ABCMdb

ABCC1 p.Arg1142Glu

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Publications

PMID: 16230346 [PubMed] Conseil G et al: "Functional importance of three basic residues clustered at the cytosolic interface of transmembrane helix 15 in the multidrug and organic anion transporter MRP1 (ABCC1)."

No. Sentence Comment

47 Substitutions of charged residues were generated in the pGEM-3Z- XmaI/MRP1 plasmid according to the manufacturer`s instructions with the following mutagenic primers (Integrated DNA Technologies, Inc., Coralville, IA), which also introduced an additional silent restriction site (substituted nucleotides are underlined): R1138E, 5Ј-CGT GGC TTC CTC CGA GCA ACT GAA GCG CCT CGA G-3Ј; R1138K, 5Ј-CGT GGC TTC CTC CAA GCA GCT TAA GCG CCT CGA G-3Ј; K1141E, 5Ј-CGG CAG CTG GAG CGC CTG GAG TCG GTC AGC-3Ј; K1141R, 5Ј-CCC GGC AGC TGC GGC GCC TCG AGT CGG-3Ј; R1142E, 5Ј-CCC GGC AGC TGA AGG AGC TCG AGT CGG TCA GCC G-3Ј;R1142K, 5Ј-CCC GGC AGC TGA AGA AGC TTG AGT CGG TCA GCC G-3Ј. Login to comment
112 Subsequent confocal microscopy studies showed that like wild-type MRP1, the Arg1138 and Arg1142 mutants are predominantly localized to the plasma membrane (Fig. 2B), although a portion of the opposite charge mutant R1142E could sometimes be found in intracellular membranes as well. Login to comment
118 A, membrane vesicles (1 ␮g of protein) prepared from HEK293T cells transfected with wild-type (WT-MRP1), and mutant (R1138E/K, K1141E/R, R1142E/K) MRP1 cDNAs were immunoblotted, and MRP1 was detected with monoclonal antibody QCRL-1. Login to comment
122 B, HEK293T cells were transfected with expression vectors encoding WT-MRP1 and MRP1 mutants R1138E, R1138K, K1141E, K1141R, R1142E, and R1142K, and 48 h later, cells were probed with monoclonal antibody QCRL-3 and processed for confocal fluorescence microscopy using Alexa Fluor 488 conjugated secondary antibody. Login to comment
136 Photolabeling of Arg1138 , Lys1141 , and Arg1142 Mutants of MRP1 with [3 H]LTC4-To ascertain whether the decrease in LTC4 uptake by the R1138E/K, K1141E, and R1142E/K mutants was related to a decrease in substrate binding, membrane vesicles enriched for wild-type or mutant MRP1s were photolabeled with [3 H]LTC4. Login to comment
139 Therefore, reduced substrate binding appeared to explain, at least in part, the loss of LTC4 transport for the R1138E and K1141E mutants but not the R1138K, R1142E, and R1142K mutants. Login to comment
163 In contrast, when the catalytic activity of MRP1 was measured at 37 °C by the orthovanadate-induced trapping of 8-azido-[␣32 P]ADP in the post-hydrolysis state (Fig. 6B), five of the six mutants (R1138E/K, R1142E/K, and K1141E) showed substantially reduced nucleotide trapping (ϳ40-60% that of wild-type MRP1). Login to comment
168 In contrast, although [3 H]LTC4 labeling of the R1142E and R1142K mutants was also eliminated by ATP alone, labeling in the orthovanadate-induced ADP trapped (low affinity) state was stronger for these two mutants than for wild-type MRP1. Login to comment
169 One possible explanation for this is that the ability of R1142E and R1142K to release LTC4 after ATP hydrolysis might be impaired, which could contribute to their reduced transport activity. Login to comment
211 As with the Arg1138 mutants and in contrast to the Lys1141 mutants, our observations with R1142K and R1142E indicated that it is necessary to preserve both the charge and the size of the cationic side chain at position 1142 for full MRP1 transport activity. This may reflect the capacity of Arg to form bidentate hydrogen bonds that Lys cannot. Login to comment
227 Wild-type (WT-MRP1) and Arg1142 mutant (R1142E, R1142K) membrane vesicles (50 ␮g of protein) were incubated in transport buffer containing 5 mM MgCl2 for 20 min in the absence (-) or presence (ϩ) of ATP (1 mM) and sodium orthovanadate (1 mM),aloneorincombination,andthenincubatedwith[3 H]LTC4 (200nM,90nCi)forafurther 30 min followed by UV cross-linking, SDS-PAGE, and fluorography. The levels of [3 H]LTC4 labeling in the presence of ATP, and ATP plus orthovanadate, are expressed relative to the labeling in the absence of both agents for each sample and are indicated by the numbers below the film. Login to comment

PMID: 21143116 [PubMed] He SM et al: "Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)."

No. Sentence Comment

744 The Arg1138Glu and Lys1141Glu mutants, but not the Arg1138Lys, Arg1142Glu, and Arg1142Lys mutants, showed a >50% decrease in binding to [3 H]LTC4. Login to comment