport of multiple organic anion substrates of MRP1. Login to comment

PMID: 16816140 [PubMed] Deeley RG et al: "Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins."

No. Sentence Comment

884 Thus Ala substitution of Trp445 (TM8), Trp553 (TM10), and Trp1198 (TM16) eliminated or dramatically reduced transport levels of a broad range of organic anion substrates. Login to comment
883 Thus Ala substitution of Trp445 (TM8), Trp553 (TM10), and Trp1198 (TM16) eliminated or dramatically reduced transport levels of a broad range of organic anion substrates. Login to comment
885 Thus Ala substitution of Trp445 (TM8), Trp553 (TM10), and Trp1198 (TM16) eliminated or dramatically reduced transport levels of a broad range of organic anion substrates. Login to comment

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

110 W445A and P448A mutations in TM8 substantially reduced the ATP-dependent transport of some MRP1 substrates, including LTC4, GSH, MTX, E217βG or E13SO4 [75, 77]. Login to comment

PMID: 19949927 [PubMed] Chang XB et al: "Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1."

No. Sentence Comment

104 Mutations of C43S in TM1 (112); P343A, K332L and K332D in TM6 (113, 114); W445A and P448A in TM8 (113, 115); T550A, T556A and P557A in TM10 (113, 116); N590A, F594A, P595A, N597A, S604A and S605A in TM11 (113, 117, 118); E1089Q, E1089A, E1089L, E1089N, K1092, S1097 and N1100 in TM14 (119, 120); R1197K in TM16 (121); Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y or R1249K in TM17 (121-124) significantly affect MRP1 function. Login to comment